**4. Effects of culture managements on Fe/Zn and Cd/Pb accumulation in rice grains**

In rice seeds, Fe localizes to dorsal vascular bundle, aleurone layer and endosperm, and it localizes to the scutellum and vascular bundle of the scutellum of embryo [78]. Zn is distributed to all parts of the seed with a significantly high value for the aleurone layer and embryo [79]. Low Fe and Zn contents in rice are often restricted due to low available pools of Fe or Zn in soil. Enriching Fe or Zn concentration in grains through either fertilization or water management is referred to as agronomic biofortification, which is a short-term strategy for complementing the breeding programs.

Fe is abundant in mineral soil, but Fe deficiency still can occur in aerobic condition [80]. The major problem with Fe uptake is solubility. Fe in the soil (usually in the form of Fe2+, either chelated or as a sulfate salt) is easily converted to unavailable Fe3+ under aerobic condition. Thus, application of Fe as fertilizer is not an effective strategy for increasing rice seed Fe [81]. Otherwise, foliar application is a better option to overcome Fe deficiency, increasing grains Fe and its bioavailability in rice [82]. In contrast, as soil changes from aerobic to anaerobic conditions after flooding, Fe-oxides are dissolved when the Fe3+ is reduced to Fe2+, which weakens the oxide stability and increases its water solubility [83]. In fact, irrigation management in rice strongly influences soil redox potential, which affects the availability of Fe, so flooded soil nearly always has sufficient Fe for rice uptake [4].

Zn status and content in soil are the dominant factors restricting Zn content of rice seeds, followed by rice genotypes and fertilizers [84]. In aerobic condition, Zn mainly presents in soil in the form of ion Zn2+. The application of Zn as fertilizer is effective in promoting rice growth and also in the fortification of rice with Zn [85, 86]. However, the availability of Zn decreases with flooding due to precipitation as insoluble zinc sulfide [87] or as insoluble carbonates mixtures [83]. Positive effects of soil Zn fertilization on grains Zn have been noticed primarily with aerobic water management [84]. In addition, foliar Zn application compared to soil Zn fertilization has been more effective in improving grains Zn concentration in flooded condition [88].

Fe uptake and accumulation. P deprivation also enhances the sensitivity to Cd in rice plants by

Improving Rice Grain Quality by Enhancing Accumulation of Iron and Zinc While Minimizing…

An increasing number of evidences show that different N fertilizer forms and content affect Cd accumulation in rice. Sarwar et al. [98] reported that enhanced N application increased biomass production and reduced Cd toxicity to some extent due to dilution effect. N application increased soluble protein that could bind mobile Cd to immobile form. Different N fertilizer forms also have relationships with Cd uptake and accumulation in rice [102]. NH4

+ - 53


+ and

+

can trigger cell membrane

http://dx.doi.org/10.5772/intechopen.72826

and proton absorption by root

inhibiting biomass accumulation and reducing PCs synthesis deprivation.

containing fertilizer is considered to contribute to enhance Cd uptake [98]. NH4

+


−

Cd-PCs complexes and being transported into vacuoles in rice cells [96, 98, 110].

water and fertilizer management on Pb accumulation in rice grains.

within plants, resulting in Cd accumulation. In addition, NH<sup>4</sup>

accumulated more Cd than NH<sup>4</sup>

depolarization and lead to influx of NH<sup>4</sup>

experiment, suggesting that effects of NH<sup>4</sup>

should be applied during the grain filling stage.

found that rice fed with excess NO3

−

contrast, NO3

−

with NO3

fertilizers acidify rhizosphere by proton excretion from root cells, exchanging with NH4

leading to low pH in soil [103]. In low pH soil, Cd moves toward root system and translocates

from root to shoot though this mechanism reduces Cd uptake in a certain way [98, 103]. In

cells, leading to high pH [99], and cell membrane polarization caused by nitrate can produce Cd detoxification mechanism [98]. Nevertheless, Xie et al. [104] found that plants supplied

and NO3

to rhizosphere pH transformation or charge distribution of cell membrane. Yang et al. [105]

uptake by upregulating the expression of *OsIRT1.* In addition, Wangstrand et al. [106] once proposed that application of N fertilizer was dependent on different growth stages and recommended that more N fertilizer should be applied at the vegetative stage while less N doses

Besides P and N, other fertilizers are also related with Cd accumulation in rice. Fe is reported to remarkably increase Cd concentration in root and shoot of rice [107]. In contrast, a peculiar mechanism against Cd stress by application of Fe fertilizer is iron plague (IP) formation [96]. IP can serve as a barrier and prevent Cd from entering into root cells, resulting in reduced Cd accumulation while enhanced Fe concentration in rice [108]. Si application can reduce mobilization of Cd due to increased pH in soil [98], and complexes formation of Si with Cd is another mechanism for alleviating Cd toxicity in rice [96]. Application of S fertilizer may decrease Cd toxicity in the form of insoluble CdS, by which reduces mobility of Cd in soil [109]. S also participates in GSH and PCs biosynthesis. S increases Cd tolerance by forming

Pb accumulation in rice was wildly reported in Southeast Asian countries, such as China. Many researchers reported the toxic effects of Pb on rice growth and mineral absorption [111], but researches on reducing Pb accumulation in rice grains by water and fertilizer managements are still limited. Hu et al. [112] reported that Selenium (Se) application reduced Pb concentration in rice tissues but had no significant effect on Pb accumulation in brown rice grains. Soil remediation methods are applied to reduce Cd/Pb toxicity to some extent, including soil removal, replacement, inversion and flooded condition before and after heading [41, 113], but it is not easy to apply. In summary, more work is still needed to explore the effect of

−

+

+

+

−

into root cells, which accelerates translocation of Cd

treatment by *Thlaspi caerulescens* in hydroponic

not only enhanced Fe uptake but also increased Cd

on Cd uptake are not simply attributed

Although foliar application of Fe or Zn is more effective than soil application for increasing Fe or Zn content in rice grains, the efficiency of foliar applied Fe or Zn also depends on the application stages [89]. Late season foliar application of Zn or Fe at flowering or at early grain filling stage is more effective in improving grain Zn or Fe, respectively, than early season application [90, 91]. Although the level of Zn in grains is positively related with Fe, research showed that foliar fertilization of combined Fe and Zn fertilizers enhanced both grain Fe and Zn content without any antagonistic effects [82], indicating that fertilization of one element does not affect the grains concentration of the others [82, 92]. Totally, in order to increase both Fe and Zn content in rice grains under anaerobic or flooded conditions, the most effective fertilization strategy is a combination of foliar Zn and Fe spray soon after flowering or at early grain filling stage [4]. HarvestZinc Fertilizer Project started in 2008 and aimed at assessing the potential of Zn fertilizer in order to increase Zn content in cereal grains, especially in wheat and rice.

N fertilizer application has been reported to be related with Fe and Zn content in rice grains. Optimized N fertilizer application could increase grains Fe and Zn content in several crop species, including rice under sufficient Zn supply [92–94]. The reason is suggested as follows: (1) N nutrition promotes protein synthesis, which is a major sink for Fe and Zn [92]; (2) N nutrition enhances the expression of Zn and Fe transporter proteins, such as ZIP family transporters [92]; (3) N nutrition enhances the production of N compounds, such as NA and DMA [95]; (4) N nutrition increases Fe and Zn accumulation time by increasing vegetative growth and grain filling periods [4]. In contrast, N fertilizer can decrease rice grains Zn content under low Zn condition by increased biomass production and enhanced biological dilution [94]. In summary, optimized N fertilization application in rice production is very important to regulate Fe and Zn accumulation.

As a result of similar physical and chemical characteristics of Zn and Cd [96], Cd is mainly present as free Cd2+ in soil under aerobic condition regardless of soil redox potential [97], and the effect of flooding on Zn mobilization is indirect rather than direct compared with Fe. Cadmium in acidic soil is ionized as Cd<sup>+</sup> [48] and moves toward root system and translocates within plants, resulting in Cd accumulation eventually [98]. In previous reports, phosphate (P) fertilizer was thought to increase rice Cd accumulation [99, 100]. Because Cd emerges in the rock phosphate used for P fertilizer production, P fertilizers generally contain significant amounts of Cd [98]. Nowadays, these relatively high-Cd phosphate rock sources have been avoided in the fertilizer. Sarwar et al. [98] reported that mono-ammonium-phosphate (MAP) could enhance Cd solubility and uptake by lowering soil pH. However, Bolan et al. [12] reported that P fertilizer can reduce Cd solubility by insoluble Cd formation such as Cd(OH)<sup>2</sup> or Cd<sup>3</sup> (PO4 ) 2 . Yang et al. [101] proposed that P deprivation decreased rice Cd uptake by competitively increasing Fe uptake and accumulation. P deprivation also enhances the sensitivity to Cd in rice plants by inhibiting biomass accumulation and reducing PCs synthesis deprivation.

Zn status and content in soil are the dominant factors restricting Zn content of rice seeds, followed by rice genotypes and fertilizers [84]. In aerobic condition, Zn mainly presents in soil in the form of ion Zn2+. The application of Zn as fertilizer is effective in promoting rice growth and also in the fortification of rice with Zn [85, 86]. However, the availability of Zn decreases with flooding due to precipitation as insoluble zinc sulfide [87] or as insoluble carbonates mixtures [83]. Positive effects of soil Zn fertilization on grains Zn have been noticed primarily with aerobic water management [84]. In addition, foliar Zn application compared to soil Zn fertilization has been more effective in improving grains Zn concentration in flooded condition [88]. Although foliar application of Fe or Zn is more effective than soil application for increasing Fe or Zn content in rice grains, the efficiency of foliar applied Fe or Zn also depends on the application stages [89]. Late season foliar application of Zn or Fe at flowering or at early grain filling stage is more effective in improving grain Zn or Fe, respectively, than early season application [90, 91]. Although the level of Zn in grains is positively related with Fe, research showed that foliar fertilization of combined Fe and Zn fertilizers enhanced both grain Fe and Zn content without any antagonistic effects [82], indicating that fertilization of one element does not affect the grains concentration of the others [82, 92]. Totally, in order to increase both Fe and Zn content in rice grains under anaerobic or flooded conditions, the most effective fertilization strategy is a combination of foliar Zn and Fe spray soon after flowering or at early grain filling stage [4]. HarvestZinc Fertilizer Project started in 2008 and aimed at assessing the potential of Zn fertilizer in order to increase Zn content in cereal grains, especially in wheat and rice.

N fertilizer application has been reported to be related with Fe and Zn content in rice grains. Optimized N fertilizer application could increase grains Fe and Zn content in several crop species, including rice under sufficient Zn supply [92–94]. The reason is suggested as follows: (1) N nutrition promotes protein synthesis, which is a major sink for Fe and Zn [92]; (2) N nutrition enhances the expression of Zn and Fe transporter proteins, such as ZIP family transporters [92]; (3) N nutrition enhances the production of N compounds, such as NA and DMA [95]; (4) N nutrition increases Fe and Zn accumulation time by increasing vegetative growth and grain filling periods [4]. In contrast, N fertilizer can decrease rice grains Zn content under low Zn condition by increased biomass production and enhanced biological dilution [94]. In summary, optimized N fertilization application in rice production is very important to regu-

As a result of similar physical and chemical characteristics of Zn and Cd [96], Cd is mainly present as free Cd2+ in soil under aerobic condition regardless of soil redox potential [97], and the effect of flooding on Zn mobilization is indirect rather than direct compared with Fe. Cadmium

plants, resulting in Cd accumulation eventually [98]. In previous reports, phosphate (P) fertilizer was thought to increase rice Cd accumulation [99, 100]. Because Cd emerges in the rock phosphate used for P fertilizer production, P fertilizers generally contain significant amounts of Cd [98]. Nowadays, these relatively high-Cd phosphate rock sources have been avoided in the fertilizer. Sarwar et al. [98] reported that mono-ammonium-phosphate (MAP) could enhance Cd solubility and uptake by lowering soil pH. However, Bolan et al. [12] reported that P fertil-

et al. [101] proposed that P deprivation decreased rice Cd uptake by competitively increasing

izer can reduce Cd solubility by insoluble Cd formation such as Cd(OH)<sup>2</sup>

[48] and moves toward root system and translocates within

or Cd<sup>3</sup>

(PO4 ) 2 . Yang

late Fe and Zn accumulation.

52 Rice Crop - Current Developments

in acidic soil is ionized as Cd<sup>+</sup>

An increasing number of evidences show that different N fertilizer forms and content affect Cd accumulation in rice. Sarwar et al. [98] reported that enhanced N application increased biomass production and reduced Cd toxicity to some extent due to dilution effect. N application increased soluble protein that could bind mobile Cd to immobile form. Different N fertilizer forms also have relationships with Cd uptake and accumulation in rice [102]. NH4 + containing fertilizer is considered to contribute to enhance Cd uptake [98]. NH4 + -containing fertilizers acidify rhizosphere by proton excretion from root cells, exchanging with NH4 + and leading to low pH in soil [103]. In low pH soil, Cd moves toward root system and translocates within plants, resulting in Cd accumulation. In addition, NH<sup>4</sup> + can trigger cell membrane depolarization and lead to influx of NH<sup>4</sup> + into root cells, which accelerates translocation of Cd from root to shoot though this mechanism reduces Cd uptake in a certain way [98, 103]. In contrast, NO3 − -containing fertilizer causes simultaneous NO3 − and proton absorption by root cells, leading to high pH [99], and cell membrane polarization caused by nitrate can produce Cd detoxification mechanism [98]. Nevertheless, Xie et al. [104] found that plants supplied with NO3 − accumulated more Cd than NH<sup>4</sup> + treatment by *Thlaspi caerulescens* in hydroponic experiment, suggesting that effects of NH<sup>4</sup> + and NO3 − on Cd uptake are not simply attributed to rhizosphere pH transformation or charge distribution of cell membrane. Yang et al. [105] found that rice fed with excess NO3 − not only enhanced Fe uptake but also increased Cd uptake by upregulating the expression of *OsIRT1.* In addition, Wangstrand et al. [106] once proposed that application of N fertilizer was dependent on different growth stages and recommended that more N fertilizer should be applied at the vegetative stage while less N doses should be applied during the grain filling stage.

Besides P and N, other fertilizers are also related with Cd accumulation in rice. Fe is reported to remarkably increase Cd concentration in root and shoot of rice [107]. In contrast, a peculiar mechanism against Cd stress by application of Fe fertilizer is iron plague (IP) formation [96]. IP can serve as a barrier and prevent Cd from entering into root cells, resulting in reduced Cd accumulation while enhanced Fe concentration in rice [108]. Si application can reduce mobilization of Cd due to increased pH in soil [98], and complexes formation of Si with Cd is another mechanism for alleviating Cd toxicity in rice [96]. Application of S fertilizer may decrease Cd toxicity in the form of insoluble CdS, by which reduces mobility of Cd in soil [109]. S also participates in GSH and PCs biosynthesis. S increases Cd tolerance by forming Cd-PCs complexes and being transported into vacuoles in rice cells [96, 98, 110].

Pb accumulation in rice was wildly reported in Southeast Asian countries, such as China. Many researchers reported the toxic effects of Pb on rice growth and mineral absorption [111], but researches on reducing Pb accumulation in rice grains by water and fertilizer managements are still limited. Hu et al. [112] reported that Selenium (Se) application reduced Pb concentration in rice tissues but had no significant effect on Pb accumulation in brown rice grains. Soil remediation methods are applied to reduce Cd/Pb toxicity to some extent, including soil removal, replacement, inversion and flooded condition before and after heading [41, 113], but it is not easy to apply. In summary, more work is still needed to explore the effect of water and fertilizer management on Pb accumulation in rice grains.

## **5. Breeding and transgenic approaches to increase Fe/Zn and reduce Cd/Pb accumulation in rice grains**

Zn transport. Ishikawa et al. [128] detected four QTLs (*qGZn*9, *qGZn*10, *qGZn*2–1, *qGZn*2–2) responsible for high Zn accumulation, and *qGZn*9 showed the best effect, which provides

Improving Rice Grain Quality by Enhancing Accumulation of Iron and Zinc While Minimizing…

http://dx.doi.org/10.5772/intechopen.72826

55

Genotype dependence has been well observed for the accumulation of Cd in rice. More Cd accumulated in shoots and grains of *indica* cultivars than *japonica* cultivars [55, 129, 130]. Recently, Cd and Pb contents of 100 top Chinese rice cultivars were determined. The results also showed that *indica* accumulated more Cd than *japonica* [131]. Studies on rice screened for Cd-free, but Fe/Zn-rich cultivars have been an important issue in agricultural field. Significant efforts have been made on breeding of low-Cd accumulating rice cultivars in Japan, where Cd accumulation in rice grains has long been recognized as serious agricultural issues [41, 132]. Ishikawa et al. [132] identified and screened three low-Cd mutants (*lcd-kmt1*, *lcd-kmt2*, and *lcd-kmt3*) with *japonica* rice cultivar, Koshihikari, which acted as parent by the way of carbon ion-beam irradiation, showing that there were lower Cd concentration in grains of the three mutants than Koshihikari wide type (WT). Such three low-Cd mutants were attributed to mutations of *OsNRAMP5* responsible for Cd transport in rice by sequence analysis [132]. The three low-Cd mutants have different mutation sites in *OsNRAMP5*. An insertion of transposon *mPingA1*, which was activated by ion beam and preferred to insert into exon of *OsNRAMP5*, was identified in *lcd-kmt1*, resulting in nonfunction of OsNRAMP5 and decreased Cd accumulation in grains [132]. Similar results were observed in *lcd-kmt2* and *lcdkmt3* due to a single-base pair deletion and a large deletion in *OsNRAMP5*, respectively [132]. Meanwhile, Ishikawa et al. [132] proposed that *lcd-kmt1* and *lcd-kmt2* were more promising for breeding program according to agronomic traits as a consequence of earlier heading and smaller plant size than Koshihikari WT in *lcd-kmt3* [132]. In addition, Abe et al. [133] developed a novel population composed of 46 chromosome segment substitution lines (CSSLs), in which LAC23 served as donor segments and were substituted into Koshihikari. LAC23 could result in lower grain-to-straw ratio than Koshihikari [133]. Therefore, cultivars containing LAC23 performed low Cd content in grains [133]. As for breeding Fe/Zn-rich cultivars, Olive et al. [134] bred high level of ferritin cultivars with rice mega variety IR64 as background and introducing ferritin into endosperm increased Fe content in grains [132, 133]. IR64 mutants obtained from sodium azide treatment were reported to have high Zn level [123]. Booyaves et al. [135] expressed Arabibopsis IRT1 (*AtIRT1*) in high-iron NFP rice lines, which expressed *NICOTIANAMINE SYNTHASE* (*AtNAS1*) and *FERRITIN*, enhancing Fe contents in both

In addition, QTL analysis was also applied to identify responsible genes for Cd transport. QTL for Cd concentration in Anjana Dhan (*indica* rice cultivar) is identified on chromosome 7, responsive gene for which is *OsHMA3* [23, 55, 126]. Abe et al. [136] introduced a nonfunctional allele of *OsHMA3* from Jarian (*indica* rice cultivar) into Koshihikari (*japonica* rice cultivar) by marker-assisted selection, and these plants showed reduced Cd uptake from soil. Suppression of *OsLCT1* expression can decrease grains Cd accumulation by RNAi without influencing nutrient accumulation. On the contrary, Fe content in the brown rice is remarkably higher [16], suggesting that RNAi-mediated *OsLCT1* suppression in rice is a promising approach to establish "high Fe but low-Cd-rice." Furthermore, T-DNA-mediated *OsLCD* knockout mutant showed reduced grain Cd accumulation while having no negative effect on grain yield. Thus, the *lcd* mutant might be a probable mutant line for further research [137].

valuable allele for breeding rice with high Zn level in grains.

unpolished and polished grains.

Since 1992, researchers at International Rice Research Institute (IRRI) have evaluated the genetic variability of Fe [114] and Zn [115] concentration in rice grains. The range in Fe and Zn contents of 939 varieties tested in one study were 7.5–24.4 μg g−1 for Fe, and 13.5–58.4 μg g−1 for Zn [116]. Among these 939 varieties, high grains Fe and Zn concentrations were identified, including Jalmagna, Zuchem, and Xua Bue Nuo [116]. HarvestPlus Challenge Program launched in 2004 supports biofortification of staple food crops, including rice for increased Fe, Zn, and vitamin A. However, iron biofortification of rice based on conventional breeding has met with only marginal success. The iron level achieved to date is still too low to address the required target level set by HarvestPlus (around 14 μg g−1), indicating that iron biofortification in rice remains a challenge [117]. In contrast, a number of varieties of biofortified zinc in rice and wheat are now available or being tested in countries all over the world, including India, Bangladesh, and Pakistan. Complementing the traditional breeding efforts, modern transgenic technology provides perspectives for efficiently improving Fe and Zn content of rice grains to dietary significant level for humans' nutrition [118].

Recent attempts on the biofortification of Fe and Zn in rice grains using transgenic techniques have shown some positive results. Overexpression of the barley NA synthase gene *HvNAS1* in rice plants caused an increase in DMA and NA concentrations in root, shoot, and seed, accompanied with enhanced Fe, Zn, and Cu concentrations in grains [119]. Zheng et al. [120] indicated that biofortified rice with NA could efficiently enhance bioavailability by overexpression *OsNAS1* in rice endosperm. Alexander et al. [121] constructed three rice populations overexpressing *OsNAS1*, *OsNAS2* and *OsNAS3*. These constitutive overexpression of the *OsNAS* genes led to increased NA level, positively correlated with enhanced Zn concentration both in unpolished and polished grains, which reduces Zn nutrient loss to some extent due to polishing process. Goto et al. [122] demonstrated that high level of Fe in rice endosperm could be acquired by overexpression of ferritin. Swamy et al. [123] suggested that overexpression of the ferritin gene *OsFer2* in basmati rice (Pusa Sugandh II) was observed to accumulate higher levels of Fe and Zn. Combination of upregulated expression of ferritin with overproduction of NA can significantly enhance Fe and Zn content [88]. In addition, manipulation of specific transporters involved in Fe/Zn uptake and translocation is also considered to be promising approach for enhancing Fe/Zn content. Ishimaru et al. [68] introduced *OsYSL2* mediated by sucrose transporter (OsSUT1) promoter into rice plants due to location of OsSUT1 around endosperm, resulting in high concentration of Fe in polished rice. Overexpression of the Fe transporter gene *OsIRT1* or *OsYSL15*, the Fe deficiency-inducible bHLH transcription factor OsIRO2, and knockdown of the vacuolar Fe transporter gene *OsVIT1* or *OsVIT2*, were regarded as an effective approaches to increase the Fe concentration of seeds [124]. Overexpression of OsHMA3 enhanced the uptake of Zn by upregulating the ZIP family genes in the root [125]. OsHMA2 was involved in loading of Zn to the developing tissues in rice [75]. Quantitative trait locus (QTL) analysis is a useful approach to identify responsible genes for the respective transport processes [126]. Anuradna et al. [127] identified QTLs and candidate genes for Fe/Zn transport in rice seeds. *OsYSL1* and *OsMTP1* are responsible for Fe transport, while *OsARD2*, *OsIRT1*, *OsNAS1*, *OsNAS2* are responsible for Zn transport. Ishikawa et al. [128] detected four QTLs (*qGZn*9, *qGZn*10, *qGZn*2–1, *qGZn*2–2) responsible for high Zn accumulation, and *qGZn*9 showed the best effect, which provides valuable allele for breeding rice with high Zn level in grains.

**5. Breeding and transgenic approaches to increase Fe/Zn and reduce** 

rice grains to dietary significant level for humans' nutrition [118].

Since 1992, researchers at International Rice Research Institute (IRRI) have evaluated the genetic variability of Fe [114] and Zn [115] concentration in rice grains. The range in Fe and Zn contents of 939 varieties tested in one study were 7.5–24.4 μg g−1 for Fe, and 13.5–58.4 μg g−1 for Zn [116]. Among these 939 varieties, high grains Fe and Zn concentrations were identified, including Jalmagna, Zuchem, and Xua Bue Nuo [116]. HarvestPlus Challenge Program launched in 2004 supports biofortification of staple food crops, including rice for increased Fe, Zn, and vitamin A. However, iron biofortification of rice based on conventional breeding has met with only marginal success. The iron level achieved to date is still too low to address the required target level set by HarvestPlus (around 14 μg g−1), indicating that iron biofortification in rice remains a challenge [117]. In contrast, a number of varieties of biofortified zinc in rice and wheat are now available or being tested in countries all over the world, including India, Bangladesh, and Pakistan. Complementing the traditional breeding efforts, modern transgenic technology provides perspectives for efficiently improving Fe and Zn content of

Recent attempts on the biofortification of Fe and Zn in rice grains using transgenic techniques have shown some positive results. Overexpression of the barley NA synthase gene *HvNAS1* in rice plants caused an increase in DMA and NA concentrations in root, shoot, and seed, accompanied with enhanced Fe, Zn, and Cu concentrations in grains [119]. Zheng et al. [120] indicated that biofortified rice with NA could efficiently enhance bioavailability by overexpression *OsNAS1* in rice endosperm. Alexander et al. [121] constructed three rice populations overexpressing *OsNAS1*, *OsNAS2* and *OsNAS3*. These constitutive overexpression of the *OsNAS* genes led to increased NA level, positively correlated with enhanced Zn concentration both in unpolished and polished grains, which reduces Zn nutrient loss to some extent due to polishing process. Goto et al. [122] demonstrated that high level of Fe in rice endosperm could be acquired by overexpression of ferritin. Swamy et al. [123] suggested that overexpression of the ferritin gene *OsFer2* in basmati rice (Pusa Sugandh II) was observed to accumulate higher levels of Fe and Zn. Combination of upregulated expression of ferritin with overproduction of NA can significantly enhance Fe and Zn content [88]. In addition, manipulation of specific transporters involved in Fe/Zn uptake and translocation is also considered to be promising approach for enhancing Fe/Zn content. Ishimaru et al. [68] introduced *OsYSL2* mediated by sucrose transporter (OsSUT1) promoter into rice plants due to location of OsSUT1 around endosperm, resulting in high concentration of Fe in polished rice. Overexpression of the Fe transporter gene *OsIRT1* or *OsYSL15*, the Fe deficiency-inducible bHLH transcription factor OsIRO2, and knockdown of the vacuolar Fe transporter gene *OsVIT1* or *OsVIT2*, were regarded as an effective approaches to increase the Fe concentration of seeds [124]. Overexpression of OsHMA3 enhanced the uptake of Zn by upregulating the ZIP family genes in the root [125]. OsHMA2 was involved in loading of Zn to the developing tissues in rice [75]. Quantitative trait locus (QTL) analysis is a useful approach to identify responsible genes for the respective transport processes [126]. Anuradna et al. [127] identified QTLs and candidate genes for Fe/Zn transport in rice seeds. *OsYSL1* and *OsMTP1* are responsible for Fe transport, while *OsARD2*, *OsIRT1*, *OsNAS1*, *OsNAS2* are responsible for

**Cd/Pb accumulation in rice grains**

54 Rice Crop - Current Developments

Genotype dependence has been well observed for the accumulation of Cd in rice. More Cd accumulated in shoots and grains of *indica* cultivars than *japonica* cultivars [55, 129, 130]. Recently, Cd and Pb contents of 100 top Chinese rice cultivars were determined. The results also showed that *indica* accumulated more Cd than *japonica* [131]. Studies on rice screened for Cd-free, but Fe/Zn-rich cultivars have been an important issue in agricultural field. Significant efforts have been made on breeding of low-Cd accumulating rice cultivars in Japan, where Cd accumulation in rice grains has long been recognized as serious agricultural issues [41, 132]. Ishikawa et al. [132] identified and screened three low-Cd mutants (*lcd-kmt1*, *lcd-kmt2*, and *lcd-kmt3*) with *japonica* rice cultivar, Koshihikari, which acted as parent by the way of carbon ion-beam irradiation, showing that there were lower Cd concentration in grains of the three mutants than Koshihikari wide type (WT). Such three low-Cd mutants were attributed to mutations of *OsNRAMP5* responsible for Cd transport in rice by sequence analysis [132]. The three low-Cd mutants have different mutation sites in *OsNRAMP5*. An insertion of transposon *mPingA1*, which was activated by ion beam and preferred to insert into exon of *OsNRAMP5*, was identified in *lcd-kmt1*, resulting in nonfunction of OsNRAMP5 and decreased Cd accumulation in grains [132]. Similar results were observed in *lcd-kmt2* and *lcdkmt3* due to a single-base pair deletion and a large deletion in *OsNRAMP5*, respectively [132]. Meanwhile, Ishikawa et al. [132] proposed that *lcd-kmt1* and *lcd-kmt2* were more promising for breeding program according to agronomic traits as a consequence of earlier heading and smaller plant size than Koshihikari WT in *lcd-kmt3* [132]. In addition, Abe et al. [133] developed a novel population composed of 46 chromosome segment substitution lines (CSSLs), in which LAC23 served as donor segments and were substituted into Koshihikari. LAC23 could result in lower grain-to-straw ratio than Koshihikari [133]. Therefore, cultivars containing LAC23 performed low Cd content in grains [133]. As for breeding Fe/Zn-rich cultivars, Olive et al. [134] bred high level of ferritin cultivars with rice mega variety IR64 as background and introducing ferritin into endosperm increased Fe content in grains [132, 133]. IR64 mutants obtained from sodium azide treatment were reported to have high Zn level [123]. Booyaves et al. [135] expressed Arabibopsis IRT1 (*AtIRT1*) in high-iron NFP rice lines, which expressed *NICOTIANAMINE SYNTHASE* (*AtNAS1*) and *FERRITIN*, enhancing Fe contents in both unpolished and polished grains.

In addition, QTL analysis was also applied to identify responsible genes for Cd transport. QTL for Cd concentration in Anjana Dhan (*indica* rice cultivar) is identified on chromosome 7, responsive gene for which is *OsHMA3* [23, 55, 126]. Abe et al. [136] introduced a nonfunctional allele of *OsHMA3* from Jarian (*indica* rice cultivar) into Koshihikari (*japonica* rice cultivar) by marker-assisted selection, and these plants showed reduced Cd uptake from soil. Suppression of *OsLCT1* expression can decrease grains Cd accumulation by RNAi without influencing nutrient accumulation. On the contrary, Fe content in the brown rice is remarkably higher [16], suggesting that RNAi-mediated *OsLCT1* suppression in rice is a promising approach to establish "high Fe but low-Cd-rice." Furthermore, T-DNA-mediated *OsLCD* knockout mutant showed reduced grain Cd accumulation while having no negative effect on grain yield. Thus, the *lcd* mutant might be a probable mutant line for further research [137].

Breeding low Pb cultivars is also considered to reduce Pb contamination. Developing rice cultivars with low Pb mobilization within root and translocation toward aerial parts to the minimum extent may be a better option to cultivate rice in Pb tainted soils. Li et al. [138] screened three cultivars (Tianyou196, Wufengyou2168, and Guinongzhan) with low Pb level in brown rice. Furthermore, Ashraf et al. [139] compared Meixiangzhan (MXZ-2), Xiangyaxiangzhan(XYXZ), Guixiangzhan (GXZ), Basmati-385 (B-385), and Nongxiang-18 (NX-18) to four different Pb concentrations, indicating that GXZ proved better able to tolerate Pb stress than all other rice cultivars, which are therefore suggested for use in future breeding programs for paddy fields contaminated by Pb.

Cd/Pb in grains based on functional QTLs or genes. These cultivars show no agriculturally or economically adverse traits and can be applied sooner. On the other hand, modern transgenic technology provides perspectives for efficiently improving Fe/Zn content and decreasing Cd/ Pb content in rice grains to dietary significant levels for humans' nutrition (As to Pb, more researches on QTL and genes still needed). Besides improving rice seeds, water and fertilizer management is also significantly related with increased Fe/Zn and decreased Cd/Pb in rice grains. More studies are still needed to optimize irrigation time, fertilizer categories, dosage, and application stages. In addition, although it is available to establish rice cultivars with high Fe or Zn content, or establish rice cultivars with low Cd or Pb separately, interactions among these metals need to be better understood, and more steps are still needed to cultivate rice with

Improving Rice Grain Quality by Enhancing Accumulation of Iron and Zinc While Minimizing…

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57

This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. LY18C130011 and LY15C130007, Science Foundation of Zhejiang Sci-Tech University, Foundation of Zhejiang Provincial Top Key Discipline of Biology, and Foundation

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, People's Republic of China

[1] Gómez-Galera S, Rojas E, Sudhakar D, Zhu C, Pelacho AM, Capell T, Christou P. Critical evaluation of strategies for mineral fortification of staple food crops. Transgenic

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all these merits and without decreasing rice production.

of Zhejiang Provincial Key Discipline of Botany.

\*Address all correspondence to: jiexiong@zju.edu.cn

Research. 2010;**19**:165-180. DOI: 10.1007/s11248-009-9311-y

**Acknowledgements**

**Author details**

**References**

Lei Gao and Jie Xiong\*

Dordrecht: Springer; 2002

fpls.2015.00121

### **6. Conclusions and perspectives**

Fe and Zn are essential nutrients for humans, but Cd and Pb are toxic at high levels for humans. All these metals accumulated in the grains of rice, a staple cereal worldwide. Compared with Pb, significant progress has been made in investigating the mechanisms for Fe, Zn, and Cd uptake and accumulation in rice grains. These basic discoveries provide us with the increasing possibility to establish high Fe/Zn and low Cd/Pb rice. Here, we summarized a strategy scheme for producing biofortified Fe/Zn but low Cd/Pb rice cultivars as follows (**Figure 2**). On the one hand, scientists have screened or bred nontransgenic rice cultivars with high Fe/Zn or/and low

**Figure 2.** Schematic diagram of strategies for enhancing accumulation of iron and zinc while minimizing cadmium and lead in rice. Based on functional QTLs or genes, scientists screen or breed nontransgenic rice cultivars with high Fe/ Zn or/and low Cd/Pb in grains. Modern transgenic technology provides perspectives for efficiently improving Fe/Zn content and decreasing Cd/Pb content in rice grains. Water and fertilizers management are also significantly related with increased Fe/Zn and decreased Cd/Pb in rice grains.

Cd/Pb in grains based on functional QTLs or genes. These cultivars show no agriculturally or economically adverse traits and can be applied sooner. On the other hand, modern transgenic technology provides perspectives for efficiently improving Fe/Zn content and decreasing Cd/ Pb content in rice grains to dietary significant levels for humans' nutrition (As to Pb, more researches on QTL and genes still needed). Besides improving rice seeds, water and fertilizer management is also significantly related with increased Fe/Zn and decreased Cd/Pb in rice grains. More studies are still needed to optimize irrigation time, fertilizer categories, dosage, and application stages. In addition, although it is available to establish rice cultivars with high Fe or Zn content, or establish rice cultivars with low Cd or Pb separately, interactions among these metals need to be better understood, and more steps are still needed to cultivate rice with all these merits and without decreasing rice production.

### **Acknowledgements**

Breeding low Pb cultivars is also considered to reduce Pb contamination. Developing rice cultivars with low Pb mobilization within root and translocation toward aerial parts to the minimum extent may be a better option to cultivate rice in Pb tainted soils. Li et al. [138] screened three cultivars (Tianyou196, Wufengyou2168, and Guinongzhan) with low Pb level in brown rice. Furthermore, Ashraf et al. [139] compared Meixiangzhan (MXZ-2), Xiangyaxiangzhan(XYXZ), Guixiangzhan (GXZ), Basmati-385 (B-385), and Nongxiang-18 (NX-18) to four different Pb concentrations, indicating that GXZ proved better able to tolerate Pb stress than all other rice cultivars, which are therefore suggested for use in future breeding

Fe and Zn are essential nutrients for humans, but Cd and Pb are toxic at high levels for humans. All these metals accumulated in the grains of rice, a staple cereal worldwide. Compared with Pb, significant progress has been made in investigating the mechanisms for Fe, Zn, and Cd uptake and accumulation in rice grains. These basic discoveries provide us with the increasing possibility to establish high Fe/Zn and low Cd/Pb rice. Here, we summarized a strategy scheme for producing biofortified Fe/Zn but low Cd/Pb rice cultivars as follows (**Figure 2**). On the one hand, scientists have screened or bred nontransgenic rice cultivars with high Fe/Zn or/and low

**Figure 2.** Schematic diagram of strategies for enhancing accumulation of iron and zinc while minimizing cadmium and lead in rice. Based on functional QTLs or genes, scientists screen or breed nontransgenic rice cultivars with high Fe/ Zn or/and low Cd/Pb in grains. Modern transgenic technology provides perspectives for efficiently improving Fe/Zn content and decreasing Cd/Pb content in rice grains. Water and fertilizers management are also significantly related with

programs for paddy fields contaminated by Pb.

**6. Conclusions and perspectives**

56 Rice Crop - Current Developments

increased Fe/Zn and decreased Cd/Pb in rice grains.

This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. LY18C130011 and LY15C130007, Science Foundation of Zhejiang Sci-Tech University, Foundation of Zhejiang Provincial Top Key Discipline of Biology, and Foundation of Zhejiang Provincial Key Discipline of Botany.

### **Author details**

Lei Gao and Jie Xiong\*

\*Address all correspondence to: jiexiong@zju.edu.cn

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, People's Republic of China

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**Chapter 5**

Provisional chapter

**Rice Production with Furrow Irrigation in the**

DOI: 10.5772/intechopen.74820

Furrow irrigated rice is an alternative method for growing rice with less water and labor than conventional flood irrigation. In the Mississippi River Delta region, layflat plastic pipe is used to supply water to furrows from irrigation wells. Different size holes are punched in pipe to optimize uniformity of water distribution. Beds are made before planting to channel water down furrows. Rice seed is planted in rows with a grain drill. Water infiltration in furrows is two-dimensional through a wetted perimeter with soil in the bottom of furrows and sidewalls of beds. An ideal field for furrow irrigation has no more than 0.1% slope with high clay content. No rice cultivars have been developed specifically for furrow irrigation but tests showed that some cultivars tolerate water stress better than others. In field trials, rice yields with furrow irrigation were lower than flooded rice with the greatest yield loss in the upper part of fields. However, results indicated that rice yields can be increased with proper timing of nitrogen fertilization

Farmers have grown rice in flooded fields for thousands of years. To survive in waterlogged soils, rice plants developed a unique plant structure. Within hours of submergence, rice plants produce aerenchyma cells to form air tubes in the stems which helps move oxygen internally from above the water to the roots [1]. This mechanism gives rice a competitive advantage over weeds that cannot survive in water. However, in the absence of flood water, rice plants lose this advantage with weeds and are not able to tolerate long periods of time without irrigation

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and irrigation and adaption of new rice herbicides for weed control.

Keywords: irrigation, furrow, beds, layflat pipe, scheduling

Rice Production with Furrow Irrigation in the

**Mississippi River Delta Region of the USA**

Mississippi River Delta Region of the USA

Gene Stevens, Matthew Rhine and Jim Heiser

Gene Stevens, Matthew Rhine and Jim Heiser

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74820

Abstract

1. Introduction


#### **Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA** Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA

DOI: 10.5772/intechopen.74820

Gene Stevens, Matthew Rhine and Jim Heiser Gene Stevens, Matthew Rhine and Jim Heiser

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74820

#### Abstract

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[139] Ashraf U, Hussain S, Anjum SA, Abbas F, Tanveer M, Noor MA, Tang XR. Alterations in growth, oxidative damage, and metal uptake of five aromatic rice cultivars under lead toxicity. Plant Physiology and Biochemistry. 2017;**115**:461-471. DOI: 10.1016/j.

Plant Molecular Biology. 2016;**90**:207-215. DOI: 10.1007/s11103-015-0404-0

from soil. Breeding Science. 2011;**61**:43-51. DOI: 10.1270/jsbbs.61.221

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DOI: 10.1073/pnas.1211132109

68 Rice Crop - Current Developments

291. DOI: 10.1270/jsbbs.63.284

DOI: 10.1007/s11032-013-9931-z

1798. DOI: 10.1016/S1001-0742(11)60972-8

plaphy.2017.04.019

Furrow irrigated rice is an alternative method for growing rice with less water and labor than conventional flood irrigation. In the Mississippi River Delta region, layflat plastic pipe is used to supply water to furrows from irrigation wells. Different size holes are punched in pipe to optimize uniformity of water distribution. Beds are made before planting to channel water down furrows. Rice seed is planted in rows with a grain drill. Water infiltration in furrows is two-dimensional through a wetted perimeter with soil in the bottom of furrows and sidewalls of beds. An ideal field for furrow irrigation has no more than 0.1% slope with high clay content. No rice cultivars have been developed specifically for furrow irrigation but tests showed that some cultivars tolerate water stress better than others. In field trials, rice yields with furrow irrigation were lower than flooded rice with the greatest yield loss in the upper part of fields. However, results indicated that rice yields can be increased with proper timing of nitrogen fertilization and irrigation and adaption of new rice herbicides for weed control.

Keywords: irrigation, furrow, beds, layflat pipe, scheduling

### 1. Introduction

Farmers have grown rice in flooded fields for thousands of years. To survive in waterlogged soils, rice plants developed a unique plant structure. Within hours of submergence, rice plants produce aerenchyma cells to form air tubes in the stems which helps move oxygen internally from above the water to the roots [1]. This mechanism gives rice a competitive advantage over weeds that cannot survive in water. However, in the absence of flood water, rice plants lose this advantage with weeds and are not able to tolerate long periods of time without irrigation

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or rainfall. Rice is less suited for aerobic soil conditions than other summer grain crops such as maize and sorghum.

2. Row beds, field slope and soil texture

water pools in low areas and flows across beds.

against gravity through small soil pores.

flood irrigated rice [20].

Before planting furrow irrigated rice, beds are made in fields to channel the flow of irrigation water in furrows down the slope in the field. Farmers typically use lister or disk hipper equipment pulled with tractors to make the beds in the fall. Winter rains firm the soil and melt soil clods into beds. Beds should be tall enough at rice planting to prevent irrigation water from breaking over bed tops. Sometimes, in place of beds, farmers can plant on flat soil and use furrow plows which cuts evenly spaced narrow trenches for water to flow. The optimum spacing of the water furrows depends on the lateral wicking or soaking properties of the soil. A common bed spacing is 76 cm (30 inch). Rice is planted parallel with beds using a grain drill in 19 cm (7.5 inch) row spacings. Depending on row spacing, water in furrows come in direct with only 20 percent of the soil in a field compared to complete soil coverage in conventional

Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA

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71

Rice plants on the tops of beds are the first to become water stressed and most prone to die in high evapotranspiration (ET) weather conditions. Water infiltration in furrow is twodimensional through a wetted perimeter with soil in the bottom of furrows and sidewalls of beds. Rice plants growing near the center of beds are the farthest from furrow water. Clay soils have smaller pores between individual particles than sand or silt. This causes clay soils to more effectively wick furrow irrigation water through small capillary pores across beds than loam soils (Figure 1). Capillary rise is the ability of water to flow in narrow spaces in opposition to

An ideal field for rice production with furrow irrigation is precision graded using lasers with no more than 0.1% slope with high clay content. For rice, a tail levee should be constructed after planting and stand establishment. This will save water and maintain near-flooded conditions in the low end of the field. At some point, a farmer will rotate rice to other crops such as soybean to disrupt disease and insect cycles. Soybeans require adequate surface drainage to avoid waterlogging and damaging roots [22]. Other crops usually need at least 0.10 to 0.15% slope to grow well. Most soils cannot adequately soak across beds for rice when slopes are greater than 0.2% because water flows too fast down the furrows. If the slope is not uniform,

Figure 1. Irrigation water infiltrates into soil below the furrow and wicks to each side and up into beds by capillary rise

gravity [21]. This is the action that allows paper towels to soak up liquid spills.

In environments where water is in short supply or pumping costs are high, producing rice with furrow irrigation saves water and fuel compared to flood irrigation. In the Mississippi River Delta Region of the United States, the main reason farmers grow furrow irrigated rice to avoid the labor needed to install and remove levees and gates [2]. This region includes the states of Louisiana, Arkansas, Missouri, Tennessee, and Mississippi. Furrow and center pivot irrigated rice grain usually has low arsenic content. In flooded soil, iron is reduced by anaerobic conditions releasing soluble arsenic for rice roots to uptake. Research in Arkansas and Missouri showed significantly less arsenic in the harvested grain from sprinkler and furrow irrigated rice compared to flooded rice [3–5].

No rice cultivars have been released by breeders developed specifically for furrow irrigation. Farmers need to plant cultivars with the best possible disease resistance. Asian rice (Oryza sativa) is divided into five groups: indica, aus, tropical japonica, temperate japonica, and aromatic [6]. The majority of rice cultivars grown in Mississippi River Delta are long-grain types selected with high amylose content for indica cooking properties. Cultivars grown here are mainly indica type but also may have one or more japonica parents in their pedigree [7]. Hybrid rice is often made by crossing indica and japonica parents which provides high heterosis vigor in offspring [8, 9]. Rice breeders typically select for progeny with increased yield potential and resistance to sheath blight [Rhizoctonia solani (Kuhn)] and blast [Pyricularia grisea (Cavara)] diseases [10, 11]. Blast which spread by wind borne spores is the main concern of farmers growing furrow irrigated rice. Sheath blight is spread by floating spores in flood water which does not apply to furrow irrigation. Blast control practices such as planting cultivars rated with good resistance or applying fungicides is not always enough to prevent the disease [12]. In flooded rice, blast disease is most severe in water stressed plants growing on top of levees or the highest part of a field where water is shallow. Blast can also be devastating in aerobic rice grown without flooding. Where rainfall and irrigation water are scarce, farmers need rice varieties to plant with improved drought tolerance and ability to resist diseases [13].

Approximately 40 million hectares of rice is grown in places around the world where water resources are limited [14]. Upland rice (Oryza glaberrima) grown in rainfed fields of sub-Sahara Africa are generally more tolerance to drought conditions than Asian rice. Using embryo rescue techniques, crosses were made between O. sativa and African upland (aerobic rainfed) rice (O. glaberrima) by scientists at the West Africa Rice Development Association [15]. These crossbred varieties are widely grown in Africa. However, when sufficient water is available either by irrigation or abundant rainfall, these cultivars often produce lower yields than their parent Asian rice lines.

Much effort has been placed in Asian countries on identifying genes in O. sativa rice responsible for tolerance to abiotic stresses such as high sodium and low soil moisture conditions [16–18]. Lee et al. [19] increased rice grain yields by 23–42% in drought stress conditions with plants overexpressing root specific OsERF71 compared to controls.

### 2. Row beds, field slope and soil texture

or rainfall. Rice is less suited for aerobic soil conditions than other summer grain crops such as

In environments where water is in short supply or pumping costs are high, producing rice with furrow irrigation saves water and fuel compared to flood irrigation. In the Mississippi River Delta Region of the United States, the main reason farmers grow furrow irrigated rice to avoid the labor needed to install and remove levees and gates [2]. This region includes the states of Louisiana, Arkansas, Missouri, Tennessee, and Mississippi. Furrow and center pivot irrigated rice grain usually has low arsenic content. In flooded soil, iron is reduced by anaerobic conditions releasing soluble arsenic for rice roots to uptake. Research in Arkansas and Missouri showed significantly less arsenic in the harvested grain from sprinkler and furrow

No rice cultivars have been released by breeders developed specifically for furrow irrigation. Farmers need to plant cultivars with the best possible disease resistance. Asian rice (Oryza sativa) is divided into five groups: indica, aus, tropical japonica, temperate japonica, and aromatic [6]. The majority of rice cultivars grown in Mississippi River Delta are long-grain types selected with high amylose content for indica cooking properties. Cultivars grown here are mainly indica type but also may have one or more japonica parents in their pedigree [7]. Hybrid rice is often made by crossing indica and japonica parents which provides high heterosis vigor in offspring [8, 9]. Rice breeders typically select for progeny with increased yield potential and resistance to sheath blight [Rhizoctonia solani (Kuhn)] and blast [Pyricularia grisea (Cavara)] diseases [10, 11]. Blast which spread by wind borne spores is the main concern of farmers growing furrow irrigated rice. Sheath blight is spread by floating spores in flood water which does not apply to furrow irrigation. Blast control practices such as planting cultivars rated with good resistance or applying fungicides is not always enough to prevent the disease [12]. In flooded rice, blast disease is most severe in water stressed plants growing on top of levees or the highest part of a field where water is shallow. Blast can also be devastating in aerobic rice grown without flooding. Where rainfall and irrigation water are scarce, farmers need rice varieties to plant with improved

Approximately 40 million hectares of rice is grown in places around the world where water resources are limited [14]. Upland rice (Oryza glaberrima) grown in rainfed fields of sub-Sahara Africa are generally more tolerance to drought conditions than Asian rice. Using embryo rescue techniques, crosses were made between O. sativa and African upland (aerobic rainfed) rice (O. glaberrima) by scientists at the West Africa Rice Development Association [15]. These crossbred varieties are widely grown in Africa. However, when sufficient water is available either by irrigation or abundant rainfall, these cultivars often produce lower yields than their

Much effort has been placed in Asian countries on identifying genes in O. sativa rice responsible for tolerance to abiotic stresses such as high sodium and low soil moisture conditions [16–18]. Lee et al. [19] increased rice grain yields by 23–42% in drought stress conditions with plants

maize and sorghum.

70 Rice Crop - Current Developments

irrigated rice compared to flooded rice [3–5].

drought tolerance and ability to resist diseases [13].

overexpressing root specific OsERF71 compared to controls.

parent Asian rice lines.

Before planting furrow irrigated rice, beds are made in fields to channel the flow of irrigation water in furrows down the slope in the field. Farmers typically use lister or disk hipper equipment pulled with tractors to make the beds in the fall. Winter rains firm the soil and melt soil clods into beds. Beds should be tall enough at rice planting to prevent irrigation water from breaking over bed tops. Sometimes, in place of beds, farmers can plant on flat soil and use furrow plows which cuts evenly spaced narrow trenches for water to flow. The optimum spacing of the water furrows depends on the lateral wicking or soaking properties of the soil. A common bed spacing is 76 cm (30 inch). Rice is planted parallel with beds using a grain drill in 19 cm (7.5 inch) row spacings. Depending on row spacing, water in furrows come in direct with only 20 percent of the soil in a field compared to complete soil coverage in conventional flood irrigated rice [20].

Rice plants on the tops of beds are the first to become water stressed and most prone to die in high evapotranspiration (ET) weather conditions. Water infiltration in furrow is twodimensional through a wetted perimeter with soil in the bottom of furrows and sidewalls of beds. Rice plants growing near the center of beds are the farthest from furrow water. Clay soils have smaller pores between individual particles than sand or silt. This causes clay soils to more effectively wick furrow irrigation water through small capillary pores across beds than loam soils (Figure 1). Capillary rise is the ability of water to flow in narrow spaces in opposition to gravity [21]. This is the action that allows paper towels to soak up liquid spills.

An ideal field for rice production with furrow irrigation is precision graded using lasers with no more than 0.1% slope with high clay content. For rice, a tail levee should be constructed after planting and stand establishment. This will save water and maintain near-flooded conditions in the low end of the field. At some point, a farmer will rotate rice to other crops such as soybean to disrupt disease and insect cycles. Soybeans require adequate surface drainage to avoid waterlogging and damaging roots [22]. Other crops usually need at least 0.10 to 0.15% slope to grow well. Most soils cannot adequately soak across beds for rice when slopes are greater than 0.2% because water flows too fast down the furrows. If the slope is not uniform, water pools in low areas and flows across beds.

Figure 1. Irrigation water infiltrates into soil below the furrow and wicks to each side and up into beds by capillary rise against gravity through small soil pores.

### 3. Layflat irrigation pipe

In the past, farmers used rigid aluminum pipe to apply furrow irrigation to crops in the Delta Region. Around 1990, rigid pipe began to be replaced by flexible, plastic layflat pipe [23]. The tubing is usually white in color and sold in large rolls. Generally, the thicker the mil of the plastic, the greater the pressure a pipe can handle without bursting. Most farmers use 6 or 10 mil thickness. A common type is 30 cm (12 inch) diameter, 10 mil thickness and rolls out to 402 m (1/4 mi) length. It costs around \$275 USD. It will handle up to 3785 liters per minute (1000 gallons per minute) and 90 millibars (1.3 pounds per square inch) pressure [24]. Layflat pipe is usually installed with a "polypipe roller" implement which is mounted on the three point hitch of a tractor. One end of the tubing is attached to a well pipe with nylon zip ties and duct tape (Figure 2). The tractor moves slowly across the end of the beds on the high end of the field. The roller has a small plow which cuts a groove in the soil and the layflat pipe is rolled out in the trench. The shallow trench help keep the tubing from shifting when irrigation water is pumped into it. It is best to install layflat pipe on a calm day to avoid empty pipe from blowing away before it can be filled with water. After the pipe has water in it, wind is usually not a problem.

The well should be started and water pumped into the pipe as soon as possible. After water reaches the open end of the pipe, a knot is tied in it. As water pressure increases in the pipe, holes are quickly punched in the plastic pipe at every furrow to avoid letting the pipe explode. To obtain even water flow across the field, small holes are punched near the well where pressure is highest. Hole sizes should be made progressively larger going away from the well. Computer programs such as PHAUCET developed by USDA-Natural Resources Conservation Service can be used to determine the optimum hole sizes to punch at each furrow [25]. In large fields, it may be difficult to maintain enough pressure in long runs of plastic pipe. To solve the problem, fields can be divided in sections with pipe gates opened and closed to irrigate one area at a time or in equal blocks in a split-set configuration using a programmable surge valve [26]. Irrigation with surge valves is usually done in two stages (Figure 3). The first stage

advances water in furrows across the field in the shortest possible time. The second stage cycles water sets to improve infiltration in soil on the upper end of a field. A tail levee helps avoid losing water to runoff. Since the crop is rice, flooding the lower end of a field is not a

Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA

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73

Linquist et al. [3] found that the reproductive stages of rice are the most sensitive to water stress. Alternating wetting and drying by irrigation in rice vegetative stages did not reduce yields if flooding was maintained from panicle initiation through harvest. In treatments where wetting and drying cycles was done the entire season methane emissions were reduced 93%

Rice is less forgiving than other crops when irrigation water is applied too late or in insufficient amounts. Most irrigation decisions by farmers are made by looking at the crops or soil. A national survey showed that 44% of farmers scheduled irrigation on fields based on visual condition of the crop and 25% checked the feel of the soil [27]. Only 3% used daily crop evapotranspiration (ET) and 3% used soil moisture sensors. Three percent of the farmers said

Irrigation scheduling programs are useful tools for improving water efficiency in furrow irrigated rice. Several state extension services have developed mobile phone apps linked to electronic weather station networks to calculate evapotranspiration (ET) used for irrigation scheduling [28–31]. Obtaining daily data is a challenge for farms located outside weather station networks. In a two year study, we compared electronic atmometers (ETgages) to weather stations [32]. The ETgages showed good accuracy at 1/10 the cost of a station for

problem unless it becomes more than .

Figure 3. Surge valve used to improve distribution of furrow irrigation in fields.

compared to continuous flooded rice.

they began irrigating when they saw their neighbor start.

4. Irrigation scheduling

supplying daily ET estimates.

Figure 2. Connecting plastic tubing to well pipe with zip ties and duct tape.

Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA http://dx.doi.org/10.5772/intechopen.74820 73

Figure 3. Surge valve used to improve distribution of furrow irrigation in fields.

advances water in furrows across the field in the shortest possible time. The second stage cycles water sets to improve infiltration in soil on the upper end of a field. A tail levee helps avoid losing water to runoff. Since the crop is rice, flooding the lower end of a field is not a problem unless it becomes more than .

Linquist et al. [3] found that the reproductive stages of rice are the most sensitive to water stress. Alternating wetting and drying by irrigation in rice vegetative stages did not reduce yields if flooding was maintained from panicle initiation through harvest. In treatments where wetting and drying cycles was done the entire season methane emissions were reduced 93% compared to continuous flooded rice.

### 4. Irrigation scheduling

3. Layflat irrigation pipe

72 Rice Crop - Current Developments

Figure 2. Connecting plastic tubing to well pipe with zip ties and duct tape.

In the past, farmers used rigid aluminum pipe to apply furrow irrigation to crops in the Delta Region. Around 1990, rigid pipe began to be replaced by flexible, plastic layflat pipe [23]. The tubing is usually white in color and sold in large rolls. Generally, the thicker the mil of the plastic, the greater the pressure a pipe can handle without bursting. Most farmers use 6 or 10 mil thickness. A common type is 30 cm (12 inch) diameter, 10 mil thickness and rolls out to 402 m (1/4 mi) length. It costs around \$275 USD. It will handle up to 3785 liters per minute (1000 gallons per minute) and 90 millibars (1.3 pounds per square inch) pressure [24]. Layflat pipe is usually installed with a "polypipe roller" implement which is mounted on the three point hitch of a tractor. One end of the tubing is attached to a well pipe with nylon zip ties and duct tape (Figure 2). The tractor moves slowly across the end of the beds on the high end of the field. The roller has a small plow which cuts a groove in the soil and the layflat pipe is rolled out in the trench. The shallow trench help keep the tubing from shifting when irrigation water is pumped into it. It is best to install layflat pipe on a calm day to avoid empty pipe from blowing away before it can be filled with water. After the pipe has water in it, wind is usually not a problem. The well should be started and water pumped into the pipe as soon as possible. After water reaches the open end of the pipe, a knot is tied in it. As water pressure increases in the pipe, holes are quickly punched in the plastic pipe at every furrow to avoid letting the pipe explode. To obtain even water flow across the field, small holes are punched near the well where pressure is highest. Hole sizes should be made progressively larger going away from the well. Computer programs such as PHAUCET developed by USDA-Natural Resources Conservation Service can be used to determine the optimum hole sizes to punch at each furrow [25]. In large fields, it may be difficult to maintain enough pressure in long runs of plastic pipe. To solve the problem, fields can be divided in sections with pipe gates opened and closed to irrigate one area at a time or in equal blocks in a split-set configuration using a programmable surge valve [26]. Irrigation with surge valves is usually done in two stages (Figure 3). The first stage

> Rice is less forgiving than other crops when irrigation water is applied too late or in insufficient amounts. Most irrigation decisions by farmers are made by looking at the crops or soil. A national survey showed that 44% of farmers scheduled irrigation on fields based on visual condition of the crop and 25% checked the feel of the soil [27]. Only 3% used daily crop evapotranspiration (ET) and 3% used soil moisture sensors. Three percent of the farmers said they began irrigating when they saw their neighbor start.

> Irrigation scheduling programs are useful tools for improving water efficiency in furrow irrigated rice. Several state extension services have developed mobile phone apps linked to electronic weather station networks to calculate evapotranspiration (ET) used for irrigation scheduling [28–31]. Obtaining daily data is a challenge for farms located outside weather station networks. In a two year study, we compared electronic atmometers (ETgages) to weather stations [32]. The ETgages showed good accuracy at 1/10 the cost of a station for supplying daily ET estimates.

Most state extension irrigation apps use the same algorithms to calculate daily soil water balances. The complex calculations are not displayed to users in most irrigation apps. The Penman-Monteith equation is usually used to estimate standardized short-grass evapotranspiration called ETo. The first version was developed in 1948 by Howard Penman and other engineers have fine-tuned it over the years [33]. ET is the combination of transpiration from the crop and evaporation of the water from the soil or plant surfaces. The University of Missouri Extension Service maintains an agricultural weather station network (mesonet) which provides weather data to farmers for managing irrigation. The weather stations must meet standards approved by the American Society of Agricultural Engineers [34]. Most of the 34 stations in the mesonet have a Campbell Scientific™ CR-1000 data logger which is programmed to calculate standardized short-grass evapotranspiration called ETo. For farmers calculating daily crop ET for irrigation scheduling, ETo is multiplied by a coefficient (Kc) specific to the crop in the field. In the Northern Hemisphere, ETo is usually highest in June, and July when days are longer. ETo varies from year to year which is a limitation for irrigation scheduling from printed charts that rely on long-term weather averages.

is that more nitrogen was lost by denitrification compared to treatments with less water. Averaged across irrigation trigger treatments, the lowest yield occurred in the upper parts of the test field. The lower part of the field had standing water part of the time because of water

Table 2. Rice yields from three locations in furrow irrigated field averaged across irrigation trigger treatments at the

Mg ha<sup>1</sup>

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Prior to the last decade, most farmers in the Mississippi River Delta region split nitrogen fertilizer between two or three applications in the season on flood irrigated rice [36]. A typical program was 100 kg N ha<sup>1</sup> applied immediately before flooding at the 5 leaf stage and 34 kg N ha<sup>1</sup> applied at internode elongation (IE) followed by 34 kg N ha<sup>1</sup> two weeks later. Now many farmers apply all the nitrogen before flooding. In 2017, a nitrogen test was conducted to evaluate timing nitrogen applications on furrow irrigated rice at four stages of growth. Total nitrogen ranged from 100 to 250 kg N ha<sup>1</sup> (Table 3). Results showed that

Treatment 5-leaf stage (5 L) Internode Elongation (IE) IE + 2 weeks Boot Total N Rice yield†

Table 3. Rice yields from nitrogen treatments at 5-leaf, internode elongation (IE), IE + 2 weeks, and boot growth stage at

 50 50 0 0 100 8.99 c 50 50 50 0 150 9.47 b 50 50 0 50 150 8.90 c 50 50 50 50 200 9.86 a 100 50 0 0 150 9.15 c 100 50 50 0 200 9.69 ab 100 50 0 50 200 9.06 c 100 50 50 50 250 9.62 ab

Yield values followed by the same letter were not significantly different at the 0.05 level.

the Missouri Rice research farm in Qulin, Missouri in 2017.

kg N ha<sup>1</sup> Mg ha<sup>1</sup>

held back by the end leeve (Table 2).

Application timing

Missouri Rice research farm in Qulin, Missouri in 2017.

Field location Rice yield†

Yield values followed by the same letter were not significantly different at the 0.05 level.

upper 8.61 c middle 9.32 b lower 10.09 a

5. Nitrogen management

†

†

Farmers do not have time to manually calculate daily crop ET and soil water deficits from weather data for their fields. The main difference between extension irrigation apps is their interface design and ease of use. Growers usually just want to know which fields on their farm need irrigation today or the coming week. Predictions such as crop growth from temperature are important but secondary. In 2015, the University of Missouri Extension Service released an irrigation app for mobile phones called the Crop Water Use app which uses daily ETo from the state mesonet [31]. Many of the equations in the Missouri program including crop coefficients were modified from the Arkansas Irrigation Scheduler. A crop coefficient for non-flooded rice was made working with scientists at University of Arkansas and USDA-Agricultural Research Service [35].

Irrigation frequency is impacted by the app setup settings by the farmer. In the Missouri program, soil available water holding capacity, rooting depth and percent allowable depletion determine the irrigation trigger. Fields with sandy soils with low available water holding capacity trigger faster and need smaller amounts of irrigation water more frequently than medium textured soils. In a field trial with furrow rice on silt loam soil, we found that setting the rooting depth at 30 cm (12 inches) in the app produced the highest grain yields in 2017 (Table 1). A possible explanation for the significantly lower yields with the 15 cm root setting


† Yield values followed by the same letter were not significantly different at the 0.05 level.

Table 1. Irrigation applications and rice yields for three root depth triggers in the crop water use app for furrow irrigated rice at the Missouri Rice research farm in Qulin, Missouri in 2017.


Table 2. Rice yields from three locations in furrow irrigated field averaged across irrigation trigger treatments at the Missouri Rice research farm in Qulin, Missouri in 2017.

is that more nitrogen was lost by denitrification compared to treatments with less water. Averaged across irrigation trigger treatments, the lowest yield occurred in the upper parts of the test field. The lower part of the field had standing water part of the time because of water held back by the end leeve (Table 2).

### 5. Nitrogen management

Most state extension irrigation apps use the same algorithms to calculate daily soil water balances. The complex calculations are not displayed to users in most irrigation apps. The Penman-Monteith equation is usually used to estimate standardized short-grass evapotranspiration called ETo. The first version was developed in 1948 by Howard Penman and other engineers have fine-tuned it over the years [33]. ET is the combination of transpiration from the crop and evaporation of the water from the soil or plant surfaces. The University of Missouri Extension Service maintains an agricultural weather station network (mesonet) which provides weather data to farmers for managing irrigation. The weather stations must meet standards approved by the American Society of Agricultural Engineers [34]. Most of the 34 stations in the mesonet have a Campbell Scientific™ CR-1000 data logger which is programmed to calculate standardized short-grass evapotranspiration called ETo. For farmers calculating daily crop ET for irrigation scheduling, ETo is multiplied by a coefficient (Kc) specific to the crop in the field. In the Northern Hemisphere, ETo is usually highest in June, and July when days are longer. ETo varies from year to year which is a limitation for irrigation

Farmers do not have time to manually calculate daily crop ET and soil water deficits from weather data for their fields. The main difference between extension irrigation apps is their interface design and ease of use. Growers usually just want to know which fields on their farm need irrigation today or the coming week. Predictions such as crop growth from temperature are important but secondary. In 2015, the University of Missouri Extension Service released an irrigation app for mobile phones called the Crop Water Use app which uses daily ETo from the state mesonet [31]. Many of the equations in the Missouri program including crop coefficients were modified from the Arkansas Irrigation Scheduler. A crop coefficient for non-flooded rice was made working with scientists at University of Arkansas and USDA-Agricultural Research

Irrigation frequency is impacted by the app setup settings by the farmer. In the Missouri program, soil available water holding capacity, rooting depth and percent allowable depletion determine the irrigation trigger. Fields with sandy soils with low available water holding capacity trigger faster and need smaller amounts of irrigation water more frequently than medium textured soils. In a field trial with furrow rice on silt loam soil, we found that setting the rooting depth at 30 cm (12 inches) in the app produced the highest grain yields in 2017 (Table 1). A possible explanation for the significantly lower yields with the 15 cm root setting

Rooting depth trigger Irrigations Total water in season Rice yield† cm number cm Mg ha<sup>1</sup> 15 15 76 8.98 c 30 11 55 9.68 a 45 7 36 9.36 b

Table 1. Irrigation applications and rice yields for three root depth triggers in the crop water use app for furrow irrigated

Yield values followed by the same letter were not significantly different at the 0.05 level.

rice at the Missouri Rice research farm in Qulin, Missouri in 2017.

scheduling from printed charts that rely on long-term weather averages.

Service [35].

74 Rice Crop - Current Developments

†

Prior to the last decade, most farmers in the Mississippi River Delta region split nitrogen fertilizer between two or three applications in the season on flood irrigated rice [36]. A typical program was 100 kg N ha<sup>1</sup> applied immediately before flooding at the 5 leaf stage and 34 kg N ha<sup>1</sup> applied at internode elongation (IE) followed by 34 kg N ha<sup>1</sup> two weeks later. Now many farmers apply all the nitrogen before flooding. In 2017, a nitrogen test was conducted to evaluate timing nitrogen applications on furrow irrigated rice at four stages of growth. Total nitrogen ranged from 100 to 250 kg N ha<sup>1</sup> (Table 3). Results showed that


† Yield values followed by the same letter were not significantly different at the 0.05 level.

Table 3. Rice yields from nitrogen treatments at 5-leaf, internode elongation (IE), IE + 2 weeks, and boot growth stage at the Missouri Rice research farm in Qulin, Missouri in 2017.


† Yield values followed by the same letter were not significantly different at the 0.05 level.

Table 4. Rice yields from nitrogen treatments at internode elongation (IE) + 2 weeks averaged across applications at other growth stages at the Missouri Rice research farm in Qulin, Missouri in 2017.


However, two hybrids exceeded yields of 10 Mg ha<sup>1</sup> in furrow irrigated rice with less than

Table 6. Rice yields from cultivars grown with furrow and flood irrigation at the Missouri Rice research farm in Qulin,

Mg ha<sup>1</sup> %

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Weed control programs for center pivot irrigated rice were discussed in an open access book chapter by Stevens (2015). Similar weed problems occur in furrow irrigated rice. The goal with all non-flooded rice is to maintain good weed control until the plants develops enough leaf

Often a difficult weed to control in non-flooded rice in the Mississippi River Delta is palmer amaranth pigweed (Amaranthus palmeri). In most fields, clomazone applied preemergence and propanil + quinclorac + halosulfuron applied when pigweed reach 2–4 leaf stage works well. If more pigweeds emerge later, another application of propanil + quinclorace or acifluorfen +

For many years, chemical companies did not released any new herbicides to control weeds in rice. Recently, saflufenacil (Sharpen™), an inhibitor of protoporphyrinogen oxidase (PPO inhibitor) was labeled by BASF to apply on rice postemergence before panicle initiation. In Missouri trials, Sharpen was effective for weed control but caused significant leaf burn at one location. Additionally, with the advent of PPO resistant Palmer amaranth, this herbicide may

A new broad spectrum arylpicolinate rice herbicide named Loyant™ (florpyrauxifen-benzyl) was released by DOW Chemical Company from a new class of chemicals after EPA approval in 2017. Missouri trials in 2015 showed that it might be a "game changer" for pigweed control and a good fit for non-flooded rice. A test in 2016 evaluated the effectiveness, crop injury, and

costs of different herbicide programs for non-flooded rice production.

15% reduction in yield compared to flood.

Missouri in 2017 (source: Nathan Goldschmidt).

Irrigation method

Permission to publish results was granted by MOARK Agricultural Research, LLC.

Cultivar Furrow Flood Difference

Diamond 9.73 11.69 1.97 20 CL272 7.31 9.32 2.02 28 Roy J 8.42 10.58 2.17 26 LaKast 8.27 10.48 2.22 27 MM17 6.15 9.22 3.07 50 Jupiter 8.01 11.89 3.88 48

canopy to shade emerging weeds.

7. Weed control

bentazon can be made.

become obsolete.

Table 5. Analysis of variance for effect of nitrogen treatment on rice yield at the Missouri Rice research farm in Qulin, Missouri in 2017.

treatments that included 50 kg N ha<sup>1</sup> applied two weeks after IE produced more rice than other treatments (Tables 4 and 5).

### 6. Rice cultivar and hybrid evaluation

A evaluation of rice cultivars and hybrids was conducted in 2017 in adjacent Missouri fields furrow and flood irrigated. Each line was randomized and replicated in each field. In every case, rice yields were higher in flooded plots compared to furrow irrigated plots (Table 6).



Permission to publish results was granted by MOARK Agricultural Research, LLC.

Table 6. Rice yields from cultivars grown with furrow and flood irrigation at the Missouri Rice research farm in Qulin, Missouri in 2017 (source: Nathan Goldschmidt).

However, two hybrids exceeded yields of 10 Mg ha<sup>1</sup> in furrow irrigated rice with less than 15% reduction in yield compared to flood.

### 7. Weed control

treatments that included 50 kg N ha<sup>1</sup> applied two weeks after IE produced more rice than

Table 5. Analysis of variance for effect of nitrogen treatment on rice yield at the Missouri Rice research farm in Qulin,

Table 4. Rice yields from nitrogen treatments at internode elongation (IE) + 2 weeks averaged across applications at other

A evaluation of rice cultivars and hybrids was conducted in 2017 in adjacent Missouri fields furrow and flood irrigated. Each line was randomized and replicated in each field. In every case, rice yields were higher in flooded plots compared to furrow irrigated plots (Table 6).

Mg ha<sup>1</sup> %

other treatments (Tables 4 and 5).

Missouri in 2017.

†

76 Rice Crop - Current Developments

6. Rice cultivar and hybrid evaluation

Irrigation method

Cultivar Furrow Flood Difference

RTXP760 10.43 11.59 1.16 11 CL153 7.51 8.77 1.26 17 RT7311 CL 10.89 12.40 1.51 14 CL XL745 9.83 11.64 1.81 18

IE +2 weeks Rice yield† kg N ha<sup>1</sup> Mg ha<sup>1</sup> 0 8.61 c 50 9.32 b

Yield values followed by the same letter were not significantly different at the 0.05 level.

growth stages at the Missouri Rice research farm in Qulin, Missouri in 2017.

N timing p Value 5 L 0.4484 IE + 2WK <0.0001 5 L\*IE + 2WK 0.4127 BT 0.7357 5 L\*BT 0.2687 IE + 2WK\*BT 0.2478 5 L\*IE + 2WK\*BT 0.2715

5 L = 5 leaf stage, IE = internode elongation, 2WK = 2 weeks, BT = boot growth stage.

Weed control programs for center pivot irrigated rice were discussed in an open access book chapter by Stevens (2015). Similar weed problems occur in furrow irrigated rice. The goal with all non-flooded rice is to maintain good weed control until the plants develops enough leaf canopy to shade emerging weeds.

Often a difficult weed to control in non-flooded rice in the Mississippi River Delta is palmer amaranth pigweed (Amaranthus palmeri). In most fields, clomazone applied preemergence and propanil + quinclorac + halosulfuron applied when pigweed reach 2–4 leaf stage works well. If more pigweeds emerge later, another application of propanil + quinclorace or acifluorfen + bentazon can be made.

For many years, chemical companies did not released any new herbicides to control weeds in rice. Recently, saflufenacil (Sharpen™), an inhibitor of protoporphyrinogen oxidase (PPO inhibitor) was labeled by BASF to apply on rice postemergence before panicle initiation. In Missouri trials, Sharpen was effective for weed control but caused significant leaf burn at one location. Additionally, with the advent of PPO resistant Palmer amaranth, this herbicide may become obsolete.

A new broad spectrum arylpicolinate rice herbicide named Loyant™ (florpyrauxifen-benzyl) was released by DOW Chemical Company from a new class of chemicals after EPA approval in 2017. Missouri trials in 2015 showed that it might be a "game changer" for pigweed control and a good fit for non-flooded rice. A test in 2016 evaluated the effectiveness, crop injury, and costs of different herbicide programs for non-flooded rice production.

Hybrid rice was drill planted under center pivots at the University of Missouri- Marsh Farm in Portageville, Missouri. Nitrogen was applied 56 kg urea-N ha<sup>1</sup> at first tiller growth stage with 112 kg N ha<sup>1</sup> split in five weekly UAN fertigations. Fungicide was applied by chemigation for blast control.

many grass weeds such as barnyardgrass and panicum species in addition to control of rice flatsedge, smallflower umbrella sedge and yellow nutsedge. Of the products evalutated in this study, only propanil offers any grass control. Propanil and Grandstand stunted or burned

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Figure 6. Plot photos from herbicide treatments for center pivot rice in 2016. DAT = Days after treatment. (a) Untreated check, (b) pigweed sprayed with Loyant (6 DAT), (c) propanil (21 DAT), (d) Loyant (21 DAT), (e) grandstand (21 DAT), (f)

sharpen (21 DAT).

pigweeds but most recovered and grew back later in the season (Figures 5 and 6).

Herbicide treatments were applied to small plots in replicated, randomized complete blocks. Chemicals were applied post-emergence on July 13 with a CO2 backpack sprayer. Treatments were: 1. Untreated check, 2. propanil, 3. Loyant, 4. Sharpen, and 5. Grandstand™. Each plot was visually rated 6 days and 21 days after treatment.

The primary weed in plots was palmer pigweed. Loyant did an excellent job of killing even large pigweeds with less crop injury than Sharpen (Figure 4). Loyant also provides control of

Figure 4. The crop water use app for mobile phones was released by the University of Missouri Extension Service in 2015.

Figure 5. Visual pigweed control on August 3, 2016 (21 days after treatment).

many grass weeds such as barnyardgrass and panicum species in addition to control of rice flatsedge, smallflower umbrella sedge and yellow nutsedge. Of the products evalutated in this study, only propanil offers any grass control. Propanil and Grandstand stunted or burned pigweeds but most recovered and grew back later in the season (Figures 5 and 6).

Hybrid rice was drill planted under center pivots at the University of Missouri- Marsh Farm in Portageville, Missouri. Nitrogen was applied 56 kg urea-N ha<sup>1</sup> at first tiller growth stage with 112 kg N ha<sup>1</sup> split in five weekly UAN fertigations. Fungicide was applied by chemigation for

Herbicide treatments were applied to small plots in replicated, randomized complete blocks. Chemicals were applied post-emergence on July 13 with a CO2 backpack sprayer. Treatments were: 1. Untreated check, 2. propanil, 3. Loyant, 4. Sharpen, and 5. Grandstand™. Each plot

The primary weed in plots was palmer pigweed. Loyant did an excellent job of killing even large pigweeds with less crop injury than Sharpen (Figure 4). Loyant also provides control of

Figure 4. The crop water use app for mobile phones was released by the University of Missouri Extension Service in 2015.

was visually rated 6 days and 21 days after treatment.

Figure 5. Visual pigweed control on August 3, 2016 (21 days after treatment).

blast control.

78 Rice Crop - Current Developments

Figure 6. Plot photos from herbicide treatments for center pivot rice in 2016. DAT = Days after treatment. (a) Untreated check, (b) pigweed sprayed with Loyant (6 DAT), (c) propanil (21 DAT), (d) Loyant (21 DAT), (e) grandstand (21 DAT), (f) sharpen (21 DAT).

### 8. Conclusions

All current cultivars and hybrid grown by farmers were bred for production with flood irrigation. Field trials showed that some lines are more productive with furrow irrigation than others. Scheduling irrigation application using weather based evapotranspiration calculation will take the guess work out of optimizing irrigation timing and rates. Applying nitrogen after internode elongation improved yields. New herbicide chemistry will help control problem weeds such as Palmer amaranth pigweeds.

[9] Ni J, Colowit P, Mackill D. Evaluation of genetic diversity in rice subspecies using

Rice Production with Furrow Irrigation in the Mississippi River Delta Region of the USA

http://dx.doi.org/10.5772/intechopen.74820

81

[10] Lee F, Rush M. Rice sheath blight: A major rice disease. Plant Disease. 1983;67:829-832

[11] Shull V, Hammer J. Genomic structure and variability in Pyricularia grisea. In: Zeigler R, Leong S, Teng P, editors. Rice Blast Disease. Wallingford, UK: International Rice Research

[12] Bonman J. Durable resistance to rice blast disease — Environmental influences. Euphytica.

[13] Lafitte H, Li Z, Vijayakumar C, Gao Y, Shi Y, Xu J, Fu B, Yu S, Ali A, Domingo J, Maghring R, Torres R, Mackill D. Improvement of rice drought tolerance through backcross breeding: Evaluation of donors and selection in drought nurseries. Field Crops

[14] Jeong B, Fuka S, Cooper M. Leaf water potential and osmotic adjustment as physilogical traits to improve drought tolerance in rice. Field Crops Research. 2002;76:153-163

[15] Linares O. African rice (Oryza glaberrima): History and future potential. Proceedings of the

[16] Jeong J, Kim Y, Baek K, Jung H, Ha S, Choi Y, Kim M, Reuzeau C, Kim J. Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field

[17] Quan R, Hu S, Zhang Z, Zhang H, Zhang Z, Huang R. Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance. Plant Biotechnology Journal.

[18] Zheng X, Chen B, Lu G, Han B. Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochemical and Biophysical Research Communications.

[19] Lee D, Jung H, Jang G, Jeong J, Kim Y, Ha S, Choi Y, Kim J. Overexpression of the OsERF71 transcription factor alters rice root structure and drought resistance. Plant

[20] United States Department of Agriculture–Natural Resources Conservation Service. Surface Irrigation. Part. Washington, DC: National Engineering Handbook; 2012. p. 623

[21] Baver L, Gardner WH, Gardner WR. Soil Physics. Fourth ed. New York: John Wiley and

[22] Rhine MD, Stevens G, Heiser JW, Vories E. Nitrogen fertilization on center pivot sprinkler

[23] Admire K, Burch H. Delivering water uniformly through layflat or rigid pipeline. American Society of Agricultural and Biological Engineers. Paper No. 93–2610. Chicago, IL. 1993

irrigated rice. Crop Manage. 2011:10. DOI: 10.1094/CM-2011-1021-01-RS

National Academy of Sciences USA. (PNAS). 2002;99:16360-16365

drought conditions. Plant Physiology. 2010;153:185-197

Physiology. 2016:175. DOI: https://doi.org/10.1104/pp.16.00379

microsatellite markers. Crop Science. 2002;42:601-607

Institute CAB Intern; 1994. pp. 65-86

1992;63:115-123

2010;8:476-488

2009;379:985-989

Sons; 1972

Research. 2006;97:77-86

### Author details

Gene Stevens\*, Matthew Rhine and Jim Heiser

\*Address all correspondence to: stevensw@missouri.edu

University of Missouri-Fisher Delta Research Center, Portageville, Missouri, USA

### References


8. Conclusions

80 Rice Crop - Current Developments

Author details

References

weeds such as Palmer amaranth pigweeds.

Gene Stevens\*, Matthew Rhine and Jim Heiser

Irrigation Science. 2002;21:139-144

Research. 2016;11:71-81

10.2134/cftm2016.12.008

Review. 2010;2:247-291

Global Change Biology 2015;21:407-417

\*Address all correspondence to: stevensw@missouri.edu

University of Missouri-Fisher Delta Research Center, Portageville, Missouri, USA

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[2] Vories E, Counce P, Keisling T. Comparison of flooded and furrow-irrigated rice on clay.

[3] Linquist B, Anders M, Adviento-Borbe M, Chaney R, Nalley L, da Rosa E, van Kessel C. Reducing greenhouse gas emissions, water use, and grain arsenic levels in rice systems.

[4] Aide M, Beighley D. Arsenic uptake by rice (Oryza sativa L.) having different irrigation regimes involving two southeastern Missouri soils. International Journal of Applied

[5] Stevens G, Rhine M, Vories E, Straatmann Z. Effect of irrigation and silicon fertilizer on total rice grain arsenic content and yield. Crop, Forage & Turfgrass Manage. 2017;3. DOI:

[6] Agrama H, Yan W, Jia M, Fjellstrom R, McClung A. Genetic structure associated with diversity and geographic distribution in the USDA rice world collection. National Science

[7] Sato Y, Nakamura I. American long-grain rice varieties belong to subspecies japonica. Rice Genetics Newsletter. 1993;10:72 Gramene. Rice Genetics Coop., Mishima, Japan [8] Virmani S, Sun Z, Mou T, Jauhar A, Mao C. Two-Line Hybrid Rice Breeding Manual. Los

Baños, Philippines: International Rice Research Institute; 2003

All current cultivars and hybrid grown by farmers were bred for production with flood irrigation. Field trials showed that some lines are more productive with furrow irrigation than others. Scheduling irrigation application using weather based evapotranspiration calculation will take the guess work out of optimizing irrigation timing and rates. Applying nitrogen after internode elongation improved yields. New herbicide chemistry will help control problem


[24] Enciso J, Peries X. Using flexible pipe (poly pipe) with surface irrigation. TX A&M Cooperate Extension Service. Bull. L-5469. College Station, TX. 2005

**Chapter 6**

**Provisional chapter**

**Rice Crop Rotation: A Solution for Weed Management**

The challenges for weed management have increased in rice cultivation due to the high number of cases of herbicide-resistant weeds, especially the widespread distribution of imidazolinone-resistant weedy rice. Therefore, there has been particular interest in preventive, physical, and cultural methods in recent decades. In this context, the adoption of the rice-soybean rotation is reported to be one of the most important factors for weed management in rice fields. Additionally, the use of a diversified crop rotation enables the implementation of a broader herbicide program, which is an important feature influencing weed population dynamics. Rice-soybean rotation has been adopted by farmers to control problematic weed species, reduce seed bank of troublesome weed species, and prevent rice grain yield and quality losses caused by its interference. This crop rotation scheme has brought several benefits when it comes to weed management; however, there are also some drawbacks when adopting this strategy such as the limited productivity of soybean and new weed species becoming problematic, such as *Conyza* species. Thus, this chapter explores the advantages and disadvantages of adopting crop rotation in Brazilian lowlands, and proposes a set of strategies to successfully implement crop rotation in

**Keywords:** rice-soybean rotation, herbicides, residual activity, weed resistance,

Weed management strategies are described as biological, cultural, chemical, or mechanical practices employed in an integrated manner to prevent and satisfactorily control weed infestations.

**Rice Crop Rotation: A Solution for Weed Management**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.75884

Ananda Scherner, Fábio Schreiber, André Andres, Germani Concenço, Matheus Bastos Martins and

Ananda Scherner, Fábio Schreiber, André Andres, Germani Concenço, Matheus Bastos Martins and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

lowland soils as a tool for weed management.

http://dx.doi.org/10.5772/intechopen.75884

Andressa Pitol

Andressa Pitol

**Abstract**

agriculture

**1. Introduction**


#### **Rice Crop Rotation: A Solution for Weed Management Rice Crop Rotation: A Solution for Weed Management**

DOI: 10.5772/intechopen.75884

Ananda Scherner, Fábio Schreiber, André Andres, Germani Concenço, Matheus Bastos Martins and Andressa Pitol Ananda Scherner, Fábio Schreiber, André Andres, Germani Concenço, Matheus Bastos Martins and Andressa Pitol

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75884

#### **Abstract**

[24] Enciso J, Peries X. Using flexible pipe (poly pipe) with surface irrigation. TX A&M

[25] Tacker P. Computer programs used for improved water management in Arkansas. American Society of Agricultural and Biological Engineers Paper. 62120, St. Joseph, MI. 2006

[26] Humpherys A. Surge irrigation: 2. Management. ICID Bulletin. 1989;38(2):49-61 International Commission on Irrigation and Drainage, 48, Nyaya Marg, Chanakyapuri, New

[27] Kebede H, Fisher D, Sui R, Reddy K. Irrigation methods and scheduling in the Delta region of Mississippi: Current status and strategies to improve irrigation efficiencies.

[28] Andales A, Arabi M, Bauder T, Wardle E, Traff K. WISE irrigation scheduler. CO State University, Soil and Crop Science, Fort Collins, CO. 2015, http://wise.colostate.edu/ [29] Migliaccio K, Morgan KT, Vellidis G, Zotarelli L, Fraisse C, Zurweller BA, Andreis JH, Crane JH, Rowland DL. Smartphone apps for irrigation scheduling. Transactions of the

[30] Peters T. Irrigation scheduler mobile user's manual and documentation. WA State University Extension Service, Pullman, WA. 2015. http://weather.wsu.edu/ism/ISMManual.

[31] Stevens G, Guinan P, Travlos J. Crop water use program for irrigation. Univ. MO. Ext.

[32] Straatmann Z, Stevens G, Vories E, Guinan P, Travlos J, Rhine M. Measuring short-crop reference evapotranspiration in a humid region using electronic atmometers. Agricultural

[33] Monteith JL. Evaporation from land surfaces: progress in analysis and prediction since 1948. In: Advances in Evapotranspiration, Proc. ASAE Conference on Evapotranspira-

[34] ASAE SW-244 Irrigation Management Subcommittee. 2004. Measurement and reporting practices for automated agricultural weather stations. ASAE EP505. St. Joseph, MI [35] Vories E, Stevens W, Rhine M, Straatmann Z. Investigating irrigation scheduling for rice using variable rate irrigation. Agricultural Water Management. 2017;179:314-323

[36] Stevens, Wrather A, Rhine M, Dunn D, Vories E. Predicting rice yield response to midseason nitrogen with plant area measurements. Agronomy Journal. 2008;100:387-392

ASABE. 2016;59(1):291-301 ISSN 2151-0032 DOI 10.13031/trans.59.11158

Cooperate Extension Service. Bull. L-5469. College Station, TX. 2005

American Journal of Plant Sciences. 2014;5:2917-2928

Delhi 110-021, India

82 Rice Crop - Current Developments

Bull. MP800. Columbia, MO. 2016

Water Management. 2018;195:180-186

tion. Chicago, IL. 1985. pp. 4-12

pdf

The challenges for weed management have increased in rice cultivation due to the high number of cases of herbicide-resistant weeds, especially the widespread distribution of imidazolinone-resistant weedy rice. Therefore, there has been particular interest in preventive, physical, and cultural methods in recent decades. In this context, the adoption of the rice-soybean rotation is reported to be one of the most important factors for weed management in rice fields. Additionally, the use of a diversified crop rotation enables the implementation of a broader herbicide program, which is an important feature influencing weed population dynamics. Rice-soybean rotation has been adopted by farmers to control problematic weed species, reduce seed bank of troublesome weed species, and prevent rice grain yield and quality losses caused by its interference. This crop rotation scheme has brought several benefits when it comes to weed management; however, there are also some drawbacks when adopting this strategy such as the limited productivity of soybean and new weed species becoming problematic, such as *Conyza* species. Thus, this chapter explores the advantages and disadvantages of adopting crop rotation in Brazilian lowlands, and proposes a set of strategies to successfully implement crop rotation in lowland soils as a tool for weed management.

**Keywords:** rice-soybean rotation, herbicides, residual activity, weed resistance, agriculture

#### **1. Introduction**

Weed management strategies are described as biological, cultural, chemical, or mechanical practices employed in an integrated manner to prevent and satisfactorily control weed infestations.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Since the introduction of herbicides, after the Second World War, the chemical approach has been the major method of weed control [1] and the reliance on herbicides, with limited diversification of mechanisms of action, has led to the appearance of increased cases of herbicideresistant weed species. Additionally, the lack of active ingredients with new mechanisms of action [2] and public concern associated with environmental and health hazards, further emphasizes the need to rethink herbicide use [3].

the seed bank size because it allows for changes in the timing of direct control strategies, such as tillage and herbicide spraying, and disrupts the germination periods of the weed species [8]. Weed germination is affected by crop cultivars and plant spacing due to changes in the canopy and thus the quality of the light that reaches the soil [9]. The incorporation of crops into rotation schemes that release allelopathic substances can be used as a tool to reduce the germination and emergence of some weed species [10]. Therefore, the right choice of crops and the sequence in which they appear is a strong tactic for preventing the establishment of several weed species in the field.

Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 85

Additionally, the use of a diversified crop rotation enables the implementation of a diversified herbicide rotation scheme. The use of herbicides is considered by some researchers to be the main factor influencing seed bank dynamics [11], as they can drastically reduce weed populations. Based on this scenario, this chapter aims to explore possibilities of crop rotation sequences in Brazilian lowlands, addressing the benefits and drawbacks of each crop sequence when it comes to weed management and crop productivity. Furthermore, the authors aim to propose a set of strategies that can be used to successfully implement crop rotation in lowland

An overview of the current resistant status of herbicide resistance and the efficiency of chemical control against weed species in Southern Brazil adds a new perspective to better understand the need of non-chemical weed management methods, such as crop rotation, as part of an IWM strategy in lowlands. **Table 1** summarizes the herbicides available for rice production in Brazil by mechanism of action, and provides the reader with valuable information about the control efficiency of these compounds against the most troublesome weed species. The problems with weed resistance in rice go far beyond weedy rice, with cases of herbicide-resistant biotypes being reported for *Echinochloa* spp. (*E. crus-galli*, *E. crus-pavonis*, and *E. colona*)*, Eleusine indica*, *Cyperus* spp. (*C. rotundus* and *C. difformis*), and *Sagittaria* spp. (*S. montevidensis*), which are mainly associated with the intensive use of ALS-inhibiting herbicides, poor crop rotation schemes, and cropping strategies such as irrigation systems. ALS-inhibiting herbicides are known to be highly efficient in low doses against a broad range of weed species and this is probably one of the main reasons associated with the great acceptance of the Clearfield® technology, reducing the use of other herbicides that were widely used before such as, pendi-

methalin, oxadiazon, oxifluorfen, thiobencarb, bentazon, propanil, and quinclorac.

*Echinochloa* spp. is also resistant to acetyl-CoA carboxylase (ACCase) inhibitors and quinclorac (AUX, auxin-mimic herbicides), while *Eleusine indica* and *Sagittaria* spp. are also resistant to ACCase and photosystem II (PS II) inhibitors, respectively. Quinclorac was widely used during the 1990s in Brazil to control *Echinochloa* spp. and *Aeschynomene* spp., and some researchers believe [12] that the first case of herbicide resistance in rice cultivation in the country is associated with this herbicide, which selected resistant plants of *Echinochloa* spp.

The current resistance problem evidences the urgent need of alternative management strategies to efficiently control these species and reduce the reliance on chemical control. The occurrence of resistant weed species, such as weedy rice, can reduce rice yields from 5 to 100%

soils as a tool for weed management.

**2. Weed resistance in Brazilian rice fields**

Brazilian rice production has changed considerably in the past decades, partially due to the availability of high-yielding varieties and improved production techniques that have increased productivity by approximately 50% in the Southern region. Considerable progress has been also achieved in terms of weed control with the introduction of the Clearfield® technology, which allowed producers to selectively control weedy rice (*Oryza sativa* L.) by using rice genotypes tolerant to the imidazolinone herbicides. The introduction of these varieties increased the yields by more than 2.5 t/ha, allowing productivity levels to be greater than 10 t/ha in these areas [4]. However, the continued monocropping exerted a selection pressure on the weed community, favoring weed species with phenotypes and phenology that are similar to rice, such as weedy rice and *Echinochloa* spp. Moreover, the intensive use of imidazolinone herbicides concomitantly with minimal alternative cultural practices being adopted, led to the appearance of resistant biotypes of these species.

Facing the widespread distribution of imidazolinone-resistant weedy rice in Brazil, there has been particular interest in preventive, physical, and cultural methods during recent decades. Weed control strategies in general should follow integrated weed management (IWM) principles, relying less on the use of the herbicides and, whenever feasible, including non-chemical methods [5]. IWM practices have not been adopted by all rice producers in Brazil and one of the greatest constraints is the pragmatic solution provided by the use of herbicides as compared to the long-term strategies used in IWM. In practice, IWM strategy is costly in short term and the biggest challenge is to persuade farmers to spend money in preventing problems, such as herbicide resistance, that they still do not have, but probably will face in their own fields in the near future. Herbicide resistance usually evolves due to a poor weed control program, based mainly on the chemical approach, which is largely under the farmer's own control. Thus, the recent cases and obstacles caused by herbicide resistance are changing farmers' perceptions, making them now more positive toward the adoption of non-chemical weed management methods as part of an IWM strategy.

In this context, a very diverse crop rotation is reported to be one of the most important factors in diversifying weed communities and affecting their seed bank dynamics. It is believed that a crop rotation scheme composed of crops with great variability in their biological traits can be the most effective tool for controlling weeds [6] and avoiding weed resistance. The variation of cropping sequences creates an unstable environment, which prevents the annual recurrence of particular weed species [7]. Crop rotation strategies may not eradicate troublesome species, but they can limit their growth and reproduction.

Factors such as the choice of crops and cultivars, plant row spacing, crop seeding rate, sowing date, and use of fertility-building measures have to be taken into account when planning crop sequences. These measures, when properly planned and implemented, can enhance a crop's competitive ability against weeds. Variation in crop sowing dates is one of the best strategies to reduce the seed bank size because it allows for changes in the timing of direct control strategies, such as tillage and herbicide spraying, and disrupts the germination periods of the weed species [8]. Weed germination is affected by crop cultivars and plant spacing due to changes in the canopy and thus the quality of the light that reaches the soil [9]. The incorporation of crops into rotation schemes that release allelopathic substances can be used as a tool to reduce the germination and emergence of some weed species [10]. Therefore, the right choice of crops and the sequence in which they appear is a strong tactic for preventing the establishment of several weed species in the field.

Additionally, the use of a diversified crop rotation enables the implementation of a diversified herbicide rotation scheme. The use of herbicides is considered by some researchers to be the main factor influencing seed bank dynamics [11], as they can drastically reduce weed populations. Based on this scenario, this chapter aims to explore possibilities of crop rotation sequences in Brazilian lowlands, addressing the benefits and drawbacks of each crop sequence when it comes to weed management and crop productivity. Furthermore, the authors aim to propose a set of strategies that can be used to successfully implement crop rotation in lowland soils as a tool for weed management.

### **2. Weed resistance in Brazilian rice fields**

Since the introduction of herbicides, after the Second World War, the chemical approach has been the major method of weed control [1] and the reliance on herbicides, with limited diversification of mechanisms of action, has led to the appearance of increased cases of herbicideresistant weed species. Additionally, the lack of active ingredients with new mechanisms of action [2] and public concern associated with environmental and health hazards, further

Brazilian rice production has changed considerably in the past decades, partially due to the availability of high-yielding varieties and improved production techniques that have increased productivity by approximately 50% in the Southern region. Considerable progress has been also achieved in terms of weed control with the introduction of the Clearfield® technology, which allowed producers to selectively control weedy rice (*Oryza sativa* L.) by using rice genotypes tolerant to the imidazolinone herbicides. The introduction of these varieties increased the yields by more than 2.5 t/ha, allowing productivity levels to be greater than 10 t/ha in these areas [4]. However, the continued monocropping exerted a selection pressure on the weed community, favoring weed species with phenotypes and phenology that are similar to rice, such as weedy rice and *Echinochloa* spp. Moreover, the intensive use of imidazolinone herbicides concomitantly with minimal alternative cultural practices being

Facing the widespread distribution of imidazolinone-resistant weedy rice in Brazil, there has been particular interest in preventive, physical, and cultural methods during recent decades. Weed control strategies in general should follow integrated weed management (IWM) principles, relying less on the use of the herbicides and, whenever feasible, including non-chemical methods [5]. IWM practices have not been adopted by all rice producers in Brazil and one of the greatest constraints is the pragmatic solution provided by the use of herbicides as compared to the long-term strategies used in IWM. In practice, IWM strategy is costly in short term and the biggest challenge is to persuade farmers to spend money in preventing problems, such as herbicide resistance, that they still do not have, but probably will face in their own fields in the near future. Herbicide resistance usually evolves due to a poor weed control program, based mainly on the chemical approach, which is largely under the farmer's own control. Thus, the recent cases and obstacles caused by herbicide resistance are changing farmers' perceptions, making them now more positive toward the adoption of non-chemical weed

In this context, a very diverse crop rotation is reported to be one of the most important factors in diversifying weed communities and affecting their seed bank dynamics. It is believed that a crop rotation scheme composed of crops with great variability in their biological traits can be the most effective tool for controlling weeds [6] and avoiding weed resistance. The variation of cropping sequences creates an unstable environment, which prevents the annual recurrence of particular weed species [7]. Crop rotation strategies may not eradicate troublesome

Factors such as the choice of crops and cultivars, plant row spacing, crop seeding rate, sowing date, and use of fertility-building measures have to be taken into account when planning crop sequences. These measures, when properly planned and implemented, can enhance a crop's competitive ability against weeds. Variation in crop sowing dates is one of the best strategies to reduce

emphasizes the need to rethink herbicide use [3].

84 Rice Crop - Current Developments

management methods as part of an IWM strategy.

species, but they can limit their growth and reproduction.

adopted, led to the appearance of resistant biotypes of these species.

An overview of the current resistant status of herbicide resistance and the efficiency of chemical control against weed species in Southern Brazil adds a new perspective to better understand the need of non-chemical weed management methods, such as crop rotation, as part of an IWM strategy in lowlands. **Table 1** summarizes the herbicides available for rice production in Brazil by mechanism of action, and provides the reader with valuable information about the control efficiency of these compounds against the most troublesome weed species. The problems with weed resistance in rice go far beyond weedy rice, with cases of herbicide-resistant biotypes being reported for *Echinochloa* spp. (*E. crus-galli*, *E. crus-pavonis*, and *E. colona*)*, Eleusine indica*, *Cyperus* spp. (*C. rotundus* and *C. difformis*), and *Sagittaria* spp. (*S. montevidensis*), which are mainly associated with the intensive use of ALS-inhibiting herbicides, poor crop rotation schemes, and cropping strategies such as irrigation systems. ALS-inhibiting herbicides are known to be highly efficient in low doses against a broad range of weed species and this is probably one of the main reasons associated with the great acceptance of the Clearfield® technology, reducing the use of other herbicides that were widely used before such as, pendimethalin, oxadiazon, oxifluorfen, thiobencarb, bentazon, propanil, and quinclorac.

*Echinochloa* spp. is also resistant to acetyl-CoA carboxylase (ACCase) inhibitors and quinclorac (AUX, auxin-mimic herbicides), while *Eleusine indica* and *Sagittaria* spp. are also resistant to ACCase and photosystem II (PS II) inhibitors, respectively. Quinclorac was widely used during the 1990s in Brazil to control *Echinochloa* spp. and *Aeschynomene* spp., and some researchers believe [12] that the first case of herbicide resistance in rice cultivation in the country is associated with this herbicide, which selected resistant plants of *Echinochloa* spp.

The current resistance problem evidences the urgent need of alternative management strategies to efficiently control these species and reduce the reliance on chemical control. The occurrence of resistant weed species, such as weedy rice, can reduce rice yields from 5 to 100%


to pose a high resistance risk [18]. In the past 30 years, more than 35 grass species have evolved resistance to ACCase-inhibiting herbicides worldwide, especially due to target-site resistance mechanism [19], which threatens the long-term use of this technology in paddy rice. Therefore, this new technology shows great potential to reduce problems with resistant grass species but to ensure the longevity and optimize its efficiency, it is necessary to carefully follow the recom-

Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 87

It is possible to observe that there is a great amount of herbicides registered for weed control in rice (**Table 1**). However, weed resistance has been reported for several molecules, especially for weedy rice and *Echinochloa* spp. There are some herbicides that provide satisfactory control of resistant biotypes when sprayed in pre-emergence, for instance, *Echinochloa* spp. resistant to ALS inhibitors can be controlled with the application of pendimethalin (MA) and clomazone (DOXP), with great control levels being reported (up to 95%) in experimental studies. However, it is likely that some plants will escape pre-emergence control and the herbicide options for post-emergence are quite limited because the species are already resistant to most

**Table 1** also shows that herbicides such as oxadiazon and oxyfluorfen (PPO) do not control *Echinochloa* spp. and weedy rice when applied in pre-emergence. However, when applied on a water layer before sowing the crop (label instructions), these herbicides can provide better control of such weeds. The application of these herbicides is quite complex and growers must follow carefully the label instructions to achieve greater control efficiency. Moreover, these herbicides are likely to contaminate the environment and can cause crop injuries, which are

It is also important to mention that the control levels given in **Table 1** for all herbicides are only expressed when they are applied following the instructions of the manufacturer, with

Based on the aforementioned facts, the need to include other control strategies, such as crop rotation that would enhance the number of molecules that can be used to control these species is evident. Nevertheless, it is important to mention that weed control levels provided by cultural measures are often meager in comparison to the efficacy of herbicides and, thus, do not reduce their need, at least in the short term. Moreover, the costs and the unpredictability of many cultural strategies are the main reasons why farmers are reluctant to adopt them, and IWM strategy will only be prioritized when the occurrence of resistant weed biotypes causes

Lowlands in Southern Brazil are mainly cultivated with rice in the summer period and kept uncultivated during the fallow season. Crop residues left on the soil surface can be used for cattle grazing and in some cases, cover crops are sown during the winter. In general, longterm crop rotation is not included in this cropping system due to the introduction of chemical fertilizers and pesticides, mechanization, and improved crop varieties [21]. However, crop

some of the reasons for their greatly reduced usage in recent years.

extreme failures and almost complete lost in herbicide efficacy [20].

**3. Crop rotation in Brazilian lowlands**

specific doses and at the correct development stage of the crop and weed.

mendations for use.

of them.

Information not available; no control; control < 50%; control 50–70%; control 71–95%; control > 95%. \*Product not registered to control the weed species. ACCase: lipid synthesis inhibition (inh. of ACCase); ALS: inhibition of ALS (branched chain amino acid synthesis); PS II: inhibition of photosynthesis PS II; PS I: PS I electron diversion; PPO: Inhibition of protoporphyrinogen oxidase; DOXP: Inhibition of DOXP (1-deoxy-d-xylulose-5-phosphate or clomazone) synthase; EPSPS: Inhibition of EPSPS (5-enolpyruvylshikimate-3-phosphate) synthase; MA: Inhibition of microtubule assembly; AUX: Synthetic auxin. Pre: Pre-emergence; Post: Post-emergence; NP: Application on needle point, glyphosate applied over the first-day emerging rice, R: Resistant. HRAC: Herbicide Resistance Action Committee. Font: SOSBAI, 2016 and Agrofit, 2017 < Available at: http://agrofit.agricultura.gov.br/agrofit\_cons/principal\_agrofit\_cons>.

**Table 1.** Application timing, control levels of the most troublesome weed species in Brazilian Rice.

[13], resulting in large economic losses [14]. Thus, greater use of cultural methods, such as crop rotation, should be taken into account to reduce the weed population, resulting in less dependence on herbicides, selection pressure, and herbicide resistance.

The majority of weed resistance cases in rice are reported for ALS inhibitors, indicating that herbicide use tends to shift to other mechanisms of action to efficiently control ALSresistant weed species. For example, clomazone and propanil provide a great control (>95%) of *Echinochloa* spp. in Brazil as indicated in **Table 1**; however, biotypes with resistance to those herbicides have been already reported in Arkansas and California due to their frequent use [15, 16]. Thus, it is likely that herbicide resistance might evolve in Brazil for these species as a consequence of the increasing frequency in which they are sprayed.

The future introduction of a new herbicide-tolerant technology for paddy rice in Brazil, the Provisia™ Rice System, includes post-emergence ACCase-inhibiting herbicides as an alternative to improve the control of resistant grass species such as weedy rice [17]. Therefore, this technology tends to increase the use of these herbicides, which is a mechanism of action considered to pose a high resistance risk [18]. In the past 30 years, more than 35 grass species have evolved resistance to ACCase-inhibiting herbicides worldwide, especially due to target-site resistance mechanism [19], which threatens the long-term use of this technology in paddy rice. Therefore, this new technology shows great potential to reduce problems with resistant grass species but to ensure the longevity and optimize its efficiency, it is necessary to carefully follow the recommendations for use.

It is possible to observe that there is a great amount of herbicides registered for weed control in rice (**Table 1**). However, weed resistance has been reported for several molecules, especially for weedy rice and *Echinochloa* spp. There are some herbicides that provide satisfactory control of resistant biotypes when sprayed in pre-emergence, for instance, *Echinochloa* spp. resistant to ALS inhibitors can be controlled with the application of pendimethalin (MA) and clomazone (DOXP), with great control levels being reported (up to 95%) in experimental studies. However, it is likely that some plants will escape pre-emergence control and the herbicide options for post-emergence are quite limited because the species are already resistant to most of them.

**Table 1** also shows that herbicides such as oxadiazon and oxyfluorfen (PPO) do not control *Echinochloa* spp. and weedy rice when applied in pre-emergence. However, when applied on a water layer before sowing the crop (label instructions), these herbicides can provide better control of such weeds. The application of these herbicides is quite complex and growers must follow carefully the label instructions to achieve greater control efficiency. Moreover, these herbicides are likely to contaminate the environment and can cause crop injuries, which are some of the reasons for their greatly reduced usage in recent years.

It is also important to mention that the control levels given in **Table 1** for all herbicides are only expressed when they are applied following the instructions of the manufacturer, with specific doses and at the correct development stage of the crop and weed.

Based on the aforementioned facts, the need to include other control strategies, such as crop rotation that would enhance the number of molecules that can be used to control these species is evident. Nevertheless, it is important to mention that weed control levels provided by cultural measures are often meager in comparison to the efficacy of herbicides and, thus, do not reduce their need, at least in the short term. Moreover, the costs and the unpredictability of many cultural strategies are the main reasons why farmers are reluctant to adopt them, and IWM strategy will only be prioritized when the occurrence of resistant weed biotypes causes extreme failures and almost complete lost in herbicide efficacy [20].

### **3. Crop rotation in Brazilian lowlands**

[13], resulting in large economic losses [14]. Thus, greater use of cultural methods, such as crop rotation, should be taken into account to reduce the weed population, resulting in less

Information not available; no control; control < 50%; control 50–70%; control 71–95%; control > 95%. \*Product not registered to control the weed species. ACCase: lipid synthesis inhibition (inh. of ACCase); ALS: inhibition of ALS (branched chain amino acid synthesis); PS II: inhibition of photosynthesis PS II; PS I: PS I electron diversion; PPO: Inhibition of protoporphyrinogen oxidase; DOXP: Inhibition of DOXP (1-deoxy-d-xylulose-5-phosphate or clomazone) synthase; EPSPS: Inhibition of EPSPS (5-enolpyruvylshikimate-3-phosphate) synthase; MA: Inhibition of microtubule assembly; AUX: Synthetic auxin. Pre: Pre-emergence; Post: Post-emergence; NP: Application on needle point, glyphosate applied over the first-day emerging rice, R: Resistant. HRAC: Herbicide Resistance Action Committee. Font: SOSBAI,

2016 and Agrofit, 2017 < Available at: http://agrofit.agricultura.gov.br/agrofit\_cons/principal\_agrofit\_cons>.

**Table 1.** Application timing, control levels of the most troublesome weed species in Brazilian Rice.

86 Rice Crop - Current Developments

The majority of weed resistance cases in rice are reported for ALS inhibitors, indicating that herbicide use tends to shift to other mechanisms of action to efficiently control ALSresistant weed species. For example, clomazone and propanil provide a great control (>95%) of *Echinochloa* spp. in Brazil as indicated in **Table 1**; however, biotypes with resistance to those herbicides have been already reported in Arkansas and California due to their frequent use [15, 16]. Thus, it is likely that herbicide resistance might evolve in Brazil for these species as a

The future introduction of a new herbicide-tolerant technology for paddy rice in Brazil, the Provisia™ Rice System, includes post-emergence ACCase-inhibiting herbicides as an alternative to improve the control of resistant grass species such as weedy rice [17]. Therefore, this technology tends to increase the use of these herbicides, which is a mechanism of action considered

dependence on herbicides, selection pressure, and herbicide resistance.

consequence of the increasing frequency in which they are sprayed.

Lowlands in Southern Brazil are mainly cultivated with rice in the summer period and kept uncultivated during the fallow season. Crop residues left on the soil surface can be used for cattle grazing and in some cases, cover crops are sown during the winter. In general, longterm crop rotation is not included in this cropping system due to the introduction of chemical fertilizers and pesticides, mechanization, and improved crop varieties [21]. However, crop rotation is one of the essential practices in sustainable agricultural systems, because of its effects on soil fertility, control of pathogens and pests, including weeds.

Yield reduction due to weed competition in rice cropping is estimated between 10 and 15% of potential production [22, 23]. Nevertheless, it was the widespread distribution of imidazolinone-resistant weedy rice that promoted the introduction of new crops in areas under rice monoculture. This strategy aims to reduce the seed bank of troublesome weed species and prevent rice grain yield and quality losses caused by weed interference. There are several mechanisms responsible for this effect, including allelopathy, changes in fauna, and disturbance patterns, which could diversify selection pressures by influencing seed bank dynamics. Some studies have shown that the seed bank of troublesome species in rice cultivation is greatly reduced when monoculture is abandoned [24, 25]. Rotation also affects species communities by determining the tillage frequency and effects attributed to cropping practices, such as herbicide programs, crop seed rate, and sowing time [26, 27].

Moreover, the introduction of other crops, such as soybean, in lowlands increases soil fertility due to nutrients cycling and reduces some disease pressure. Even though the positive effects of a very diverse crop rotation scheme that includes legumes and cereals are well recognized, the greatest constraints for the introduction of this strategy in lowlands are the drainage problems and the absence of species that endure long periods of water surplus in the soil. The poor natural drainage in these areas is usually the result of the flat relief associated with a shallow soil profile and impermeable sub-surface layer [28]. The physicochemical characteristics of the soils and the low natural fertility are other factors that affect crops performance in these fields [29].

the production can be a real obstacle to its wide cultivation. Moreover, the performance of this crop in lowland soils is highly associated with the choice of cultivar, adequate sowing date,

**Figure 1.** Number of seeds of *Echinochloa* spp. (a) and *Urochloa plantaginea* (b) under two tillage systems (conventional and direct drilling) and herbicide application (H- with or T-without) as a function of two crop rotation schemes: R-SOY rice-soybean and R-SOR—rice-sorghum, two tillage systems (C- conventional and D- direct drilling) and herbicide

application (with or without). Treatment means were compared on the basis of 95% confidence intervals.

From the crop rotation point of view, the introduction of maize and sorghum in areas under rice monoculture brings several benefits for weed management; however, the inclusion of a legume species adds much more diversity to this system. Thus, soybean is probably the most promising crop to be used in a crop rotation scheme with rice, allowing farmers to increase their income, control weeds more efficiently by diversifying herbicide mechanisms of action of the herbicides and cultural practices. Moreover, leguminous species increase nitrogen (N)

Studies evaluating the performance of soybean in Brazilian lowlands showed that this crop may be highly productive in these soils, reaching more than 4.000 kg ha−1 [32]. However, soybean is still less profitable than rice in lowland soils because the crop is quite sensitive to water excess, especially during germination and emergence. Water surplus in soil during flowering and grain filling can also affect soybean productivity, though the crop is slightly less sensitive to this stress at those development stages [33]. Thus, it is clear that the feasibility of the ricesoybean rotation depends on the progress of research works for the adaptation of different genotypes to flooding and poor drainage conditions. Moreover, the compaction of lowland soils and reduced nitrogen fixation due to low rhizobium activity are other limiting factors to soybean productivity. Nevertheless, soybean has been shown to be a valuable tool in controlling weedy rice and aquatic weed problems as well as reducing some disease pressure.

In a study conducted at Embrapa Temperate Agriculture (Pelotas-Brazil), evaluating the effects of two crop rotation systems: rice-soybean (R-SOY) and rice-sorghum (R-SOR); two tillage systems: conventional and direct drilling; and herbicide application: with or without; on the seed bank of *Echinochloa* spp. and *Urochloa plantaginea* (**Figure 1**), it was reported that in


Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 89

and the use nitrogen fertilizers [30].

availability in the soil due to symbiotic N<sup>2</sup>

crop and increasing the yields of cereals grown in succession [31].

Several studies aimed to evaluate the performance of various summer and winter crops to be used in a rotation scheme with rice in lowlands and will be explored in more detail in the following sub-sections.

#### **3.1. Summer crops**

Summer crops such as maize (*Zea mays*), sorghum (*Sorghum bicolor*), and soybean (*Glycine max*) have been explored in a crop rotation scheme with rice. Researchers have been trying to identify cultivars of these crops that can adapt to lowlands [30].

The performance of maize in these soils is quite limited because their physicochemical features do not favor the development and productivity of this crop. Lowlands soils in Southern Brazil are generally acidic, with low pH (ranging from 4.5 to 5.4), and maize plants develop better in soils with pH close to 7. Therefore, liming the soil is an essential practice in these soils to allow maize cultivation [31]. The choice of a maize cultivar with vigorous stalk, adequate height, low spike insertion, reduced lodging, and breaking resistance is another aspect that has to be considered when including this crop in a crop rotation system with rice [30].

On the other hand, sorghum is a species that adapts well in lowland soils because it has a great tolerance to drought periods and water excess when in the advanced stages of development (more than five leaves), producing up to 70 t/ha of biomass that can be used for cattle grazing. Therefore, the introduction of this species in a rotation system with rice can be a tool to reduce the seed bank of troublesome species in these areas (**Figure 1**), though trading of

rotation is one of the essential practices in sustainable agricultural systems, because of its

Yield reduction due to weed competition in rice cropping is estimated between 10 and 15% of potential production [22, 23]. Nevertheless, it was the widespread distribution of imidazolinone-resistant weedy rice that promoted the introduction of new crops in areas under rice monoculture. This strategy aims to reduce the seed bank of troublesome weed species and prevent rice grain yield and quality losses caused by weed interference. There are several mechanisms responsible for this effect, including allelopathy, changes in fauna, and disturbance patterns, which could diversify selection pressures by influencing seed bank dynamics. Some studies have shown that the seed bank of troublesome species in rice cultivation is greatly reduced when monoculture is abandoned [24, 25]. Rotation also affects species communities by determining the tillage frequency and effects attributed to cropping practices,

Moreover, the introduction of other crops, such as soybean, in lowlands increases soil fertility due to nutrients cycling and reduces some disease pressure. Even though the positive effects of a very diverse crop rotation scheme that includes legumes and cereals are well recognized, the greatest constraints for the introduction of this strategy in lowlands are the drainage problems and the absence of species that endure long periods of water surplus in the soil. The poor natural drainage in these areas is usually the result of the flat relief associated with a shallow soil profile and impermeable sub-surface layer [28]. The physicochemical characteristics of the soils and the low natural fertility are other factors that affect crops performance in these fields [29]. Several studies aimed to evaluate the performance of various summer and winter crops to be used in a rotation scheme with rice in lowlands and will be explored in more detail in the

Summer crops such as maize (*Zea mays*), sorghum (*Sorghum bicolor*), and soybean (*Glycine max*) have been explored in a crop rotation scheme with rice. Researchers have been trying to

The performance of maize in these soils is quite limited because their physicochemical features do not favor the development and productivity of this crop. Lowlands soils in Southern Brazil are generally acidic, with low pH (ranging from 4.5 to 5.4), and maize plants develop better in soils with pH close to 7. Therefore, liming the soil is an essential practice in these soils to allow maize cultivation [31]. The choice of a maize cultivar with vigorous stalk, adequate height, low spike insertion, reduced lodging, and breaking resistance is another aspect that

has to be considered when including this crop in a crop rotation system with rice [30].

On the other hand, sorghum is a species that adapts well in lowland soils because it has a great tolerance to drought periods and water excess when in the advanced stages of development (more than five leaves), producing up to 70 t/ha of biomass that can be used for cattle grazing. Therefore, the introduction of this species in a rotation system with rice can be a tool to reduce the seed bank of troublesome species in these areas (**Figure 1**), though trading of

effects on soil fertility, control of pathogens and pests, including weeds.

such as herbicide programs, crop seed rate, and sowing time [26, 27].

identify cultivars of these crops that can adapt to lowlands [30].

following sub-sections.

88 Rice Crop - Current Developments

**3.1. Summer crops**

**Figure 1.** Number of seeds of *Echinochloa* spp. (a) and *Urochloa plantaginea* (b) under two tillage systems (conventional and direct drilling) and herbicide application (H- with or T-without) as a function of two crop rotation schemes: R-SOY rice-soybean and R-SOR—rice-sorghum, two tillage systems (C- conventional and D- direct drilling) and herbicide application (with or without). Treatment means were compared on the basis of 95% confidence intervals.

the production can be a real obstacle to its wide cultivation. Moreover, the performance of this crop in lowland soils is highly associated with the choice of cultivar, adequate sowing date, and the use nitrogen fertilizers [30].

From the crop rotation point of view, the introduction of maize and sorghum in areas under rice monoculture brings several benefits for weed management; however, the inclusion of a legume species adds much more diversity to this system. Thus, soybean is probably the most promising crop to be used in a crop rotation scheme with rice, allowing farmers to increase their income, control weeds more efficiently by diversifying herbicide mechanisms of action of the herbicides and cultural practices. Moreover, leguminous species increase nitrogen (N) availability in the soil due to symbiotic N<sup>2</sup> -fixation, lowering fertilizer needs for the following crop and increasing the yields of cereals grown in succession [31].

Studies evaluating the performance of soybean in Brazilian lowlands showed that this crop may be highly productive in these soils, reaching more than 4.000 kg ha−1 [32]. However, soybean is still less profitable than rice in lowland soils because the crop is quite sensitive to water excess, especially during germination and emergence. Water surplus in soil during flowering and grain filling can also affect soybean productivity, though the crop is slightly less sensitive to this stress at those development stages [33]. Thus, it is clear that the feasibility of the ricesoybean rotation depends on the progress of research works for the adaptation of different genotypes to flooding and poor drainage conditions. Moreover, the compaction of lowland soils and reduced nitrogen fixation due to low rhizobium activity are other limiting factors to soybean productivity. Nevertheless, soybean has been shown to be a valuable tool in controlling weedy rice and aquatic weed problems as well as reducing some disease pressure.

In a study conducted at Embrapa Temperate Agriculture (Pelotas-Brazil), evaluating the effects of two crop rotation systems: rice-soybean (R-SOY) and rice-sorghum (R-SOR); two tillage systems: conventional and direct drilling; and herbicide application: with or without; on the seed bank of *Echinochloa* spp. and *Urochloa plantaginea* (**Figure 1**), it was reported that in general, the number of seeds of both species in the seed bank was higher under rice-soybean rotation than in rice-sorghum rotation.

the control plot (test) due to the high infestation of *Echinochloa* spp. The control of *Echinochloa* spp. was satisfactory (> 80%) in all treatments, except when clethodim was sprayed, which showed similar results with the untreated plots (test). Clethodim resulted in lower control percentage for weedy rice as well, which were not statistically different from the control plots. Postemergence applications of glyphosate demonstrated good control for weedy rice. Moreover, a single application of s-metolachlor (DG2, **Figure 2**) provides more than 80% control for the three species. Thus, to avoid the pressure selection and future cases of weed resistance in soybean, the application of a pre-emergent followed by a post-emergent herbicide is a good control strategy in this scenario. Based on the results of this study, it is possible to observe that the herbicide rotation scheme is made viable by the inclusion of soybean into a crop rotation system in lowland soils, and can greatly reduce weed occurrence and consequently, the seed bank of

Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 91

Nowadays, soybean is considered the best option in a crop rotation scheme with rice in lowland soils, although it presents some obstacles. The variation of cropping sequences with the inclusion of soybean creates an unstable environment for most weeds, which prevents the annual recurrence of particular weed species that are promoted by rice cultivation. Crop rotation, in general, adds more diversity into the systems; however, a monotonous rotation scheme composed only of rice and soybean can exert a selection pressure on the weed community, favoring species most adapted to both crop environments. Therefore, weed species such as *Conyza* spp. that were not problematic in these areas when under rice monoculture, can be favored due to the introduction of soybean in the system. Moreover, *Echinochloa* spp. and weedy rice can become problematic for soybean cultivation if their control is not satisfactory and end up evolving resistance to frequently used herbicides such as glyphosate.

Winter cover crops, which are grown during an otherwise fallow period, are a possible means of improving weed control in rice cultivation. Cover crops are well known to improve nutrient dynamics, soil organic matter content, microbial activity, water retention, and prevent nitrate leaching [34]. Moreover, returning of crop straws has been suggested to improve overall soil conditions, reduce the requirement for N fertilizers, and support sustainable rice productivity. However, while the benefits of cover crops for nutrient management are well

Rice demands high amount of potassium (K), which is mainly accumulated in the straw residues, and is easily lost by leaching and surface runoff after crop harvesting. Therefore, the inclusion of cover crops composed of grass species that tend to produce a great amount of biomass and absorb nutrients such as nitrogen and potassium, are a great strategy for nutrient

Moreover, pertinent choices of cover crop species can suppress the growth of serious weeds and protect the soil during winter, resulting in a better soil structure as opposed to leaving soil bare. Italian ryegrass (*Lolium multiflorum*) is a grass species that has been widely used during winter in paddy soils and is a good option for cattle grazing and as cover crop. The species has great biomass production and high nutritional value for animals, as well as impressive

some troublesome species in rice cultivation.

documented, weed effects are less verified.

cycling, substantially avoiding nutrient losses [35].

**3.2. Winter crops**

The seed bank of both species under R-SOR rotation was not affected by tillage systems and herbicide treatment. In R-SOY rotation, the number of seeds of *Echinochloa* spp. was higher in direct drilling than in conventional tilling in the control treatments (without herbicides). Moreover, the inclusion of herbicides reduced the seed number of this species in both tillage systems under R-SOY rotation. On the other hand, the soil seed bank of *U. plantaginea* in a R-SOY rotation in the control plots was not affected by tillage, but the inclusion of herbicides reduced the number of seeds per m2 in direct-drilling plots. These results showed that rice-sorghum rotation is a good option to reduce the seed bank of *Echinochloa* spp. and *U. plantaginea* independently of tillage system and herbicide treatment. The success of a rice-soybean rotation to reduce the seed bank of these species depends on the tillage system and the inclusion of herbicides. When this system is not well manipulated, there is a risk of increasing the number of seeds in the seed bank as seen in the combination of this crop rotation (R-SOY) with direct drilling.

Another study, conducted at the same institution, tested several herbicide treatments that can be considered when soybean is introduced into a crop rotation system with rice to control troublesome species such as weedy rice, *Echinochloa* spp. and *U. plantaginea* (**Figure 2**). The results showed that all treatments efficiently controlled *U. plantaginea*, which was also suppressed in

**Figure 2.** Control (%) of *Echinochloa* spp., *Urochloa plantaginea*, and *Oryza sativa* (weedy rice) with different herbicides treatments, considering a rice-soybean rotation. Gly-one post-emergence application of 3 L ha−1 of glyphosate; clethodim—one post-emergence application of 600 mL ha−1 of clethodim; Gly/Gly—two post-emergence application of 3 L ha−1 of glyphosate; DG2- one pre-emergence application of s-metolachlor (dual gold); DG2/Gly- one pre-emergence application of s-metolachlor and one post-emergence application of 3 L ha−1 of glyphosate; DG2/Gly/Gly- one preemergence application of s-metolachlor and two post-emergence application of 3 L ha−1 of glyphosate. Treatment means were compared on the basis of 95% confidence intervals.

the control plot (test) due to the high infestation of *Echinochloa* spp. The control of *Echinochloa* spp. was satisfactory (> 80%) in all treatments, except when clethodim was sprayed, which showed similar results with the untreated plots (test). Clethodim resulted in lower control percentage for weedy rice as well, which were not statistically different from the control plots. Postemergence applications of glyphosate demonstrated good control for weedy rice. Moreover, a single application of s-metolachlor (DG2, **Figure 2**) provides more than 80% control for the three species. Thus, to avoid the pressure selection and future cases of weed resistance in soybean, the application of a pre-emergent followed by a post-emergent herbicide is a good control strategy in this scenario. Based on the results of this study, it is possible to observe that the herbicide rotation scheme is made viable by the inclusion of soybean into a crop rotation system in lowland soils, and can greatly reduce weed occurrence and consequently, the seed bank of some troublesome species in rice cultivation.

Nowadays, soybean is considered the best option in a crop rotation scheme with rice in lowland soils, although it presents some obstacles. The variation of cropping sequences with the inclusion of soybean creates an unstable environment for most weeds, which prevents the annual recurrence of particular weed species that are promoted by rice cultivation. Crop rotation, in general, adds more diversity into the systems; however, a monotonous rotation scheme composed only of rice and soybean can exert a selection pressure on the weed community, favoring species most adapted to both crop environments. Therefore, weed species such as *Conyza* spp. that were not problematic in these areas when under rice monoculture, can be favored due to the introduction of soybean in the system. Moreover, *Echinochloa* spp. and weedy rice can become problematic for soybean cultivation if their control is not satisfactory and end up evolving resistance to frequently used herbicides such as glyphosate.

### **3.2. Winter crops**

**Figure 2.** Control (%) of *Echinochloa* spp., *Urochloa plantaginea*, and *Oryza sativa* (weedy rice) with different herbicides treatments, considering a rice-soybean rotation. Gly-one post-emergence application of 3 L ha−1 of glyphosate; clethodim—one post-emergence application of 600 mL ha−1 of clethodim; Gly/Gly—two post-emergence application of 3 L ha−1 of glyphosate; DG2- one pre-emergence application of s-metolachlor (dual gold); DG2/Gly- one pre-emergence application of s-metolachlor and one post-emergence application of 3 L ha−1 of glyphosate; DG2/Gly/Gly- one preemergence application of s-metolachlor and two post-emergence application of 3 L ha−1 of glyphosate. Treatment means

general, the number of seeds of both species in the seed bank was higher under rice-soybean

The seed bank of both species under R-SOR rotation was not affected by tillage systems and herbicide treatment. In R-SOY rotation, the number of seeds of *Echinochloa* spp. was higher in direct drilling than in conventional tilling in the control treatments (without herbicides). Moreover, the inclusion of herbicides reduced the seed number of this species in both tillage systems under R-SOY rotation. On the other hand, the soil seed bank of *U. plantaginea* in a R-SOY rotation in the control plots was not affected by tillage, but the inclusion of herbicides reduced the

is a good option to reduce the seed bank of *Echinochloa* spp. and *U. plantaginea* independently of tillage system and herbicide treatment. The success of a rice-soybean rotation to reduce the seed bank of these species depends on the tillage system and the inclusion of herbicides. When this system is not well manipulated, there is a risk of increasing the number of seeds in the seed

Another study, conducted at the same institution, tested several herbicide treatments that can be considered when soybean is introduced into a crop rotation system with rice to control troublesome species such as weedy rice, *Echinochloa* spp. and *U. plantaginea* (**Figure 2**). The results showed that all treatments efficiently controlled *U. plantaginea*, which was also suppressed in

bank as seen in the combination of this crop rotation (R-SOY) with direct drilling.

in direct-drilling plots. These results showed that rice-sorghum rotation

rotation than in rice-sorghum rotation.

number of seeds per m2

90 Rice Crop - Current Developments

were compared on the basis of 95% confidence intervals.

Winter cover crops, which are grown during an otherwise fallow period, are a possible means of improving weed control in rice cultivation. Cover crops are well known to improve nutrient dynamics, soil organic matter content, microbial activity, water retention, and prevent nitrate leaching [34]. Moreover, returning of crop straws has been suggested to improve overall soil conditions, reduce the requirement for N fertilizers, and support sustainable rice productivity. However, while the benefits of cover crops for nutrient management are well documented, weed effects are less verified.

Rice demands high amount of potassium (K), which is mainly accumulated in the straw residues, and is easily lost by leaching and surface runoff after crop harvesting. Therefore, the inclusion of cover crops composed of grass species that tend to produce a great amount of biomass and absorb nutrients such as nitrogen and potassium, are a great strategy for nutrient cycling, substantially avoiding nutrient losses [35].

Moreover, pertinent choices of cover crop species can suppress the growth of serious weeds and protect the soil during winter, resulting in a better soil structure as opposed to leaving soil bare. Italian ryegrass (*Lolium multiflorum*) is a grass species that has been widely used during winter in paddy soils and is a good option for cattle grazing and as cover crop. The species has great biomass production and high nutritional value for animals, as well as impressive ability to re-grow after grazing, being highly competitive for nutrients, water, and sunlight [36]. Italian ryegrass can naturally establish itself from soil seed bank after the first year of cultivation in a given area, reducing costs with its plantation [37]; besides, it is well adapted to lowland soils. Moreover, when this species is cultivated, weed growth and development are inhibited due to the alellopathic substances released by the crop [38, 39].

**Figure 3** shows how important the inclusion of cover crops is to hamper weed infestations. In the left side of the picture, the area was left uncultivated favoring weed establishment, especially *Conyza* spp., whereas in the right side, Italian ryegrass was sown, which greatly reduced the infestation weeds.

Moreover, **Figure 4** gives the density of the weed flora (plants per m−2) in a given area cropped with soybean for 2 years. In winter, the first half of the area was left fallow, while ryegrass was grown in the other half. The weed flora was mainly composed of *Conyza* spp, *Soliva* spp., and *Richardia brasiliensis*. Weed density was assessed in the beginning of spring, showing a clear reduction in the number of plants where the cover crop was established in comparison to leaving the soil bare. It was also possible to observe that weed density in fallow plots was approximately 10 times bigger than the one assessed in the plots with Italian ryegrass. Similar results were obtained by other authors who found that this species is an effective choice of cover crop to suppress weed communities due to its great biomass production and alellopathic properties [38, 40, 41].

Glyphosate is normally used to control this species prior to sowing the summer crop due to its broad spectrum of action, efficiency, and low cost. However, the intensive use of this herbicide has left resistant biotypes of Italian ryegrass, which are becoming more frequent [42]. Other herbicides, such as the popularly known graminicides that inhibit the ACCase enzyme can be used to efficiently control biotypes resistant to glyphosate. These herbicides can be used in a mixture with glyphosate or with another herbicide with broad spectrum of action such as paraquat or ammonium-glufosinate [43, 44]. However, it is important to consider that biotypes of Italian ryegrass resistant to ACCase inhibitors have been already reported and, therefore, require careful use.

**Figure 4.** Number of plants (m−2) of *Conyza* spp, *Soliva* spp., and *Richardia brasiliensis* in plots without (fallow) and with

Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 93

The efficiency of these herbicides is highly dependent on the development stage in which they are applied. Nevertheless, the main constraint in the use of these compounds in lowland soils is associated with the negative effects that they can cause in rice plants due to their residual activity. Residual herbicides tend to dissipate slowly in paddy soils due to poor drainage, and when the interval between spraying and sowing rice is short, crop establishment is likely to

Therefore, it is essential to determine the correct sowing and desiccation time of the cover crops, because the decomposition of crop residues can be quite slow in lowland soils, due to the great soil moisture and physical-chemical features of this type of soil. Thus, the herbicides used can affect the establishment and consequently the yield of the following crop. The ideal timing should be set according to cover crop traits, cultivation density, developmental stage,

Another option for winter rotation is the mixture of grass species and legumes, which has high nutritional value for animals if the crop is used for grazing; it can benefit rice cultivation due to the nitrogen (N) input in the soil and a great improvement of the physical-chemical properties of the soil. Among the legumes species that can be introduced in a crop rotation

soil cultivation technique adopted, and level plus type of herbicide used [45].

be affected.

*Lollium multiflorum* as a cover crop in Southern Brazil.

It is clear that the introduction of Italian ryegrass into a crop rotation system with rice has many benefits in lowland soils. Nevertheless, the inadequate management of the crop residues can jeopardize the establishment of rice in succession due to the great amount of biomass that is kept on the soil surface. When the amount of crop residues left on the soil surface is greater than 30 t ha−1 it is difficult for the seed drill to cut the straw as the residues act as a physical barrier [40], resulting in the poor establishment of the following crop. Moreover, the alellopathic properties of this species can be considered another drawback for its inclusion in a crop rotation scheme, especially in no-tillage systems, affecting rice germination and emergence [41].

**Figure 3.** Weed infestation in experimental plots with (right) and without (left) Italian ryegrass (*Lollium multiflorum*) in southern Brazil.

ability to re-grow after grazing, being highly competitive for nutrients, water, and sunlight [36]. Italian ryegrass can naturally establish itself from soil seed bank after the first year of cultivation in a given area, reducing costs with its plantation [37]; besides, it is well adapted to lowland soils. Moreover, when this species is cultivated, weed growth and development are

**Figure 3** shows how important the inclusion of cover crops is to hamper weed infestations. In the left side of the picture, the area was left uncultivated favoring weed establishment, especially *Conyza* spp., whereas in the right side, Italian ryegrass was sown, which greatly reduced

Moreover, **Figure 4** gives the density of the weed flora (plants per m−2) in a given area cropped with soybean for 2 years. In winter, the first half of the area was left fallow, while ryegrass was grown in the other half. The weed flora was mainly composed of *Conyza* spp, *Soliva* spp., and *Richardia brasiliensis*. Weed density was assessed in the beginning of spring, showing a clear reduction in the number of plants where the cover crop was established in comparison to leaving the soil bare. It was also possible to observe that weed density in fallow plots was approximately 10 times bigger than the one assessed in the plots with Italian ryegrass. Similar results were obtained by other authors who found that this species is an effective choice of cover crop to suppress weed communities due to its great biomass production and alello-

It is clear that the introduction of Italian ryegrass into a crop rotation system with rice has many benefits in lowland soils. Nevertheless, the inadequate management of the crop residues can jeopardize the establishment of rice in succession due to the great amount of biomass that is kept on the soil surface. When the amount of crop residues left on the soil surface is greater than 30 t ha−1 it is difficult for the seed drill to cut the straw as the residues act as a physical barrier [40], resulting in the poor establishment of the following crop. Moreover, the alellopathic properties of this species can be considered another drawback for its inclusion in a crop rotation scheme, especially in no-tillage systems, affecting rice germination and

**Figure 3.** Weed infestation in experimental plots with (right) and without (left) Italian ryegrass (*Lollium multiflorum*) in

inhibited due to the alellopathic substances released by the crop [38, 39].

the infestation weeds.

92 Rice Crop - Current Developments

pathic properties [38, 40, 41].

emergence [41].

southern Brazil.

**Figure 4.** Number of plants (m−2) of *Conyza* spp, *Soliva* spp., and *Richardia brasiliensis* in plots without (fallow) and with *Lollium multiflorum* as a cover crop in Southern Brazil.

Glyphosate is normally used to control this species prior to sowing the summer crop due to its broad spectrum of action, efficiency, and low cost. However, the intensive use of this herbicide has left resistant biotypes of Italian ryegrass, which are becoming more frequent [42]. Other herbicides, such as the popularly known graminicides that inhibit the ACCase enzyme can be used to efficiently control biotypes resistant to glyphosate. These herbicides can be used in a mixture with glyphosate or with another herbicide with broad spectrum of action such as paraquat or ammonium-glufosinate [43, 44]. However, it is important to consider that biotypes of Italian ryegrass resistant to ACCase inhibitors have been already reported and, therefore, require careful use.

The efficiency of these herbicides is highly dependent on the development stage in which they are applied. Nevertheless, the main constraint in the use of these compounds in lowland soils is associated with the negative effects that they can cause in rice plants due to their residual activity. Residual herbicides tend to dissipate slowly in paddy soils due to poor drainage, and when the interval between spraying and sowing rice is short, crop establishment is likely to be affected.

Therefore, it is essential to determine the correct sowing and desiccation time of the cover crops, because the decomposition of crop residues can be quite slow in lowland soils, due to the great soil moisture and physical-chemical features of this type of soil. Thus, the herbicides used can affect the establishment and consequently the yield of the following crop. The ideal timing should be set according to cover crop traits, cultivation density, developmental stage, soil cultivation technique adopted, and level plus type of herbicide used [45].

Another option for winter rotation is the mixture of grass species and legumes, which has high nutritional value for animals if the crop is used for grazing; it can benefit rice cultivation due to the nitrogen (N) input in the soil and a great improvement of the physical-chemical properties of the soil. Among the legumes species that can be introduced in a crop rotation system in lowland soils, common bird's foot trefoil (*Lotus corniculatus* L.) and white clover (*Trifolium repens*) seem to be good alternatives for Brazilian lowland scenarios, as they survive to some degree in soils with poor drainage. However, little is known about the benefits of these species in relation to weed management when introduced into a crop rotation system in lowland areas, due to the lack of more detailed studies.

market in Southern Brazil that perform better in lowland soils in terms of productivity and could be an option for producers, even though they do not tolerate waterlogging periods. For instance, among others, BMX Apolo, BMX Ícone, BRS Taura RR, BR IRGA 6070 RR, and BR IRGA 1642 IPRO are good choices of soybean cultivars, whereas P30F53H, 2B655Hx, and 2B688Hx are maize cultivars that are most promising to show high yields in lowlands [48]. Even though highyielding cultivars of these crops are already available in the market, it is important to mention that new cultivars aiming for greater yields and stress tolerance are frequently launched. Thus,

It is important to mention that there is no magic recipe to ensure the success of crops such as maize, soybean, or sorghum in lowland soils as each field has some peculiar attributes that should be taken into account and climatic conditions change all the time. Moreover, the introduction of a diverse crop rotation system alone is not sufficient to guarantee that the density of troublesome weed species will be reduced. However, the introduction of this strategy allow farmers to diversify herbicides (with different mechanisms of action), soil cultivation type, and timing and sowing dates, that together are capable of disturbing the ecosystem and

The most important thing to consider in the real world when establishing new cropping systems in a farm is to plan and introduce it in small areas in the beginning, allowing the necessary cultural modifications to be applied. This will make the crop rotation functional and productive, avoiding a possible economical drawback in case of problems in the first tests of

, André Andres<sup>2</sup>

[1] Owen MDK. Diverse approaches to herbicide-resistant weed management. Weed Science.

[2] Green JM. Current state of herbicide in herbicide-resistant crops. Pest Management Science.

[3] Grundy AC. Predicting weed emergence: A review of approaches and future challenges.

\*, Germani Concenço2

,

Rice Crop Rotation: A Solution for Weed Management http://dx.doi.org/10.5772/intechopen.75884 95

producers and professionals in this sector must keep themselves informed.

hampering the establishment of recurrent weed species.

, Fábio Schreiber1

1 Federal University of Pelotas, Pelotas, Brazil

2016;(Special Issue):570-584

Weed Research. 2002;**43**:1-11

2014;**70**:1351-1357

2 Embrapa Temperate Agriculture, Pelotas, Brazil

\*Address all correspondence to: andre.andres@embrapa.br

and Andressa Pitol<sup>1</sup>

the new crop rotation scheme.

**Author details**

Ananda Scherner<sup>1</sup>

**References**

Matheus Bastos Martins1

## **4. Recommendations to successfully implement crop rotation in lowlands**

In the last decade, there has been an increasing interest in the introduction of new crops in lowland soils in Southern Brazil, which are mainly cultivated with rice in summer and used for cattle grazing in winter. This interest is driven by several factors such as to increase the profitability of the production system and reduce the problems that have been caused by herbicide-resistant weeds. As mentioned in the chapter, sorghum, maize, and soybean are the main crop choices to be included in a crop rotation scheme with rice; however, the success of these crops is highly dependent on manipulating the ecosystem according to their needs, especially making sure that poor soil drainage and fertility will not hamper the productivity of these crops. Moreover, ensuring good soil drainage during the winter as well, would allow farmers to introduce other grass species, such as *Avena sativa,* that can be very useful for cattle grazing for instance but are quite sensitive to waterlogging.

There are several strategies that can be adopted to enhance the natural drainage in these areas, even though they are quite flat. The use of furrows works quite well and can be used as an irrigation system as well [46]. Drought periods are quite common over the summer in Southern Brazil, and considering the water requirements of maize and soybean, irrigation might be needed to ensure crops productivity in this region. The digital elevation model (DEM) is another technique that can be used for the design and allocation of drains in the area, enhancing the natural soil drainage. The DEM can be obtained with geodesic data collected by a global navigation satellite system (GNSS), with accuracy improved by a real-time kinematic (RTK) positioning. This system provides a detailed survey of the area and through the analysis of the water flow, the drainage system is designed [46].

On the other hand, instead of only manipulating the environment to meet the needs of sorghum, maize, and soybean plants, the development of crop cultivars that tolerate periods of water surplus in the soil would be a great tool to ensure their adaptation to lowland soils. To date, several genes that control the behavior of plants under water stress have been already identified and characterized. However, the information gathered so far is not enough for the development of crop cultivars that would tolerate water excess in the soil; there is still a long way to go. To enhance this knowledge and amend the strategies that have been used for plant breeding, researchers are developing high-performance sequencers and making use of statistical and transformation techniques [47]. Therefore, farmers should expect in near future the introduction of high-yield crop varieties of soybean and maize that are well adapted to the paddy soils ecosystem. Nevertheless, there are some varieties of soybean and maize available in the market in Southern Brazil that perform better in lowland soils in terms of productivity and could be an option for producers, even though they do not tolerate waterlogging periods. For instance, among others, BMX Apolo, BMX Ícone, BRS Taura RR, BR IRGA 6070 RR, and BR IRGA 1642 IPRO are good choices of soybean cultivars, whereas P30F53H, 2B655Hx, and 2B688Hx are maize cultivars that are most promising to show high yields in lowlands [48]. Even though highyielding cultivars of these crops are already available in the market, it is important to mention that new cultivars aiming for greater yields and stress tolerance are frequently launched. Thus, producers and professionals in this sector must keep themselves informed.

It is important to mention that there is no magic recipe to ensure the success of crops such as maize, soybean, or sorghum in lowland soils as each field has some peculiar attributes that should be taken into account and climatic conditions change all the time. Moreover, the introduction of a diverse crop rotation system alone is not sufficient to guarantee that the density of troublesome weed species will be reduced. However, the introduction of this strategy allow farmers to diversify herbicides (with different mechanisms of action), soil cultivation type, and timing and sowing dates, that together are capable of disturbing the ecosystem and hampering the establishment of recurrent weed species.

The most important thing to consider in the real world when establishing new cropping systems in a farm is to plan and introduce it in small areas in the beginning, allowing the necessary cultural modifications to be applied. This will make the crop rotation functional and productive, avoiding a possible economical drawback in case of problems in the first tests of the new crop rotation scheme.

### **Author details**

system in lowland soils, common bird's foot trefoil (*Lotus corniculatus* L.) and white clover (*Trifolium repens*) seem to be good alternatives for Brazilian lowland scenarios, as they survive to some degree in soils with poor drainage. However, little is known about the benefits of these species in relation to weed management when introduced into a crop rotation system in

In the last decade, there has been an increasing interest in the introduction of new crops in lowland soils in Southern Brazil, which are mainly cultivated with rice in summer and used for cattle grazing in winter. This interest is driven by several factors such as to increase the profitability of the production system and reduce the problems that have been caused by herbicide-resistant weeds. As mentioned in the chapter, sorghum, maize, and soybean are the main crop choices to be included in a crop rotation scheme with rice; however, the success of these crops is highly dependent on manipulating the ecosystem according to their needs, especially making sure that poor soil drainage and fertility will not hamper the productivity of these crops. Moreover, ensuring good soil drainage during the winter as well, would allow farmers to introduce other grass species, such as *Avena sativa,* that can be very useful for cattle

There are several strategies that can be adopted to enhance the natural drainage in these areas, even though they are quite flat. The use of furrows works quite well and can be used as an irrigation system as well [46]. Drought periods are quite common over the summer in Southern Brazil, and considering the water requirements of maize and soybean, irrigation might be needed to ensure crops productivity in this region. The digital elevation model (DEM) is another technique that can be used for the design and allocation of drains in the area, enhancing the natural soil drainage. The DEM can be obtained with geodesic data collected by a global navigation satellite system (GNSS), with accuracy improved by a real-time kinematic (RTK) positioning. This system provides a detailed survey of the area and through

On the other hand, instead of only manipulating the environment to meet the needs of sorghum, maize, and soybean plants, the development of crop cultivars that tolerate periods of water surplus in the soil would be a great tool to ensure their adaptation to lowland soils. To date, several genes that control the behavior of plants under water stress have been already identified and characterized. However, the information gathered so far is not enough for the development of crop cultivars that would tolerate water excess in the soil; there is still a long way to go. To enhance this knowledge and amend the strategies that have been used for plant breeding, researchers are developing high-performance sequencers and making use of statistical and transformation techniques [47]. Therefore, farmers should expect in near future the introduction of high-yield crop varieties of soybean and maize that are well adapted to the paddy soils ecosystem. Nevertheless, there are some varieties of soybean and maize available in the

**4. Recommendations to successfully implement crop rotation in** 

lowland areas, due to the lack of more detailed studies.

grazing for instance but are quite sensitive to waterlogging.

the analysis of the water flow, the drainage system is designed [46].

**lowlands**

94 Rice Crop - Current Developments

Ananda Scherner<sup>1</sup> , Fábio Schreiber1 , André Andres<sup>2</sup> \*, Germani Concenço2 , Matheus Bastos Martins1 and Andressa Pitol<sup>1</sup>


### **References**


[4] Merroto A Jr, Goulart ICGR, Nunes AL, Kalsing A, Markus C, Menezes VG, Wander AE. Evolutionary and social consequences of introgression of nontransgenic herbicide resistance from rice to weedy rice in Brazil. Evolutionary Applications. 2016;**7**:837-836

[20] Chauvel B, Guillemin JP, Colbach N, Gasquez J. Evaluation of cropping systems for management of herbicide-resistant populations of blackgrass (*Alopecurus myosuroides*

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[22] Moody K. Weed control in wet-seeded rice. Experimental Agronomy. 1993;**29**:393-403

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[25] Andres A, Avila LA, Marchezan E, Menezes V. Rotação de culturas e pousio do solo na redução do banco de sementes de arroz vermelho em solo de várzea. Revista Brasileira

[26] Smith RG, Gross KL, Januchowski S. Earthworms and weed seed distribution in annual

[27] Scherner A, Melander B, Kudsk P. Vertical distribution and composition of weed seeds within the plough layer after eleven years of contrasting crop rotation and tillage

[28] Pinto LFS, Miguel P, Pauletto EA. Solos de várzea e terras baixas. In: Cultivo de soja e milho em terras baixas do Rio Grande do Sul. Pelotas -RS, Brazil: Embrapa; 2017. pp. 23-43

[29] Sousa RO, Camargo FA, Vahl LC. 2006. Solos alagados: reação de redox. In: Meurer EJ (Org). Fundamentos de química do solo. 3rd ed. Porto Alegre: Evangraf, p. 185-211

[30] Gomes AS, Magalhães AM Jr. Arroz irrigado no sul do Brasil. Brasília-DF: Embrapa

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[32] Timm PA, Campos ADS, Bueno MV, Aires T, Silva JT, Schreiber F, Parfitt JMB, Scvittaro WB, Timm LC. Avaliação de cultivares de Soja produzida em sistema de camalhão em terras baixas. In: Proceedings of the X Congress Do Arroz Irrigado, Gramado – RS. Brazil; 2017

[33] Oliveira ACB. Cultivares de Soja. In: Cultivo de soja e milho em terras baixas do Rio

[34] Wittwer RA, Dorn B, Jossi W, van der Heijden MGA. Cover crops support ecological

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[13] Burgos NR, Singh V, Tseng TM, et al. The impact of herbicide-resistant rice technology on phenotypic diversity and population structure of United States weedy rice. Plant

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[15] Norsworthy JK, Scott R, Smith K, Still J, Estorninos LE Jr, Bangarwa S. Confirmation and management of clomazone-resistant barnyardgrass in rice. In: Proceedings of the 62nd

[16] Yasuor H, TenBrook PL, Tjeerdema RS, Fischer AJ. Responses to clomazone and 5-ketoclomazone by *Echinochloa phyllopogon* resistant to multiple herbicides in Californian rice

[17] Webster EP, Linscombe SB, Bergeron EA, McKnight BM, Fish JC. Provisia rice: A future option in rice. 2017. Web page: http://wssaabstracts.com/public/29/abstract-271.html

[18] Heap I. International Survey of Herbicide Resistant Weeds. 2006. Web page: http://www.

[19] Délye C, Zhang X-Q, Michel S, Matéjicek S, Powles SB. Molecular bases for sensitivity to acetyl-coenzyme A carboxylase inhibitors in blackgrass. Plant Physiology. 2005;**137**:794-806

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[36] Lopes MLT, Carvalho PCDF, Anghinoni I, et al. Sistema de integração lavoura-pecuária: desempenho e qualidade da carcaça de novilhos superprecoces terminados em pastagem de aveia e azevém manejada sob diferentes alturas. Ciência Rural. 2008;**38**:178-184

**Chapter 7**

**Provisional chapter**

**Microwave Weed and Soil Treatment in Rice**

**Microwave Weed and Soil Treatment in Rice** 

DOI: 10.5772/intechopen.77952

Herbicides resistance has challenged sustainable rice productivity. Consequently, interest in chemical-free weed management has increased to overcome this constraint. This chapter has demonstrated the effect of pre-sowing microwave soil heating as a new alternative to chemicals for confirmed herbicide resistant weeds of the Australian rice production system. Microwave can superheat weed plants, creating micro-steam explosions in the plant structures to kill weeds. This requires the least amount of energy to achieve weed control and can be likened to a 'knock down' herbicide treatment. Considerably, more microwave energy can be applied to the soil to achieve weed seed bank deactivation; however, there is growing evidence that this strategy also changes the soil biota and nutrient profile in favour of substantial increases in crop yield, when crops are planted into this microwave-treated soil. An energy application of approximately 400–500 J cm−2 gave approximately 70–80% reduction in weed establishment in three field trials conducted at two agro-ecological zones of the Australia. In addition, there was a 10 times higher nitrogen use efficiency, and a 37% higher water use efficiency was achieved through this aspect of the microwave technology. There is also evidence that the soil treatment strategy provides persistent effects, beyond a single season; therefore, the rice

production is better than when using conventional weed control methods. **Keywords:** weeds, herbicide resistance, microwave, soil health, crop health

Rice (*Oryza sativa* L.) is the staple food of 60% of the world's population [1], performs a significant role in the socio-economic constancy of the world, and is grown in a vast range of agro-ecological conditions. In Australia, rice farming is done in the Murray-Darling Basin, on

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Muhammad Jamal Khan and Graham Ian Brodie

Muhammad Jamal Khan and Graham Ian Brodie

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77952

**Production**

**Abstract**

**1. Introduction**

**Production**


#### **Microwave Weed and Soil Treatment in Rice Production Microwave Weed and Soil Treatment in Rice Production**

DOI: 10.5772/intechopen.77952

Muhammad Jamal Khan and Graham Ian Brodie Muhammad Jamal Khan and Graham Ian Brodie

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77952

#### **Abstract**

[36] Lopes MLT, Carvalho PCDF, Anghinoni I, et al. Sistema de integração lavoura-pecuária: desempenho e qualidade da carcaça de novilhos superprecoces terminados em pastagem de aveia e azevém manejada sob diferentes alturas. Ciência Rural. 2008;**38**:178-184

[37] Trezzi MM. Antagonismo das associações de clodinafop-propargyl com metsulfuronmethyl e 2,4-d no controle de azevém (*Lolium multiflorum*). Planta Daninha. 2007;**25**:

[38] Moraes PVD et al. Manejo de plantas de cobertura no controle de plantas daninhas na

[39] Soares GLG, Vieira TR. Inibição da germinação e do crescimento radicular de alface (cv. "Grand rapids") por extratos aquosos de cinco espécies de Gleicheniaceae. Revista

[40] Rizzardi MA, Silva LF.Influência das coberturas vegetais de aveia-preta e nabo forrageiro na época de controle de plantas daninhas em milho. Planta Daninha. 2006;**24**:669-675 [41] Trezzi MM, Vidal RA. Potencial de utilização de cobertura vegetal de sorgo e milheto na supressão de plantas daninhas em condição de campo: II – efeitos da cobertura morta.

[42] Powles SB, Yu Q. Evolution in action: Plants resistant to herbicides. Annual Reviews in

[43] Vargas L, Roman ES, Rizzardi MA, Toledo EB. Manejo de azevémresistente ao glyphosate em pomares de maçã com herbicida select (clethodim). Revista Brasileira de Herbicidas.

[44] Spader V, Cristiane É, Lopes P, Geraldo E. Residual activity of ACCaseinhibitor herbicides applied at pre-sowing of corn. Revista Brasileira de Herbicidas. 2012;**11**:42-48 [45] Dabney SM, Delgado JA, Reeves DW. Using winter cover crops to improve soil and water quality. Communications in Soil Science and Plant Analysis. 2001;**32**:1221-1250

[46] Parfitt JMB, Winkler AS, Pinto MAB, Silva JT, Timm LC. Irrigação e drenagem para cultivo de soja e milho. In: Cultivo de soja e milho em terras baixas do Rio Grande do Sul.

[47] Mertz-Henning LM, Nepomuceno AL, Nakayama TJ, Neumaier N, Farias JRB. Avanços biotecnológicos para o desenvolvimento da tolerâncias de soja e milho ao estresse por encharcamento do solo. In: Cultivo de soja e milho em terras baixas do Rio Grande do

[48] Emygdio BM. Cultivares de Milho. In: Cultivo de soja e milho em terras baixas do Rio

cultura do milho. Planta Daninha. 2009;**27**:289-296

Floresta e Ambiente. 2000;**7**:180-197

Planta Daninha. 2004;**22**:1-10

Plant Biology. 2010;**61**:317-347

Pelotas -RS, Brazil: Embrapa; 2017. pp. 45-78

Sul. Pelotas -RS, Brazil: Embrapa; 2017. pp. 317-336

Grande do Sul. Pelotas -RS, Brazil: Embrapa; 2017. pp. 141-162

2006;**1**:30-36

839-847

98 Rice Crop - Current Developments

Herbicides resistance has challenged sustainable rice productivity. Consequently, interest in chemical-free weed management has increased to overcome this constraint. This chapter has demonstrated the effect of pre-sowing microwave soil heating as a new alternative to chemicals for confirmed herbicide resistant weeds of the Australian rice production system. Microwave can superheat weed plants, creating micro-steam explosions in the plant structures to kill weeds. This requires the least amount of energy to achieve weed control and can be likened to a 'knock down' herbicide treatment. Considerably, more microwave energy can be applied to the soil to achieve weed seed bank deactivation; however, there is growing evidence that this strategy also changes the soil biota and nutrient profile in favour of substantial increases in crop yield, when crops are planted into this microwave-treated soil. An energy application of approximately 400–500 J cm−2 gave approximately 70–80% reduction in weed establishment in three field trials conducted at two agro-ecological zones of the Australia. In addition, there was a 10 times higher nitrogen use efficiency, and a 37% higher water use efficiency was achieved through this aspect of the microwave technology. There is also evidence that the soil treatment strategy provides persistent effects, beyond a single season; therefore, the rice production is better than when using conventional weed control methods.

**Keywords:** weeds, herbicide resistance, microwave, soil health, crop health

### **1. Introduction**

Rice (*Oryza sativa* L.) is the staple food of 60% of the world's population [1], performs a significant role in the socio-economic constancy of the world, and is grown in a vast range of agro-ecological conditions. In Australia, rice farming is done in the Murray-Darling Basin, on

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an area of 70,000 ha, with an annual grain production capacity of 0.69 M t [2]. Direct seeding is a common sowing strategy of rice in Australia due to high labour costs associated with transplanted rice systems. Weeds are one of the major biological constraints to increasing rice yield. Oerke [3] estimated that globally 34% crop productivity losses are due to weeds. However, the global decline in the production of rice, due to weeds, is estimated to be 10.2% [4]. Yield loss in a direct seeded rice crops is high, compared to transplanted rice [5]. Productivity losses of rice crops generally depend on climatic conditions, weed types, weed population density, rice variety, sowing methods and weed management practices.

The troublesome weeds of the Australian rice growing belt are barnyard grass (*Echinochloa crus-galli*), dirty dora (*Cyperus difformis*), arrowhead (*Sagittaria montevidensis*), and starfruit (*Damasonium minus*). Among all the weeds, Barnyard grass (*Echinochloa crus-galli* L.) is the major problematic bio-agent of rice [6] and is also considered to be the main weed of several semi-aquatic cropping systems [7]. It follows the C4 photosynthetic pathway [5] and has indistinguishable morphology to rice at seedling stage, which makes it extremely competitive with the rice crop. A 57% reduction in rice yield was documented, with a barnyard grass population density of 9 plants m−2 [8]. Additionally, higher densities of barnyard grass may remove up to 80% of the soil nitrogen, especially during its vegetative growth stages [7]. Seed production is the key element of long-standing weed population dynamics [9]. The average seed production capacity of barnyard grass ranged from 20,000 to 73,000 seeds per plant [10] and 60% of these seed could become part of the weed seed bank. Therefore, effective weed management depends on reducing the soil weed seed bank [11].

#### **1.1. Herbicide resistance**

Globally, there are 400 weed species that have developed resistance to herbicides and annually nine new weed biotypes are reported as being herbicide resistant [12]. The overall number of herbicide resistant weed species in various crops is illustrated in **Figure 1**. Cross-resistance in weed flora is described as resistant to two or more weedicides of the same or different chemistry because of one resistant mechanism (RM) [13]. However, multiple resistances in individual weed species are generally characterized by the presence of two or more RMs. These mechanisms might be the mutation at the site of action (SOA) of herbicides (target site) or change in metabolism and translocation (nontarget site), which reduces the phytotoxic effect of herbicides on their SOA [14]. Of particular concern, the numbers of weed species, which have become resistance to glyphosate in Australian agricultural systems are shown in **Figures 2** and **3**. Metabolic resistance is more commonly found in monocot (grasses) than in dicot (broadleaf) weeds [14]. Herbicide resistance in weeds is the greatest threat to sustainable productivity of agricultural commodities in industrialized countries. Therefore, there is a present need of an alternative weed management strategy in exiting cropping system. A series of experiments have been conducted, at Dookie Campus of the University of Melbourne, to assess the effects of microwave energy as an alternative of chemical weed control.

#### **1.2. Water use efficiency**

Higher grain production per unit application of water is needed to enhance sustainable rice production for future demands. Australia is the driest inhabited continent on the planet and the Australian Academy of Technology and Engineering (ATSE) reported that 62% of Australia's water was consumed by the agriculture sector in 2013–2014. Effective water use, to improve crop yield, can save the sector's water for future generation. The cost of water in Australia is about AU\$ 200–300 per Ml, which is consequently increasing the cost of rice production in Australia, independent of direct-drill farming, which postpones permanent

**Figure 2.** Confirmed glyphosate resistance summer weeds of Australian crop production systems. Source: [15].

**Figure 1.** The overall herbicides resistant scenario of weed species in crops. Source: [12].

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 101

flooding of the crop for almost 35 days.

**Figure 1.** The overall herbicides resistant scenario of weed species in crops. Source: [12].

an area of 70,000 ha, with an annual grain production capacity of 0.69 M t [2]. Direct seeding is a common sowing strategy of rice in Australia due to high labour costs associated with transplanted rice systems. Weeds are one of the major biological constraints to increasing rice yield. Oerke [3] estimated that globally 34% crop productivity losses are due to weeds. However, the global decline in the production of rice, due to weeds, is estimated to be 10.2% [4]. Yield loss in a direct seeded rice crops is high, compared to transplanted rice [5]. Productivity losses of rice crops generally depend on climatic conditions, weed types, weed population density,

The troublesome weeds of the Australian rice growing belt are barnyard grass (*Echinochloa crus-galli*), dirty dora (*Cyperus difformis*), arrowhead (*Sagittaria montevidensis*), and starfruit (*Damasonium minus*). Among all the weeds, Barnyard grass (*Echinochloa crus-galli* L.) is the major problematic bio-agent of rice [6] and is also considered to be the main weed of sev-

indistinguishable morphology to rice at seedling stage, which makes it extremely competitive with the rice crop. A 57% reduction in rice yield was documented, with a barnyard grass population density of 9 plants m−2 [8]. Additionally, higher densities of barnyard grass may remove up to 80% of the soil nitrogen, especially during its vegetative growth stages [7]. Seed production is the key element of long-standing weed population dynamics [9]. The average seed production capacity of barnyard grass ranged from 20,000 to 73,000 seeds per plant [10] and 60% of these seed could become part of the weed seed bank. Therefore, effective weed

Globally, there are 400 weed species that have developed resistance to herbicides and annually nine new weed biotypes are reported as being herbicide resistant [12]. The overall number of herbicide resistant weed species in various crops is illustrated in **Figure 1**. Cross-resistance in weed flora is described as resistant to two or more weedicides of the same or different chemistry because of one resistant mechanism (RM) [13]. However, multiple resistances in individual weed species are generally characterized by the presence of two or more RMs. These mechanisms might be the mutation at the site of action (SOA) of herbicides (target site) or change in metabolism and translocation (nontarget site), which reduces the phytotoxic effect of herbicides on their SOA [14]. Of particular concern, the numbers of weed species, which have become resistance to glyphosate in Australian agricultural systems are shown in **Figures 2** and **3**. Metabolic resistance is more commonly found in monocot (grasses) than in dicot (broadleaf) weeds [14]. Herbicide resistance in weeds is the greatest threat to sustainable productivity of agricultural commodities in industrialized countries. Therefore, there is a present need of an alternative weed management strategy in exiting cropping system. A series of experiments have been conducted, at Dookie Campus of the University of Melbourne, to

assess the effects of microwave energy as an alternative of chemical weed control.

Higher grain production per unit application of water is needed to enhance sustainable rice production for future demands. Australia is the driest inhabited continent on the planet

photosynthetic pathway [5] and has

rice variety, sowing methods and weed management practices.

eral semi-aquatic cropping systems [7]. It follows the C4

management depends on reducing the soil weed seed bank [11].

**1.1. Herbicide resistance**

100 Rice Crop - Current Developments

**1.2. Water use efficiency**

**Figure 2.** Confirmed glyphosate resistance summer weeds of Australian crop production systems. Source: [15].

and the Australian Academy of Technology and Engineering (ATSE) reported that 62% of Australia's water was consumed by the agriculture sector in 2013–2014. Effective water use, to improve crop yield, can save the sector's water for future generation. The cost of water in Australia is about AU\$ 200–300 per Ml, which is consequently increasing the cost of rice production in Australia, independent of direct-drill farming, which postpones permanent flooding of the crop for almost 35 days.

population of ammonia oxidizer bacteria and archaea, studied during this pot trial, showed no significant response to the heating effect of microwave treatment time. The abundance of beneficial microbes is a direct indicator of soil fertility, suggesting that microwave technology

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 103

In Australia, weed management in organically produced rice offers extra challenges for the farmer. Sod seeding, where rice is directly sown into a pasture or legume stand, is a common establishment practices in southern New South Wales. The preexisting pasture of legume crop somehow suppresses aquatic weeds like dirty dora, starfruit, arrowhead and water plantain, but has little to no effect on barnyard grass. Another nonchemical weed control strategy is propane gas flame weeder; however, it costs about AU\$ 12,000–15,000 ha−1, along with careful water management and post-emergent harrowing [26]. Considering this, organic rice producers are still looking for a nonchemical weed control approach, which could control barnyard

Microwave frequencies occupy the portion of the electromagnetic spectrum (300 MHz to 300 GHz) that lies between VHF radiowaves and thermal infrared. Their application falls into two categories, depending on whether the wave is used to transmit information or energy. The first category includes terrestrial and satellite communication links, radar, radioastronomy, microwave thermography, material permittivity measurements, and so on [27]. The second category of applications is associated with microwave heating and wireless power transmission. In case of microwave heating, there is usually no signal modulation and the

*"It has long been known that an insulating material can be heated by applying energy to it in the form of high frequency electromagnetic waves"*([28], pp. 5). Industrial microwave heating has been used since the 1940s ([28], pp. 5). The initial experiments with microwave heating were conducted by Dr. Percy Spencer in 1946, following a serendipitous discovery while he was testing a magnetron [29]. Although Spencer was not the first to observe that microwave energy could impart heat to materials, he was the first to systematically study it. Since then, many heating,

One key benefit of microwave heating, over conventional convective heating, is speed. The origin of this speed is the volumetric interactions between the microwave's electric field and the material. In contrast, convective heat transfer propagates from the surface into the material, with the final temperature profile depending on the material's thermal diffusion properties [32] and the influence of moisture transport, which often hinders the convective heating

The factors that contribute to microwave heating include: the physical and chemical structure of the heated material; the frequency of the microwaves [34]; in some cases, such as wood, the orientation of the electrical field relative to the structure of the dielectric material is also

electromagnetic wave interacts directly with solid or liquid materials.

drying, thawing [30] and medical applications [31] have been developed.

can sustain soil health over a long period.

grass and sustain organic production.

**2.1. Essentials of microwave heating**

process [33].

**2. Microwave energy**

**Figure 3.** Confirmed glyphosate resistance winter weeds of Australian crop production systems. Source: [15].

Rice production in Australia consumes approximately 2.5–3 Ml ha−1 with an average grain harvest of about 1.4 t Ml−1; however, microwave soil treatment increases the production to about 1.92 t Ml−1 [16], which is around 37% higher water use efficiency compared to untreated control plots. Globally, an average of 0.003–0.005 Ml of water is required to produce 1 kg of rice (i.e. , a yield of between 0.2 and 0.3 t Ml−1), which is about 2–3 times higher than the water consumption of other cereal crops [17, 18]. Considering this, numerous studies have reported the effects of irrigation water volume on crop yield [19–21]. Interestingly, with a single crop management strategy, it is hard to harvest multiple benefits, including water use efficiency. In the following field studies, we achieved about 37% greater irrigation water use efficiency, where we treated the soil with microwave energy for weed seed bank depletion under field conditions. Therefore, microwave soil heating may also promote effective and efficient water use in Australian rice production systems, in addition to weed management. This assumption needs further research for validation under field conditions.

#### **1.3. Organic rice production**

Soil health is the key element in organic farming and as per worldwide agreement; soil fertility in organic farming system should be maintained on a long-term basis. Intensified rice farming has been deteriorating the soil quality [22] in Asian rice growing regions. However, in Australia, limited studies have reported that intensive organic farming enhanced soil fertility as compared to conventional agriculture practices [23]. It has been reported that microwave treatment of soil enhances the humification of soil organic matter [24] and has some positive effects on soil nitrogen availability for crop plants. In a pot trial, Khan et al. [25] reported a persistent effect of microwave soil heating on the second season wheat crop with better grain production benefits than in the first season after a once off microwave treatment, which suggests that there is a persistent effect of this technology on soil health. In addition, the population of ammonia oxidizer bacteria and archaea, studied during this pot trial, showed no significant response to the heating effect of microwave treatment time. The abundance of beneficial microbes is a direct indicator of soil fertility, suggesting that microwave technology can sustain soil health over a long period.

In Australia, weed management in organically produced rice offers extra challenges for the farmer. Sod seeding, where rice is directly sown into a pasture or legume stand, is a common establishment practices in southern New South Wales. The preexisting pasture of legume crop somehow suppresses aquatic weeds like dirty dora, starfruit, arrowhead and water plantain, but has little to no effect on barnyard grass. Another nonchemical weed control strategy is propane gas flame weeder; however, it costs about AU\$ 12,000–15,000 ha−1, along with careful water management and post-emergent harrowing [26]. Considering this, organic rice producers are still looking for a nonchemical weed control approach, which could control barnyard grass and sustain organic production.

### **2. Microwave energy**

Rice production in Australia consumes approximately 2.5–3 Ml ha−1 with an average grain harvest of about 1.4 t Ml−1; however, microwave soil treatment increases the production to about 1.92 t Ml−1 [16], which is around 37% higher water use efficiency compared to untreated control plots. Globally, an average of 0.003–0.005 Ml of water is required to produce 1 kg of rice (i.e. , a yield of between 0.2 and 0.3 t Ml−1), which is about 2–3 times higher than the water consumption of other cereal crops [17, 18]. Considering this, numerous studies have reported the effects of irrigation water volume on crop yield [19–21]. Interestingly, with a single crop management strategy, it is hard to harvest multiple benefits, including water use efficiency. In the following field studies, we achieved about 37% greater irrigation water use efficiency, where we treated the soil with microwave energy for weed seed bank depletion under field conditions. Therefore, microwave soil heating may also promote effective and efficient water use in Australian rice production systems, in addition to weed management. This assumption

**Figure 3.** Confirmed glyphosate resistance winter weeds of Australian crop production systems. Source: [15].

Soil health is the key element in organic farming and as per worldwide agreement; soil fertility in organic farming system should be maintained on a long-term basis. Intensified rice farming has been deteriorating the soil quality [22] in Asian rice growing regions. However, in Australia, limited studies have reported that intensive organic farming enhanced soil fertility as compared to conventional agriculture practices [23]. It has been reported that microwave treatment of soil enhances the humification of soil organic matter [24] and has some positive effects on soil nitrogen availability for crop plants. In a pot trial, Khan et al. [25] reported a persistent effect of microwave soil heating on the second season wheat crop with better grain production benefits than in the first season after a once off microwave treatment, which suggests that there is a persistent effect of this technology on soil health. In addition, the

needs further research for validation under field conditions.

**1.3. Organic rice production**

102 Rice Crop - Current Developments

Microwave frequencies occupy the portion of the electromagnetic spectrum (300 MHz to 300 GHz) that lies between VHF radiowaves and thermal infrared. Their application falls into two categories, depending on whether the wave is used to transmit information or energy. The first category includes terrestrial and satellite communication links, radar, radioastronomy, microwave thermography, material permittivity measurements, and so on [27]. The second category of applications is associated with microwave heating and wireless power transmission. In case of microwave heating, there is usually no signal modulation and the electromagnetic wave interacts directly with solid or liquid materials.

### **2.1. Essentials of microwave heating**

*"It has long been known that an insulating material can be heated by applying energy to it in the form of high frequency electromagnetic waves"*([28], pp. 5). Industrial microwave heating has been used since the 1940s ([28], pp. 5). The initial experiments with microwave heating were conducted by Dr. Percy Spencer in 1946, following a serendipitous discovery while he was testing a magnetron [29]. Although Spencer was not the first to observe that microwave energy could impart heat to materials, he was the first to systematically study it. Since then, many heating, drying, thawing [30] and medical applications [31] have been developed.

One key benefit of microwave heating, over conventional convective heating, is speed. The origin of this speed is the volumetric interactions between the microwave's electric field and the material. In contrast, convective heat transfer propagates from the surface into the material, with the final temperature profile depending on the material's thermal diffusion properties [32] and the influence of moisture transport, which often hinders the convective heating process [33].

The factors that contribute to microwave heating include: the physical and chemical structure of the heated material; the frequency of the microwaves [34]; in some cases, such as wood, the orientation of the electrical field relative to the structure of the dielectric material is also important ([35], pp. 13-17); reflections from the interfacial surface of the heated material [27]; electric field strength [34]; the geometry of the microwave applicator [28]; the geometry, size, electrical and thermal properties of the dielectric material [36–38]; the exposure time; and the moisture content of the dielectric material [33, 35].

**Microwave frequency**

**Energy level Irradiation** 

2.45 GHz 600 W 60 s Pre-emergence

2.45 GHz 600 W 8 s Post

2.45 GHz 2000 & 4000 W Varying

2.45 GHz 45–720 J cm−2 No

2.45 GHz 0.1–1.5 kW Varying

**duration (s)**

39 MHz **—** 4–37 s Pre-emergence Hard red winter

exposure time (not mention properly)

information

exposure time

2.45 GHz — 360 s Pre-emergence *Brassica* 

2.45 GHz 100–750 W 120–1200 s Pre-emergence Clover and Turnip 60–78%

Preemergence of seeds in soil

**Treatment scenario**

(Dry, 4 h soaked and 46 h germinated seeds)

emergence (Aquatic weed)

Pre and post emergences

wheat

*Zea mays, Arachis hypogaea, Prosopis juliflora, Cucumis sativus, Brassica sp., Rumex crispus, Echinochloa colonum, Amaranthus sp., Gossypium hirsutum, Glycine max, Sorghum vulgare and Triticum vulgare*

Duckweed *(Wolffia punctata)*

Johnsongrass Morningglory Redroot Pigweed Texas panicum Barnyardgrass Sunflower London rocket Rigseed euphorbia

Pre-emergence London rocket

(13 cm deep in soil profile) and Sunflower (2.5 cm seeded depth)

Black medic, Barnyard grass, Foxtail purslane, redroot pigweed, large crabgrass,

*napus, Linum usitatissimum, Avena fatua*

**Target species Percent weed-**

**seed destruction**

17% reduction in germination in dry seeds but 100% in case of moist seeds at 10 s of exposure

50% Champ *et al.* [43]

50% seed mortality

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952

> For postemergence MW treatment 309 J/

 energy was required for 100% control (field conditions) while for preemergence MW weed control 73 J/cm2

 gave 85–100% control (glass house conditions)

87% for London rocket and 93% for sunflower

reduction in seeds germination

50% Rice and

85–95% Bhartia *et al.*

[48]

cm<sup>2</sup>

**Reference**

105

Nelson and Walker [41]

Davis *et al.* [42]

Wayland *et al.* [44]

Menges and Wayland, [45]

Hightower *et al.* [46]

Putnam [47]

#### **2.2. ISM band applications**

Because microwaves are also used in the communication, navigation and defence industries, and their use in thermal heating is restricted to a small subset of the available frequency bands. A small number of frequencies have been set aside for Industrial, Scientific and Medical (ISM) applications [39]. The main frequencies of interest for industrial applications are 915 ± 13 MHz and 2450 ± 50 MHz [39].

### **3. Microwave weed treatment**

In Australian agricultural industries, the total estimated cost of weed management and loss in crop productivity due to weeds is about AU\$4 billion annually [40]. Microwave weed management is an alternative method of weed control in modern agriculture systems. The history of microwave-based weed management is given in **Table 1**. The efficacious application of microwave heating in agricultural systems can substitute for the sometimes hazardous, toxic and environmentally unsafe chemicals that are used to kill weeds [60]. Interest in the use of microwave energy as a tool to weeds control is mainly because of herbicide resistance of various weed species [61] and their long-lasting persistence in the environment [54, 62]. Microwave heating is not influenced by wind direction and speed, therefore prolonging the application periods compared to traditional methods of herbicides spraying [51].

Ayappa et al. [63] reported that the most important features of microwave heating are its accurate control, diminutive start-up time and volumetric heating. Microwave energy density is the most important factor in plant mortality rather than exposure time; therefore, two options for weed management, using microwave energy, become evident: long exposure to diffuse microwave energy; or deliberate application of a strongly focused microwave pulse to quickly debilitate the plants [58].

Microwave radiation, which triggers dielectric heating in plant tissues, is induced by the microwave's electric field. This internal heating ultimately kills or debilitates the plant [54]. Bigu-Del-Blanco et al. [49] treated 2-day-old seedling of maize with microwave energy at a frequency of 9 GHz for 22–24 h. The authors revealed that more exposure time to microwaves even at very low energy densities significantly dehydrated the maize plants and retarded their growth.

In contrast, the recent research on fleabane and paddy melon [58] has concluded that a short exposure (≤ 5 s) of high-intensity microwave heating was enough to hinder plant growth. The plant tissues, which were subjected to microwaves, rapidly dehydrated. Whatley et al. [64] stated that low moisture levels in soil attenuated the microwave transmission less than high moisture content. The authors suggested that pre-emergence microwave treatment for weed control should be worked out when the top soil layer (1–2 cm) contains relatively low moisture.


important ([35], pp. 13-17); reflections from the interfacial surface of the heated material [27]; electric field strength [34]; the geometry of the microwave applicator [28]; the geometry, size, electrical and thermal properties of the dielectric material [36–38]; the exposure time; and the

Because microwaves are also used in the communication, navigation and defence industries, and their use in thermal heating is restricted to a small subset of the available frequency bands. A small number of frequencies have been set aside for Industrial, Scientific and Medical (ISM) applications [39]. The main frequencies of interest for industrial applications

In Australian agricultural industries, the total estimated cost of weed management and loss in crop productivity due to weeds is about AU\$4 billion annually [40]. Microwave weed management is an alternative method of weed control in modern agriculture systems. The history of microwave-based weed management is given in **Table 1**. The efficacious application of microwave heating in agricultural systems can substitute for the sometimes hazardous, toxic and environmentally unsafe chemicals that are used to kill weeds [60]. Interest in the use of microwave energy as a tool to weeds control is mainly because of herbicide resistance of various weed species [61] and their long-lasting persistence in the environment [54, 62]. Microwave heating is not influenced by wind direction and speed, therefore prolonging the

Ayappa et al. [63] reported that the most important features of microwave heating are its accurate control, diminutive start-up time and volumetric heating. Microwave energy density is the most important factor in plant mortality rather than exposure time; therefore, two options for weed management, using microwave energy, become evident: long exposure to diffuse microwave energy; or deliberate application of a strongly focused microwave pulse to

Microwave radiation, which triggers dielectric heating in plant tissues, is induced by the microwave's electric field. This internal heating ultimately kills or debilitates the plant [54]. Bigu-Del-Blanco et al. [49] treated 2-day-old seedling of maize with microwave energy at a frequency of 9 GHz for 22–24 h. The authors revealed that more exposure time to microwaves even at very low energy densities significantly dehydrated the maize plants and retarded their growth.

In contrast, the recent research on fleabane and paddy melon [58] has concluded that a short exposure (≤ 5 s) of high-intensity microwave heating was enough to hinder plant growth. The plant tissues, which were subjected to microwaves, rapidly dehydrated. Whatley et al. [64] stated that low moisture levels in soil attenuated the microwave transmission less than high moisture content. The authors suggested that pre-emergence microwave treatment for weed control should be worked out when the top soil layer (1–2 cm) contains relatively low moisture.

application periods compared to traditional methods of herbicides spraying [51].

moisture content of the dielectric material [33, 35].

are 915 ± 13 MHz and 2450 ± 50 MHz [39].

**3. Microwave weed treatment**

quickly debilitate the plants [58].

**2.2. ISM band applications**

104 Rice Crop - Current Developments


and concluded that susceptibility of young seedlings to microwave heating was highly correlated with moisture content and absorption of energy. Davis et al. [67] proposed that the specific mass and volume of crop seeds were positively correlated to seed mortality during microwave heating. This might be due to the "radar cross-section" [68] attainable by seeds to transmitting microwave. More radar cross-section enables the seed to interrupt, and thus

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 107

The use of electromagnetic radiations for post-emergence control of broad leaves and grasses is the least energy-consuming process available for microwave weed control [70]. Brodie et al. [57] stated that, based on microwave energy calculation for seeds and plants on the sandy soil surface, far more energy was required to kill dry seeds as compared to the previously emerged plants. The actual energy requirement on a large scale would depend on plant density and three-dimensional microwave distribution. Hence, the total energy required for weed management might be significantly reduced if weed seeker systems [71] are employed to

Thermal runway, due to the resonance of electromagnetic field inside the structure of dielectric material, is common in dielectric heating [72, 73]. Total energy and time exposure could be dramatically reduced if thermal runway can be induced in weed plants throughout microwave irradiation treatment; therefore, analogous energy requirements to those related with traditional chemical weed control method could be achieved. This temperature-time exposure scenario can only be discovered and understood through more research into the microwave

Based on previous findings and the results of recent studies reported by Khan et al. and Brodie et al. [16, 25]; pre-sowing microwave irradiation of soil for 120 s in first field trial and 60 s in two other field trials, in rice crops, gave significant reduction in weed emergence (**Table 2**). It is possible to reduce weed pressure in direct-seeded rice systems through microwave irradiation of soil in Australia; however, more consolidated research efforts are needed to understand the long-term effects of microwave irradiation and weed control in rice.

It has been confirmed that microwave energy can debilitate emerged weed plants with a very short exposure time [25, 56, 58, 59]. Some specific microwave energy dose responses are

Soil is a complex three-dimensional living substance. The propagation of microwave energy

bulk density, organic matter content [75], soil texture [57] and specific heat of soil. Among them, the soil moisture content has three major impacts on microwave heating: (1) moisture increases the soil surface reflectivity [76], which ultimately reduced the microwave penetration into the soil [28]; (2) moist soil readily absorbs the microwave energy to generate heat [28] thus less total microwave energy propagated into the soil; and (3) moisture is also responsible

) and volumetric (θ<sup>v</sup>

) moisture content [74],

absorbs more microwave energy. This seems to be the cause of death [69].

control the activation of the microwave unit.

heating of biological materials.

**3.1. Killing emerged plants**

through soil depends upon the gravimetric (θ<sup>g</sup>

for heat-diffusing phenomena in the soil profile [77].

shown in **Figure 4**.

**3.2. Soil treatment**

**Table 1.** History of microwave weed management in different scenarios.

Van Wambeke et al. [65] and Benz et al. [66] reported that seeds, fungi and nematodes could be effectively controlled with a short exposure to microwave treatment; however, the efficacy of this short exposure was highly influenced by soil texture, exposure time (sec), soil depth and soil moisture content. Davis et al. [42] conducted an experiment to evaluate the effects of microwave on the seedling survival percentage of twelve species. They described that the seedling (48 h germination) exhibited no survival after short exposure of microwave energy and concluded that susceptibility of young seedlings to microwave heating was highly correlated with moisture content and absorption of energy. Davis et al. [67] proposed that the specific mass and volume of crop seeds were positively correlated to seed mortality during microwave heating. This might be due to the "radar cross-section" [68] attainable by seeds to transmitting microwave. More radar cross-section enables the seed to interrupt, and thus absorbs more microwave energy. This seems to be the cause of death [69].

The use of electromagnetic radiations for post-emergence control of broad leaves and grasses is the least energy-consuming process available for microwave weed control [70]. Brodie et al. [57] stated that, based on microwave energy calculation for seeds and plants on the sandy soil surface, far more energy was required to kill dry seeds as compared to the previously emerged plants. The actual energy requirement on a large scale would depend on plant density and three-dimensional microwave distribution. Hence, the total energy required for weed management might be significantly reduced if weed seeker systems [71] are employed to control the activation of the microwave unit.

Thermal runway, due to the resonance of electromagnetic field inside the structure of dielectric material, is common in dielectric heating [72, 73]. Total energy and time exposure could be dramatically reduced if thermal runway can be induced in weed plants throughout microwave irradiation treatment; therefore, analogous energy requirements to those related with traditional chemical weed control method could be achieved. This temperature-time exposure scenario can only be discovered and understood through more research into the microwave heating of biological materials.

Based on previous findings and the results of recent studies reported by Khan et al. and Brodie et al. [16, 25]; pre-sowing microwave irradiation of soil for 120 s in first field trial and 60 s in two other field trials, in rice crops, gave significant reduction in weed emergence (**Table 2**). It is possible to reduce weed pressure in direct-seeded rice systems through microwave irradiation of soil in Australia; however, more consolidated research efforts are needed to understand the long-term effects of microwave irradiation and weed control in rice.

### **3.1. Killing emerged plants**

It has been confirmed that microwave energy can debilitate emerged weed plants with a very short exposure time [25, 56, 58, 59]. Some specific microwave energy dose responses are shown in **Figure 4**.

### **3.2. Soil treatment**

Van Wambeke et al. [65] and Benz et al. [66] reported that seeds, fungi and nematodes could be effectively controlled with a short exposure to microwave treatment; however, the efficacy of this short exposure was highly influenced by soil texture, exposure time (sec), soil depth and soil moisture content. Davis et al. [42] conducted an experiment to evaluate the effects of microwave on the seedling survival percentage of twelve species. They described that the seedling (48 h germination) exhibited no survival after short exposure of microwave energy

**Microwave frequency**

106 Rice Crop - Current Developments

**Energy level Irradiation** 

9 GHz 10–30 mW/cm2 22–24 h Post

2.45 GHz 900 W 4, 8, 16, 32, 64,

2.45 GHz 800 W 120, 240, 420

2.45 GHz 700 W 120, 240, 320

2.45 GHz 750 W 5, 15, 30 and

2.45 GHz 2 kW 5, 10, 15, 30,

2.45 GHz 0.10–1.24 kWh m−2

**duration (s)**

2.45 GHz 1.2 kW 5–45 s Pre-emergence *Trifolium and* 

2.45 GHz 120 s Pre-emergence Avena sativa and

128 and 256 s

and 960 s

and 720 s

60 s

60 s

**Table 1.** History of microwave weed management in different scenarios.

**Treatment scenario**

emergence

2.45 GHz 1.5 kW 0, 10, 20 and 30 Pre-emergence Wild oat & wheat 90–100% Lal and

2.45 GHz 500 W 30 s Pre-emergence *Avena fatua* 60% (based on

Post emergence

30–300 s Pre and post

emergence

Pre and post emergence

Preemergence treatment of soil

Post emergence **Target species Percent weed-**

*Zea mays* 100% growth

*Medicago*

native weed seeds

Parthenium and Bellyache bush

*Malva parviflora and Triticum astevium*

*Lolium perenne and Lolium rigidum*

Prickly paddy melon

radish

Ryegrass and wild

*Abutilon theophrasti*, Panicum miliaceum, Lucerne and Rapeseed

Pre-emergence Rubber vine,

**seed destruction**

85% reduction in germination

seed moisture)

Reduced weed seeds emergence

Complete dehydrating of plants

88% (Rubber vine), 67% (Parthenium) and 94% (Bellyache bush) mortality at 960 s irradiation

100% destruction of tested specie at 0.65 kWh m−2

100% debilitation of plants

100% mortality Brodie and

100% seed mortality was achieved at 240 s of MW irradiation

inhibitions

**Reference**

Bigu-del-Blanco *et al.* [49]

Crawford [50]

Diprose *et al.* [51]

Reed, [52]

Barker and Craker, [53]

Sartorato *et al.* [54]

Bebawi *et al.* [55]

Brodie *et al.* [56]

Brodie *et al.* [57]

Brodie *et al.* [58]

Hollins [59]

Soil is a complex three-dimensional living substance. The propagation of microwave energy through soil depends upon the gravimetric (θ<sup>g</sup> ) and volumetric (θ<sup>v</sup> ) moisture content [74], bulk density, organic matter content [75], soil texture [57] and specific heat of soil. Among them, the soil moisture content has three major impacts on microwave heating: (1) moisture increases the soil surface reflectivity [76], which ultimately reduced the microwave penetration into the soil [28]; (2) moist soil readily absorbs the microwave energy to generate heat [28] thus less total microwave energy propagated into the soil; and (3) moisture is also responsible for heat-diffusing phenomena in the soil profile [77].

It has been reported that the dielectric constant (*ε'*) of known soil at known θ<sup>g</sup> is proportional to the bulk density of soil. The dependence of soil dielectric constant on bulk density is described by the direct dependence of bulk density on fraction of soil moisture volume [78]. The textural composition of soil (particles sizes distribution) affects the dielectric constant (*ε'*). The higher percentage of clay particles (with bulk density range of 1.0–1.6 g cm−3) increases the dielectric constant of soil [79]. This might be due to higher water holding capacity of clay particles. Therefore, this will increase the absorption of microwave energy by soil for its synchronized functions.

The temperature profile is dependent on the microwave electric field strength (E) within the soil. Brodie [56] has extensively studied the temperature distribution in soil due to microwave energy application through a horn antenna. The temperature profile can be described by Eq. (1). The Nomenclature of Eq. (1) is presented in **Table 3**.

$$\left[\frac{\tau}{\tau}\right]^T = \frac{\tau^2 \cos^2 \left(e^{\tau \tau \omega^2 t} - 1\right)}{4k\alpha^4} \left[e^{-\tau \omega \omega} + \left(\frac{h}{k} + 2\alpha\right) \cos^{-\nu^2/\omega q}\right] \left[\tau \left(\frac{\eta}{\tau \frac{\eta}{\ell}}\right) \left(\frac{\eta}{\tau \frac{\eta}{\ell}} \cos\left(\frac{\eta}{\lambda}\kappa'\right)\right) \frac{\tau^2 \sin^2 \left(\frac{\eta}{\ell}\kappa + \frac{\eta}{\ell}\right) \omega^2 \left(\frac{\eta}{\ell}\right) + \left(\frac{h}{k} + 2\alpha\right) \cos^2 \left(\frac{\eta}{\lambda}\right) \omega^2 \left(\frac{\eta}{\ell}\right)}{\left(\frac{\eta}{\lambda} - \kappa^2\right) \left(\frac{\eta}{\lambda}\right)}\right] \tag{1}$$

**Figure 5** compares measured temperature distributions with those predicted by Eq. (1). The highest temperature in the microwave-treated soil was along the centre line of horn antenna and between the 0.02 and 0.05 m below the soil surface. **Figure 6** illustrates the effects of microwave soil treatment using a different system configuration and treatment scenario.

#### **3.3. Effect on soil**

#### *3.3.1. Effect of microwave energy on nutritional dynamic of soil*

The dynamic of soil key nutrients (Carbon, Nitrogen, Phosphorus, Potassium and Sulphur) is explained by the knowledge of size and turnover rate of plant biopolymers such as C-N compounds, cellulose and hemicellulose and lignin [81]. The soil-microwave interaction is the function of various soil properties such as texture, moisture, salinity, bulk density and temperature [58, 78, 79]. Cooper and Brodie [82] investigated the effect of different durations

of microwave treatment and soil depth on soil nutrient status and pH. They found that microwave treatment of soil had no significant effect on nitrogen, phosphorus, potassium and sulphate concentrations in all the treatment combination, but they reported an increase in nitrite concentration after 120 s of microwave treatment of soil. The nitrate reduction in the

**Figure 4.** Dose-response curves for microwave treatment of four herbicides resistance weed species using a horn

Speir et al. [84] examined the effect of microwave energy on low fertility soil (100 randomly selected cores at depth of 50 mm), microbial biomass, N, phosphorus and phosphatase activity. They reported that an increase in microwave treatment duration (90 s) dramatically increased the N level (106 μg N g−1 soils) but the phosphorus concentration declined as treatment time increased. The higher flush in soil N is of microbial origin as microwave has a biocidal effect

[87]. Kittrick [88] hypothesized that the ion fixation in the clay lattice could be described by the expanding and contracting forces in the interlayer position. The contraction is due to electrostatic force of attraction between negatively charged clay mineral and positively charged ions and ion hydration causes the expansion. Fixation occurred when the force of attraction

in soil by inorganic colloids has been well documented

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 109

irradiated soil could be the principle cause of nitrite formation [83], in their study.

[85, 86]. The fixation of NH4

antenna. Source: Khan et al. [25].

+ or K<sup>+</sup>


**Table 2.** Effect of microwave energy application for weed seedbank depletion in direct seeded-rice crop under filed conditions in Australia (adapted from [16]).

It has been reported that the dielectric constant (*ε'*) of known soil at known θ<sup>g</sup>

for its synchronized functions.

108 Rice Crop - Current Developments

scenario.

**3.3. Effect on soil**

(1). The Nomenclature of Eq. (1) is presented in **Table 3**.

*3.3.1. Effect of microwave energy on nutritional dynamic of soil*

**Weed parameters Treatments LSD**

Weed density (plants plot−1) 17.6<sup>a</sup> 94.8<sup>b</sup> 37.7 −80% Weed fresh weight (g plot−1) 156.4<sup>a</sup> 612.8<sup>b</sup> 426.6 −74.6% Weed dr. Weight (g plot−1) 21.6<sup>a</sup> 122.6<sup>b</sup> 69.6 −82%

**treatment**

conditions in Australia (adapted from [16]).

tional to the bulk density of soil. The dependence of soil dielectric constant on bulk density is described by the direct dependence of bulk density on fraction of soil moisture volume [78]. The textural composition of soil (particles sizes distribution) affects the dielectric constant (*ε'*). The higher percentage of clay particles (with bulk density range of 1.0–1.6 g cm−3) increases the dielectric constant of soil [79]. This might be due to higher water holding capacity of clay particles. Therefore, this will increase the absorption of microwave energy by soil

The temperature profile is dependent on the microwave electric field strength (E) within the soil. Brodie [56] has extensively studied the temperature distribution in soil due to microwave energy application through a horn antenna. The temperature profile can be described by Eq.

**Figure 5** compares measured temperature distributions with those predicted by Eq. (1). The highest temperature in the microwave-treated soil was along the centre line of horn antenna and between the 0.02 and 0.05 m below the soil surface. **Figure 6** illustrates the effects of microwave soil treatment using a different system configuration and treatment

The dynamic of soil key nutrients (Carbon, Nitrogen, Phosphorus, Potassium and Sulphur) is explained by the knowledge of size and turnover rate of plant biopolymers such as C-N compounds, cellulose and hemicellulose and lignin [81]. The soil-microwave interaction is the function of various soil properties such as texture, moisture, salinity, bulk density and temperature [58, 78, 79]. Cooper and Brodie [82] investigated the effect of different durations

**control microwave** 

**Untreated control**

**Table 2.** Effect of microwave energy application for weed seedbank depletion in direct seeded-rice crop under filed

**(p = 0.05)**

**Percentage change from** 

is propor-

(1)

**Figure 4.** Dose-response curves for microwave treatment of four herbicides resistance weed species using a horn antenna. Source: Khan et al. [25].

of microwave treatment and soil depth on soil nutrient status and pH. They found that microwave treatment of soil had no significant effect on nitrogen, phosphorus, potassium and sulphate concentrations in all the treatment combination, but they reported an increase in nitrite concentration after 120 s of microwave treatment of soil. The nitrate reduction in the irradiated soil could be the principle cause of nitrite formation [83], in their study.

Speir et al. [84] examined the effect of microwave energy on low fertility soil (100 randomly selected cores at depth of 50 mm), microbial biomass, N, phosphorus and phosphatase activity. They reported that an increase in microwave treatment duration (90 s) dramatically increased the N level (106 μg N g−1 soils) but the phosphorus concentration declined as treatment time increased. The higher flush in soil N is of microbial origin as microwave has a biocidal effect [85, 86]. The fixation of NH4 + or K<sup>+</sup> in soil by inorganic colloids has been well documented [87]. Kittrick [88] hypothesized that the ion fixation in the clay lattice could be described by the expanding and contracting forces in the interlayer position. The contraction is due to electrostatic force of attraction between negatively charged clay mineral and positively charged ions and ion hydration causes the expansion. Fixation occurred when the force of attraction


**Table 3.** Nomenclature of mathematical terms.

dominated the cations' hydration energy. Zagal [89] pointed out that the mechanical effect induced by microwave irradiation can stimulate the dispersion of inorganic colloids. This stimulation can increase the decomposition of non-biomass organic matter in soil and release the fixed NH4<sup>+</sup> . Yang et al. [90] tested the nutrient extractability effect of microwave energy on soil. When fresh soil was exposed to microwave energy, a dramatic increase in the NH<sup>4</sup> + -N concentration was observed for an extended treatment of 120 s. They concluded that this effect was partially from nonmicrobial processes, either from site exchange or from fixed position in inorganic collides (clay minerals).

Soil organic matter (SOM) is an aggregate of organic residues in soil at different degrees of humification [97]. Various biopolymers are serially transferred to humus (fulvic acid, humic acid and humin) in soil through geological SOM development processes such as humification [98]. Protein is the basic structural component of cell and cellular enzymes [99]. Approximately 5–25% of organic inputs are expected to accumulate in soil as proteins, peptides and free amino acids [100]. Amino acids typically incorporate about 10–20% of soil organic carbon and 30–40% of soil org-N [100]. Thermal denaturation of biopolymers induced by microwave irradiation could increase the concentrations of free amino acids for succeeding turnover to

**Figure 6.** Estimated change in soil temperature treated from the 2 kW microwave system after 30 s with horn antenna at

**Figure 5.** Comparison of expected soil temperature profile with measured soil temperature profile (left) and for the

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 111

can enhance the binding efficiency of hydrophobic organic containments with SOM. They irradiated 5 g samples of soil in plastic tubes in aerobic and anaerobic conditions with activated C for 600 s in a lab-scale microwave oven (2.45 GHz) operated at 700 W. They pointed out that MW irradiation significantly alters the physical and chemical properties of SOM and increased its humification. Kim and Kim [24] studied the influences of microwave irradiation

. Hur et al. [101] demonstrated that microwave irradiation of soil

CO<sup>2</sup>

and ammonia pool NH<sup>4</sup>

2 cm above the soil. Adapted from [56].

+

750 W prototype microwave unit after 150 s of heating. Adapted from [56].

Alphei and Scheu [91] evaluated the effects of various biocidal treatments on mull-structured soil biota and nutritional dynamics. They reported the survival of soil microorganisms; in particular, higher concentration of ammonium nitrogen and phosphorus was observed when soil was subjected to microwave treatment at high power. The increase in soil C and N mineralization [89] and NH<sup>4</sup> + -N and sulphur oxidation was reported by Wainwright et al. [92]. In contrast, numerous studies documented that the effect of ionizing irradiation (γ-rays) on soil effectively increased the mineralization of NH4 + -N [93, 94]. They proposed three possible pathways which may be responsible for the release of NH<sup>4</sup> + -N from soil through irradiation: (1) ammonia could be produced by the chemical action of ionizing radiation through a variety of biochemical processes from nitrogenous organic compounds, particularly deamination of amino acid [95] and proteins, (2) several enzymes were functional in irradiated soil including urease, which is active during decomposition and produces ammonia and (3) release of N from dead organisms due to subsequent cell lysis by irradiation [96].

**Figure 5.** Comparison of expected soil temperature profile with measured soil temperature profile (left) and for the 750 W prototype microwave unit after 150 s of heating. Adapted from [56].

dominated the cations' hydration energy. Zagal [89] pointed out that the mechanical effect induced by microwave irradiation can stimulate the dispersion of inorganic colloids. This stimulation can increase the decomposition of non-biomass organic matter in soil and release

n Scaling factor for simultaneous heat and moisture movement [80]

γ Combined diffusivity for simultaneous heat and moisture transfer

x, y, z Cartesian coordinates of a point in front of the horn antenna (m) x', y' Cartesian coordinates of a point in the aperture of the antenna (m)

ω Angular frequency of electromagnetic wave (rad s−1)

τ Transmission coefficient of the soil surface

α Field attenuation factor in the soil (m−1)

k Thermal conductivity of the composite material (W m−1 °C−1)

ε<sup>o</sup> Permittivity of free space κ" Dielectric loss factor

E Electric field strength (V m−1)

Α Width of antenna aperture (m) Β Height of antenna aperture (m)

Ro Length of antenna (m)

on soil. When fresh soil was exposed to microwave energy, a dramatic increase in the NH<sup>4</sup>

concentration was observed for an extended treatment of 120 s. They concluded that this effect was partially from nonmicrobial processes, either from site exchange or from fixed posi-

Alphei and Scheu [91] evaluated the effects of various biocidal treatments on mull-structured soil biota and nutritional dynamics. They reported the survival of soil microorganisms; in particular, higher concentration of ammonium nitrogen and phosphorus was observed when soil was subjected to microwave treatment at high power. The increase in soil C and N min-

In contrast, numerous studies documented that the effect of ionizing irradiation (γ-rays) on

(1) ammonia could be produced by the chemical action of ionizing radiation through a variety of biochemical processes from nitrogenous organic compounds, particularly deamination of amino acid [95] and proteins, (2) several enzymes were functional in irradiated soil including urease, which is active during decomposition and produces ammonia and (3) release of N

+

. Yang et al. [90] tested the nutrient extractability effect of microwave energy


+



+ -N

the fixed NH4<sup>+</sup>

eralization [89] and NH<sup>4</sup>

tion in inorganic collides (clay minerals).

**Parameter Meaning**

110 Rice Crop - Current Developments

t Time (s)

**Table 3.** Nomenclature of mathematical terms.

+

soil effectively increased the mineralization of NH4

pathways which may be responsible for the release of NH<sup>4</sup>

from dead organisms due to subsequent cell lysis by irradiation [96].

**Figure 6.** Estimated change in soil temperature treated from the 2 kW microwave system after 30 s with horn antenna at 2 cm above the soil. Adapted from [56].

Soil organic matter (SOM) is an aggregate of organic residues in soil at different degrees of humification [97]. Various biopolymers are serially transferred to humus (fulvic acid, humic acid and humin) in soil through geological SOM development processes such as humification [98]. Protein is the basic structural component of cell and cellular enzymes [99]. Approximately 5–25% of organic inputs are expected to accumulate in soil as proteins, peptides and free amino acids [100]. Amino acids typically incorporate about 10–20% of soil organic carbon and 30–40% of soil org-N [100]. Thermal denaturation of biopolymers induced by microwave irradiation could increase the concentrations of free amino acids for succeeding turnover to CO<sup>2</sup> and ammonia pool NH<sup>4</sup> + . Hur et al. [101] demonstrated that microwave irradiation of soil can enhance the binding efficiency of hydrophobic organic containments with SOM. They irradiated 5 g samples of soil in plastic tubes in aerobic and anaerobic conditions with activated C for 600 s in a lab-scale microwave oven (2.45 GHz) operated at 700 W. They pointed out that MW irradiation significantly alters the physical and chemical properties of SOM and increased its humification. Kim and Kim [24] studied the influences of microwave irradiation on the SOM properties. They reported that thermal cracking induced by irradiation scenario potentially alters the molecular composition (C, H, O and N), chemical structure and humification of SOM. The results of these studies suggest that microwave soil heating has potential to maximize the crop yield.

*3.3.3. Influence of microwave soil heating on soil microbes*

sterilization was achieved through 120 s of irradiation.

**3.4. Effect on crop growth**

Soil biota is known to survive under severe physio-chemical environmental changes [120– 122]. Microwave heating of soil can eradicate soil-borne fungi with minimal reduction of prokaryotic organisms [123]. Microbial cell response to microwave irradiation depends on the location, power density, time, frequency, pulses and physiology of cells. The nitrogen fixing bacteria persist, even after relatively high energy dosages. Vela and Wyss conducted a microwave heating experiment on soil *Azotobacter* and found that they survived microwave exposure of 480 s in very moist soil while, they were inactivated after only 20 s of treatment in laboratory culture conditions. Vela et al. [124] found that soil-nitrifying bacteria were highly resistant to microwave energy applied at the rate 40,000 J cm−2 to the soil surface. However, nitrifiers (mesophilic) are much more sensitive to high temperature than ammonifying (thermophilic) bacteria. This implies that native habitat and intrinsic environment are the most important factors in resistance of soil organisms to microwave irradiation [125]. Soil bacterial communities are resistant to microwave energy; some scientists concluded that the soil shelters microflora, while others discovered that the rate of proliferation causes resistance. This rate of proliferation is determined by nutrient concentrations. The heat-shock activation of the soil bacterial community was reported by Vela et al. [124]. Bacteria can form various thermal-resistance structure (i.e., spore and cysts), which keep them resistant against harmful effects of physical environments [126]. Based on work done by Hollins [127], she reported that a sharp reduction in colony forming unit of *E. colie* with 10 s of treatment of 2.3 kg soil (**Figure 7**), treated through 2 kW microwave system under horn antenna and complete soil

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 113

Rice productivity is strongly influenced through weed management strategies. Recently, a field experiment was conducted to evaluate the effect of pre-cropping microwave soil heating for weed seedbank depletion in direct-seeded rice crop based on the above soil heating methodology [16]. In addition to weed suppression (**Table 1**), the application of microwave energy (2.45 GHz; 120 s; 560 J cm−2) into soil significantly (P = 0.05; **Table 4**) increased the tiller density (419 m−2), dry biomass yield (27.8 t ha−1) and grain yield (9.0 t ha−1) of rice, compared to the untreated control scenario 292 m−2, 22.8 t ha−1 and 6.7 t ha−1, respectively. These results are strongly supported with findings of Brodie [128], who found that in pot trial maximum rice grain yield was attained with energy application of 600 J cm−2 to soil before crop sowing. The higher crop productivity could be attributed to 70–80% reduction in weed establishment achieved through microwave irradiation of soil, ultimately leaving more room for crop growth. Thermal devitalisation of weed seedbanks in the vertical soil profile may be the possible cause of minimum weed interference with the rice crop. This was evidenced by Vidotto et al. [129] who explored the effectiveness of high temperature on seed viability of six weed species including *Echinochloa crus-galli*: the problematic weed of rice growing regions globally. They stated that 80–100% germination reduction was achieved through raising the soil temperature to 79.6°C. The same temperature regime (70–80°C) that was acquired by microwave irradiation of the soil in the present study. This effectively induced an inhibitory

effect on the weed population and therefore increased the rice crop yield.

### *3.3.2. Enzyme activity as a function of microwave soil heating*

Enzymes are essential to ecosystem processes because they arbitrate innumerable reactions that have biogeochemical importance in soil [102]. It has been demonstrated that *in vitro* exposure of microbial cells to microwave energy increased cell membrane permeability [103], released DNA and protein [104], soluble carbohydrate concentration [105] and inhibited growth of cells [106]. The enzymatic activity, selectivity and stability could be improved through highfrequency electromagnetic energy in an aqueous medium [107]. d'Ambrosio et al. [108] found that acid phosphatase was highly stable to the microwave deactivation energy of 280 mW g−1. The hydration state and polarity of the reaction medium directly influenced the enzyme functionality under microwave irradiation. Notably, Carrillo-Munoz et al. [109] performed two lipases esterification reactions in a mono-mode microwave system at temperature of 100°C. They found that a 2–9% higher yield and 2.1–2.5-fold increment increase in protease activity [110] were obtained in microwave conditions compared to conventional heating in the hydration state. Furthermore, Yadav and Lahi [111] investigated the influence of microwave on lipase activity in a highly polar solvent and concluded that microwave noticeably accelerated the enzymatic reaction with an increase in hydrophobicity. Pirogova et al. [112] tested the effect of low frequency microwave energy, in the range of 500–900 MHz and at various power levels (1, 0.1 and 0.01 μW) on the activity of l-lactate dehydrogenase in solution for 300 s. They found a 73% increase in the bioactivity of the studied enzyme in microwaveirradiated samples compared to nonirradiated samples. Asadi et al. [113] tested the physiology of cyanobacterium (*Schizothrix mexicana*) against low power microwave modulation of various frequencies; they found that 9.685 GHz significantly increased growth metabolisms. Dreyfuss and Chipley [114] documented that metabolic enzyme activity of *S. aureus* increased after microwave irradiation. The cell biopolymer excitation induced by MW exposure was suggested to alter the enzymes' functionality.

Kothari et al. [115] studied the effect of low-power microwave on protease and urease activity of nine microorganisms (Bacteria, yeast and fungi). They treated enzyme cultures for different durations (0, 120, 240 and 360 s) in a microwave oven and concluded that the significant increase in the enzymes' activity was an athermal effect of microwave energy on the metabolism of the organisms. Numerous previous studies have shown higher enzymatic activity of industrial importance as a function of microwave in various reaction media at a temperature range of 70–110°C [107] and soil enzymes that are resistant to denaturation stress by heat [116, 117]. In contrast, Yeargers et al. [118] investigated the effect of microwave and conventional heating methods on the sensitivity of two enzyme (lysozyme and trypsin) solutions and found no discernible difference in enzyme activity, but the lysozyme was slightly more heat resistant than trypsin. Elzobair et al. [119] reported that microwave energy of 800 J g−1 of soil decreased (˂10 nmolg−1 h−1) dehydrogenase enzyme activity but 3200 J g−1 increased (˃20 nmolg−1 h−1) its functionality.

#### *3.3.3. Influence of microwave soil heating on soil microbes*

on the SOM properties. They reported that thermal cracking induced by irradiation scenario potentially alters the molecular composition (C, H, O and N), chemical structure and humification of SOM. The results of these studies suggest that microwave soil heating has potential

Enzymes are essential to ecosystem processes because they arbitrate innumerable reactions that have biogeochemical importance in soil [102]. It has been demonstrated that *in vitro* exposure of microbial cells to microwave energy increased cell membrane permeability [103], released DNA and protein [104], soluble carbohydrate concentration [105] and inhibited growth of cells [106]. The enzymatic activity, selectivity and stability could be improved through highfrequency electromagnetic energy in an aqueous medium [107]. d'Ambrosio et al. [108] found that acid phosphatase was highly stable to the microwave deactivation energy of 280 mW g−1. The hydration state and polarity of the reaction medium directly influenced the enzyme functionality under microwave irradiation. Notably, Carrillo-Munoz et al. [109] performed two lipases esterification reactions in a mono-mode microwave system at temperature of 100°C. They found that a 2–9% higher yield and 2.1–2.5-fold increment increase in protease activity [110] were obtained in microwave conditions compared to conventional heating in the hydration state. Furthermore, Yadav and Lahi [111] investigated the influence of microwave on lipase activity in a highly polar solvent and concluded that microwave noticeably accelerated the enzymatic reaction with an increase in hydrophobicity. Pirogova et al. [112] tested the effect of low frequency microwave energy, in the range of 500–900 MHz and at various power levels (1, 0.1 and 0.01 μW) on the activity of l-lactate dehydrogenase in solution for 300 s. They found a 73% increase in the bioactivity of the studied enzyme in microwaveirradiated samples compared to nonirradiated samples. Asadi et al. [113] tested the physiology of cyanobacterium (*Schizothrix mexicana*) against low power microwave modulation of various frequencies; they found that 9.685 GHz significantly increased growth metabolisms. Dreyfuss and Chipley [114] documented that metabolic enzyme activity of *S. aureus* increased after microwave irradiation. The cell biopolymer excitation induced by MW exposure was

Kothari et al. [115] studied the effect of low-power microwave on protease and urease activity of nine microorganisms (Bacteria, yeast and fungi). They treated enzyme cultures for different durations (0, 120, 240 and 360 s) in a microwave oven and concluded that the significant increase in the enzymes' activity was an athermal effect of microwave energy on the metabolism of the organisms. Numerous previous studies have shown higher enzymatic activity of industrial importance as a function of microwave in various reaction media at a temperature range of 70–110°C [107] and soil enzymes that are resistant to denaturation stress by heat [116, 117]. In contrast, Yeargers et al. [118] investigated the effect of microwave and conventional heating methods on the sensitivity of two enzyme (lysozyme and trypsin) solutions and found no discernible difference in enzyme activity, but the lysozyme was slightly more heat resistant than trypsin. Elzobair et al. [119] reported that microwave energy of 800 J g−1 of soil decreased (˂10 nmolg−1 h−1) dehydrogenase enzyme activity but 3200 J g−1 increased (˃20

to maximize the crop yield.

112 Rice Crop - Current Developments

*3.3.2. Enzyme activity as a function of microwave soil heating*

suggested to alter the enzymes' functionality.

nmolg−1 h−1) its functionality.

Soil biota is known to survive under severe physio-chemical environmental changes [120– 122]. Microwave heating of soil can eradicate soil-borne fungi with minimal reduction of prokaryotic organisms [123]. Microbial cell response to microwave irradiation depends on the location, power density, time, frequency, pulses and physiology of cells. The nitrogen fixing bacteria persist, even after relatively high energy dosages. Vela and Wyss conducted a microwave heating experiment on soil *Azotobacter* and found that they survived microwave exposure of 480 s in very moist soil while, they were inactivated after only 20 s of treatment in laboratory culture conditions. Vela et al. [124] found that soil-nitrifying bacteria were highly resistant to microwave energy applied at the rate 40,000 J cm−2 to the soil surface. However, nitrifiers (mesophilic) are much more sensitive to high temperature than ammonifying (thermophilic) bacteria. This implies that native habitat and intrinsic environment are the most important factors in resistance of soil organisms to microwave irradiation [125]. Soil bacterial communities are resistant to microwave energy; some scientists concluded that the soil shelters microflora, while others discovered that the rate of proliferation causes resistance. This rate of proliferation is determined by nutrient concentrations. The heat-shock activation of the soil bacterial community was reported by Vela et al. [124]. Bacteria can form various thermal-resistance structure (i.e., spore and cysts), which keep them resistant against harmful effects of physical environments [126]. Based on work done by Hollins [127], she reported that a sharp reduction in colony forming unit of *E. colie* with 10 s of treatment of 2.3 kg soil (**Figure 7**), treated through 2 kW microwave system under horn antenna and complete soil sterilization was achieved through 120 s of irradiation.

#### **3.4. Effect on crop growth**

Rice productivity is strongly influenced through weed management strategies. Recently, a field experiment was conducted to evaluate the effect of pre-cropping microwave soil heating for weed seedbank depletion in direct-seeded rice crop based on the above soil heating methodology [16]. In addition to weed suppression (**Table 1**), the application of microwave energy (2.45 GHz; 120 s; 560 J cm−2) into soil significantly (P = 0.05; **Table 4**) increased the tiller density (419 m−2), dry biomass yield (27.8 t ha−1) and grain yield (9.0 t ha−1) of rice, compared to the untreated control scenario 292 m−2, 22.8 t ha−1 and 6.7 t ha−1, respectively. These results are strongly supported with findings of Brodie [128], who found that in pot trial maximum rice grain yield was attained with energy application of 600 J cm−2 to soil before crop sowing. The higher crop productivity could be attributed to 70–80% reduction in weed establishment achieved through microwave irradiation of soil, ultimately leaving more room for crop growth. Thermal devitalisation of weed seedbanks in the vertical soil profile may be the possible cause of minimum weed interference with the rice crop. This was evidenced by Vidotto et al. [129] who explored the effectiveness of high temperature on seed viability of six weed species including *Echinochloa crus-galli*: the problematic weed of rice growing regions globally. They stated that 80–100% germination reduction was achieved through raising the soil temperature to 79.6°C. The same temperature regime (70–80°C) that was acquired by microwave irradiation of the soil in the present study. This effectively induced an inhibitory effect on the weed population and therefore increased the rice crop yield.

**Figure 7.** Assessment of E. coli survival in top 2 cm of soil as a function of applied microwave energy (Source: [127]).

For further validation of this yield changing effect with microwave soil heating, two field trials were conducted during October, 2016 to April, 2017 in a randomized complete block design with five replications at two different locations. The first location Dookie Campus of the University of Melbourne (36.395°S, 145.703°E) is a central grain growing region of the Goulburn Valley, which is in north of the state of Victoria, Australia; part of this region grows temperate rice. This region has a temperate climate with an average annual rainfall of 575 mm and an average monthly temperature range of 9.4–20.9°C (Australian Bureau of Meteorology). Soil at this experimental site is medium clay and classified as an Upotipotpon Clay [130] or an Orthic Basic Rudosol [131]. Historically, the same paddock has since been used for sheep grazing and highly invaded with a numerous grass species. The second location Old Coree, Jerilderie, New South Vales (35.210 °S, 145.440 °E) is the rice research farm a totally owned property of the Rice Research Australia Pty. Ltd. – SunRice™. Soil was treated using a prototype 2 kW microwave system, it has four independently controlled, 2 kW microwave generators operating at 2.45 GHz. The trailer is powered from two on-board 7 kVA, three-phase electrical generators [25]. Treatment was applied for 60 s and the temperature achieved through microwave energy application into soil was about 70–75°C in top soil layer (0–5 cm) at both study locations. Brodie reported that the microwave energy application to soil of about 400–500 J cm−2 gave 1.2–1.5 t extra grain yield compared to untreated control soil (**Figure 8**). The same range of microwave energy has used to treat the soil in the above field experiments. Therefore, the microwave soil treatment for preemergence weed suppression gave substantial increase in rice crop yield at both study location (**Table 4**; **Figures 9** and **10**). This is an additional benefit of soil heating through MW energy; we assumed that temperature has influenced on the soil nutrient profile particularly nitrogen.

#### **3.5. Evaluation of rice crop production potential**

Sustainable production of rice crop is the present need of the agriculture sector to fulfil increasing demand. In general, herbicide resistance, lower water use efficiency and **Rice parameters**

**Dookie location 1**

**Treatments** **Microwave** 

**Untreated** 

**treated**

387 a 16.90 a

3.88 a 22.3 a

17.4 a

8.08

0.19

6.1%

46.77 a

44.59 a

4.29

0.26

6.1%

2.56 a

1.76

0.12

51.5%

9.21 a

7.63 b

0.65

˂0.001

20.7%

14.0 a

4.2

0.14

20.7%

19.80 a

17.05 b

0.57

˂0.001

16.1%

268 b

62.0

44.4%

480 a

418 a

145.2

0.29

44.4%

Number of tillers (m−2)

Dry biomass weight (t

Grain yield (t ha−1)

Harvest Index ¥Water use efficiency (t Ml−1)

ᴪPartial factor productivity of

nitrogen (kg rice grain per kg

application of N)

1.3 31.04

20.42

—

—

—

Note: different letters in a row reflecting a significant difference at 5% probability level.Note: different letters in a row reflecting a significant difference at 5% probability level.

ᴪPartial factor productivity of nitrogen (PFP) , change in crop yield with nitrogen application was calculated based on work done by [132]. Note: Applied

nitrogen during cropping period was 125

 kg recommendation of Ricegrowers Association of Australia.

**Table 4.**

N

ha−1 at both study locations.

¥Water use efficiency was calculated based on the change in grain yield per unit application of water. Note: Irrigation water volume was about 3 Ml ha−1 as per

Influence of pre-sowing microwave soil heating for weed seedbank depletion on rice productivity at two different agro-ecological zones of the Australia.

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 115

73.68

61.04

—

—

—

0.85

—

—

—

3.07

2.54

—

—

—

 ha−1)

**control**

**LSD**

**P-value**

**Percentage** 

**Treatments** **Microwave** 

**Untreated** 

**treated**

**control**

**LSD**

**P-Value**

**Percentage** 

**change**

**(p = 0.05)**

**change**

**(p = 0.05)**

**Jerilderie location 2**


For further validation of this yield changing effect with microwave soil heating, two field trials were conducted during October, 2016 to April, 2017 in a randomized complete block design with five replications at two different locations. The first location Dookie Campus of the University of Melbourne (36.395°S, 145.703°E) is a central grain growing region of the Goulburn Valley, which is in north of the state of Victoria, Australia; part of this region grows temperate rice. This region has a temperate climate with an average annual rainfall of 575 mm and an average monthly temperature range of 9.4–20.9°C (Australian Bureau of Meteorology). Soil at this experimental site is medium clay and classified as an Upotipotpon Clay [130] or an Orthic Basic Rudosol [131]. Historically, the same paddock has since been used for sheep grazing and highly invaded with a numerous grass species. The second location Old Coree, Jerilderie, New South Vales (35.210 °S, 145.440 °E) is the rice research farm a totally owned property of the Rice Research Australia Pty. Ltd. – SunRice™. Soil was treated using a prototype 2 kW microwave system, it has four independently controlled, 2 kW microwave generators operating at 2.45 GHz. The trailer is powered from two on-board 7 kVA, three-phase electrical generators [25]. Treatment was applied for 60 s and the temperature achieved through microwave energy application into soil was about 70–75°C in top soil layer (0–5 cm) at both study locations. Brodie reported that

to treat the soil in the above field experiments. Therefore, the microwave soil treatment for preemergence weed suppression gave substantial increase in rice crop yield at both study location (**Table <sup>4</sup>**; **Figures <sup>9</sup>** and **10**). This is an additional benefit of soil heating through MW energy; we assumed that temperature has influenced on the soil nutrient profile particularly nitrogen.

Sustainable production of rice crop is the present need of the agriculture sector to ful

fil increasing demand. In general, herbicide resistance, lower water use efficiency and

J cm−2 gave 1.2–1.5 t extra grain yield


**8**). The same range of microwave energy has used

2 cm of soil as a function of applied microwave energy (Source: [127]).

the microwave energy application to soil of about 400–500

compared to untreated control soil (**Figure**

**Figure 7.** Assessment of E. coli survival in top

114 Rice Crop - Current Developments

**3.5. Evaluation of rice crop production potential**

Note: different letters in a row reflecting a significant difference at 5% probability level.Note: different letters in a row reflecting a significant difference at 5% probability level. ᴪPartial factor productivity of nitrogen (PFP) , change in crop yield with nitrogen application was calculated based on work done by [132]. Note: Applied 

nitrogen during cropping period was 125 kg N ha−1 at both study locations.

¥Water use efficiency was calculated based on the change in grain yield per unit application of water. Note: Irrigation water volume was about 3 Ml ha−1 as per recommendation of Ricegrowers Association of Australia.

**Table 4.** Influence of pre-sowing microwave soil heating for weed seedbank depletion on rice productivity at two different agro-ecological zones of the Australia.

**Figure 8.** Infrared thermal images of microwave treated plot for weed seedbank depletion in rice crop under field conditions.

consumption, the microwave system used in their study was quite comparable or better than soil fumigation and soil steaming treatment done by Samtani et al. [135]. Higher rice crop productivity without soil nutrient depletion has been confirmed with microwave soil heating methodology with an average of 20–50% increase under field conditions (**Table 4**). The microwave soil heating did not significantly alter the grain mineral concentration of rice (**Figure 11**), which suggests that higher yield producing crops effectively utilize the yield-changing nutrients from the soil. Based on this estimate, the profitability of rice production through this technology is better than conventional weed control technology. In other domain, however, soil health and persistence effects of the treatment for up to two growing seasons give an additional productivity advantage to rice farming

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952 117

**Figure 10.** Relative increase in rice grain yield as a function of applied microwave energy. Source: [128].

**Figure 11.** Effect of microwave soil heating on quality related parameter of rice grain. Adapted from [16].

community.

**Figure 9.** Comparison of early growth establishment of rice crop. Plants on left collected from microwave treated plot and plants on right collected from untreated control plot. (Left image taken from Dookie Trial Site and right image taken from Old Coree, Jerilderie site).

nitrogen use efficiency are key sustainability limiting factors, globally. For herbicide resistant weed suppression, Khan et al. [16] compared a microwave energy cost in rice crop with pre-sowing soil fumigation [133, 134] and reported that in terms of fuel

**Figure 10.** Relative increase in rice grain yield as a function of applied microwave energy. Source: [128].

consumption, the microwave system used in their study was quite comparable or better than soil fumigation and soil steaming treatment done by Samtani et al. [135]. Higher rice crop productivity without soil nutrient depletion has been confirmed with microwave soil heating methodology with an average of 20–50% increase under field conditions (**Table 4**). The microwave soil heating did not significantly alter the grain mineral concentration of rice (**Figure 11**), which suggests that higher yield producing crops effectively utilize the yield-changing nutrients from the soil. Based on this estimate, the profitability of rice production through this technology is better than conventional weed control technology. In other domain, however, soil health and persistence effects of the treatment for up to two growing seasons give an additional productivity advantage to rice farming community.

**Figure 11.** Effect of microwave soil heating on quality related parameter of rice grain. Adapted from [16].

nitrogen use efficiency are key sustainability limiting factors, globally. For herbicide resistant weed suppression, Khan et al. [16] compared a microwave energy cost in rice crop with pre-sowing soil fumigation [133, 134] and reported that in terms of fuel

**Figure 9.** Comparison of early growth establishment of rice crop. Plants on left collected from microwave treated plot and plants on right collected from untreated control plot. (Left image taken from Dookie Trial Site and right image taken

**Figure 8.** Infrared thermal images of microwave treated plot for weed seedbank depletion in rice crop under field

conditions.

116 Rice Crop - Current Developments

from Old Coree, Jerilderie site).

### **4. Discussion**

Weed seedbank is a resting place of dormant seeds in the top soil horizon. Various biotic and abiotic factors have a tremendous effect on seed viability. Among the many abiotic factors, however, soil temperature has an ability to debilitate weed seeds *in situ* [136, 137]. Therefore, it was hypothesized that the projection of non-ionizing energy into a top soil layer through a horn antenna may cause thermal devitalisation of weed seeds. The results of field studies have strongly supported our hypothesis and achieved about 70–80% reduction in rice weeds establishment. Overall, energy of a microwave system for weed management program has a direct relationship with application duration. Therefore, for a pre-emergence weed control under field conditions, Wayland et al. [44] reported an energy level of 80 ˂ J cm−2 ˂ 160, which is quite low compared to the present investigation. In contrast, for a post-emergence weed control Sartorato et al. [54] tested the efficacy of microwave energy on seedling of *Abutilon theophrasti* and *Panicum miliaceum*. They reported that an energy range of 101.5 ˂ J cm−2 ˂ 343.3 gave significant reduction in dry weight of about 90%. However, it is highly unlikely that certain set of MW energy may give a same control spectrum, because soil moisture [74] and seed geometry [38, 67] have a considerable influence on microwave absorption. These vary according to cropping system.

microwave irradiation of soil can enhance the binding efficiency of hydrophobic organic containments with soil organic matter. They irradiated 5 g samples of soil in plastic tubes in aerobic and anaerobic conditions with activated C for 600 s in a lab-scale microwave oven (2.45 GHz) operated at 700 W. They pointed out that MW irradiation significantly alters the physical and chemical properties of soil organic matter and increased its humification. In another study, Kim and Kim [24] studied the influences of microwave irradiation on the soil organic matter properties. They reported that thermal cracking induced by irradiation potentially alters the molecular composition (C, H, O and N), chemical structure and humification of soil organic matter. Based on these previous findings, we assumed that thermal denaturation of recalcitrant humic substance induced by microwave irradiation may increase the concentrations of free

stantially increased wheat productivity in the present investigation. Moreover, microwave soil heating gave 10 times higher nitrogen use efficiency and about 20–50% higher irrigation water use efficiency in those field experiment conducted to manage the herbicides resistance weeds.

Based on these experiments, we conclude that microwave weed and soil treatment can be implemented as an alternative method of weed control in direct-seeded rice crop. Additional benefit of this technology has prompted a motivation for further research in this area to

Faculty of Veterinary and Agricultural Sciences, Dookie Campus, The University of

Advances in Weed Management. New York: Springer; 2014. pp. 125-153

[1] Mahajan G, Chauhan BS, Kumar V. Integrated weed management in rice. In: Recent

[2] ABS. Agricultural commodities, Australia. 2016. Accessed from: http://www.abs.gov.au

[4] Gianessi LP. The increasing importance of herbicides in worldwide crop production.

[5] Rao AN, Johnson DE, Sivaprasad B, Ladha JK, Mortimer AM. Weed management in

[3] Oerke EC. Crop losses to pests. The Journal of Agricultural Science. 2006;**144**:31-43

and ammonia pool NH<sup>4</sup>

+

Microwave Weed and Soil Treatment in Rice Production http://dx.doi.org/10.5772/intechopen.77952

, which might have sub-

119

amino acids for succeeding turnover to CO<sup>2</sup>

enhance sustainability in agricultural industry.

Muhammad Jamal Khan and Graham Ian Brodie\*

Melbourne, Melbourne, Australia

\*Address all correspondence to: grahamb@unimelb.edu.au

Pest Management Science. 2013;**69**:1099-1105

direct-seeded rice. Advances in Agronomy. 2007;**93**:153-255

**5. Conclusion**

**Author details**

**References**

Independent of soil heating methodology for weed control; various studies also reported the profound effect of high temperature on weed establishment. Gay et al. [133] reported on a soil steaming experiment with various duration (0, 6, 8 and 10 min) in a soil, to depths of about 1.5–16.5 cm, giving a temperature gradient of 100–37°C (decreasing with depth), in a lettuce crop for weed control. They found an average weed density of less than 50 plants m−2 in the case of soil steam treated plots compared to untreated control plots (400 plants m−2). Vidotto et al. [129] found that exposure of a soil-seed mixture to high temperature gradually decreased seed germination. Almost all the tested weed species seeds were completely devitalized through soil thermal treatment at a temperature between 70°C and 80°C.

The same temperature distribution was achieved through microwave application in the present study, which might have a degrading effect on the weed seedbank and ultimately led to a significant weed reduction. Therefore, based on previous findings and the results of this study, it may be possible to minimize the weed pressure through microwave irradiation of soil in no-till wheat production systems of Australia. However, a further research effort is needed to understand the long-term effects of microwave soil irradiation for weed control in crops. Furthermore, the fuel cost associated with a pre-sowing microwave weed management has been previously estimated by Khan et al. [16], therefore, about 0.98 L diesel m−2 were consumed in their experiment. Samtani et al. [135] calculated the fuel cost for pre-sowing steam treatment for weed control and reported a diesel consumption of between 0.81 and 2.16 L m−2. Considering the fuel consumption, the MW system used in the present investigation for soil heating was comparable or even better than soil steaming used by Gay et al. [133] and Samtani et al. [135].

In addition to weed suppression, a few previous studies have reported the supplementary effect of microwave energy on soil nutrient dynamics; Yang et al. [90] tested the nutrient extractability effect of microwave on soil. When fresh soil was exposed to microwave energy a dramatic increase in the NH<sup>4</sup> + -N concentration was observed for an extended treatment of 120 s. They concluded that this effect was partially from nonmicrobial processes, either from site exchange or from fixed position in inorganic collides (clay minerals). Hur et al. [101] demonstrated that microwave irradiation of soil can enhance the binding efficiency of hydrophobic organic containments with soil organic matter. They irradiated 5 g samples of soil in plastic tubes in aerobic and anaerobic conditions with activated C for 600 s in a lab-scale microwave oven (2.45 GHz) operated at 700 W. They pointed out that MW irradiation significantly alters the physical and chemical properties of soil organic matter and increased its humification. In another study, Kim and Kim [24] studied the influences of microwave irradiation on the soil organic matter properties. They reported that thermal cracking induced by irradiation potentially alters the molecular composition (C, H, O and N), chemical structure and humification of soil organic matter. Based on these previous findings, we assumed that thermal denaturation of recalcitrant humic substance induced by microwave irradiation may increase the concentrations of free amino acids for succeeding turnover to CO<sup>2</sup> and ammonia pool NH<sup>4</sup> + , which might have substantially increased wheat productivity in the present investigation. Moreover, microwave soil heating gave 10 times higher nitrogen use efficiency and about 20–50% higher irrigation water use efficiency in those field experiment conducted to manage the herbicides resistance weeds.

### **5. Conclusion**

**4. Discussion**

118 Rice Crop - Current Developments

increase in the NH<sup>4</sup>

+

Weed seedbank is a resting place of dormant seeds in the top soil horizon. Various biotic and abiotic factors have a tremendous effect on seed viability. Among the many abiotic factors, however, soil temperature has an ability to debilitate weed seeds *in situ* [136, 137]. Therefore, it was hypothesized that the projection of non-ionizing energy into a top soil layer through a horn antenna may cause thermal devitalisation of weed seeds. The results of field studies have strongly supported our hypothesis and achieved about 70–80% reduction in rice weeds establishment. Overall, energy of a microwave system for weed management program has a direct relationship with application duration. Therefore, for a pre-emergence weed control under field conditions, Wayland et al. [44] reported an energy level of 80 ˂ J cm−2 ˂ 160, which is quite low compared to the present investigation. In contrast, for a post-emergence weed control Sartorato et al. [54] tested the efficacy of microwave energy on seedling of *Abutilon theophrasti* and *Panicum miliaceum*. They reported that an energy range of 101.5 ˂ J cm−2 ˂ 343.3 gave significant reduction in dry weight of about 90%. However, it is highly unlikely that certain set of MW energy may give a same control spectrum, because soil moisture [74] and seed geometry [38, 67] have a considerable influence on microwave absorption. These vary according to cropping system.

Independent of soil heating methodology for weed control; various studies also reported the profound effect of high temperature on weed establishment. Gay et al. [133] reported on a soil steaming experiment with various duration (0, 6, 8 and 10 min) in a soil, to depths of about 1.5–16.5 cm, giving a temperature gradient of 100–37°C (decreasing with depth), in a lettuce crop for weed control. They found an average weed density of less than 50 plants m−2 in the case of soil steam treated plots compared to untreated control plots (400 plants m−2). Vidotto et al. [129] found that exposure of a soil-seed mixture to high temperature gradually decreased seed germination. Almost all the tested weed species seeds were completely devi-

The same temperature distribution was achieved through microwave application in the present study, which might have a degrading effect on the weed seedbank and ultimately led to a significant weed reduction. Therefore, based on previous findings and the results of this study, it may be possible to minimize the weed pressure through microwave irradiation of soil in no-till wheat production systems of Australia. However, a further research effort is needed to understand the long-term effects of microwave soil irradiation for weed control in crops. Furthermore, the fuel cost associated with a pre-sowing microwave weed management has been previously estimated by Khan et al. [16], therefore, about 0.98 L diesel m−2 were consumed in their experiment. Samtani et al. [135] calculated the fuel cost for pre-sowing steam treatment for weed control and reported a diesel consumption of between 0.81 and 2.16 L m−2. Considering the fuel consumption, the MW system used in the present investigation for soil heating was comparable or even better than soil steaming used by Gay et al. [133] and Samtani et al. [135]. In addition to weed suppression, a few previous studies have reported the supplementary effect of microwave energy on soil nutrient dynamics; Yang et al. [90] tested the nutrient extractability effect of microwave on soil. When fresh soil was exposed to microwave energy a dramatic

concluded that this effect was partially from nonmicrobial processes, either from site exchange or from fixed position in inorganic collides (clay minerals). Hur et al. [101] demonstrated that


talized through soil thermal treatment at a temperature between 70°C and 80°C.

Based on these experiments, we conclude that microwave weed and soil treatment can be implemented as an alternative method of weed control in direct-seeded rice crop. Additional benefit of this technology has prompted a motivation for further research in this area to enhance sustainability in agricultural industry.

### **Author details**

Muhammad Jamal Khan and Graham Ian Brodie\*

\*Address all correspondence to: grahamb@unimelb.edu.au

Faculty of Veterinary and Agricultural Sciences, Dookie Campus, The University of Melbourne, Melbourne, Australia

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126 Rice Crop - Current Developments


**Chapter 8**

**Provisional chapter**

**Phosphorus Efficient Phenotype of Rice**

**Phosphorus Efficient Phenotype of Rice**

DOI: 10.5772/intechopen.75642

The ideal phenotype to cope with P deficiency is suggested to be a larger root system, both in terms of length and foraging area, coupled with a high capacity for P solubilization from compounds exuded from roots. Greater soil exploration results in a large number of roots in the top soil, longer roots in general with more cortical aerenchyma, more and longer root hairs, and a shift in mycorrhizal and bacterial colonization. However, these assumptions often result from experiments in highly controlled, sterile and soil-free conditions using model plants or single ecotypes where results are then extrapolated to all genotypes and plant species. In recent years this generalization has been questioned. Here, we summarize recent rice research analyzing the natural diversity of rice root systems under P deficiency. Interestingly, while some of the high yielding genotypes do show the expected, large root system phenotype, some have a surprisingly small root system—as little as a quarter of that of the large root system varieties—but achieve similar yield and P uptake under P deficiency. This effect has recently been termed root efficiency, which we discuss in this chapter in conjunction with root foraging traits.

Rice (*Oryza sativa*) is the most important source of calories for millions of people [1] and, like all crops, its growth and yield is enabled by taking up water and nutrients from the soil through its root system. Yield can be constrained by many abiotic and biotic factors, including drought, nutrient deficiencies, and pathogen infections. Second, only to nitrogen (N), phosphorus (P)

**Keywords:** phosphorus deficiency, root system, root hair, root type,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Josefine Kant, Takuma Ishizaki, Juan Pariasca-Tanaka, Terry Rose, Matthias Wissuwa and Michelle Watt

Josefine Kant, Takuma Ishizaki, Juan Pariasca-Tanaka, Terry Rose, Matthias Wissuwa and Michelle Watt

http://dx.doi.org/10.5772/intechopen.75642

**Abstract**

xyloglucantransferase

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Chapter 8 Provisional chapter**

#### **Phosphorus Efficient Phenotype of Rice Phosphorus Efficient Phenotype of Rice**

DOI: 10.5772/intechopen.75642

Josefine Kant, Takuma Ishizaki, Juan Pariasca-Tanaka, Terry Rose, Matthias Wissuwa and Michelle Watt Josefine Kant, Takuma Ishizaki, Juan Pariasca-Tanaka, Terry Rose, Matthias Wissuwa and Michelle Watt

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75642

#### **Abstract**

The ideal phenotype to cope with P deficiency is suggested to be a larger root system, both in terms of length and foraging area, coupled with a high capacity for P solubilization from compounds exuded from roots. Greater soil exploration results in a large number of roots in the top soil, longer roots in general with more cortical aerenchyma, more and longer root hairs, and a shift in mycorrhizal and bacterial colonization. However, these assumptions often result from experiments in highly controlled, sterile and soil-free conditions using model plants or single ecotypes where results are then extrapolated to all genotypes and plant species. In recent years this generalization has been questioned. Here, we summarize recent rice research analyzing the natural diversity of rice root systems under P deficiency. Interestingly, while some of the high yielding genotypes do show the expected, large root system phenotype, some have a surprisingly small root system—as little as a quarter of that of the large root system varieties—but achieve similar yield and P uptake under P deficiency. This effect has recently been termed root efficiency, which we discuss in this chapter in conjunction with root foraging traits.

**Keywords:** phosphorus deficiency, root system, root hair, root type, xyloglucantransferase

### **1. Introduction**

Rice (*Oryza sativa*) is the most important source of calories for millions of people [1] and, like all crops, its growth and yield is enabled by taking up water and nutrients from the soil through its root system. Yield can be constrained by many abiotic and biotic factors, including drought, nutrient deficiencies, and pathogen infections. Second, only to nitrogen (N), phosphorus (P)

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

is an essential major nutrient necessary for biosynthesis of nucleotides, proteins, bio membranes, and energy metabolism such as the provision of ATP. In contrast to most N forms, P is highly sorbed to soil particles and is therefore considered to be an immobile nutrient in soils [2]. Estimates suggest that more than half of all agricultural soils are P deficient [3] which substantially affects plant growth, both below and above ground, to the extent that P is often the most yield-limiting nutrient in resource-poor farming systems in south-east Asia and Africa. In developed nations, P and N deficiencies are generally rectified by the application of mineral fertilizers to the soil, often in excess of that taken up by the crop. This leads to rapid leaching of N, more slowly also of P, into the ground water, eventually leading to eutrophication of water systems which leads to algal blooms, anaerobic water, and death of fish and other fauna [4]. In addition, mineral phosphate fertilizers are usually produced by mining rock phosphate and, although several predictions with variable numbers are present, these resources are limited and might be depleted in 50–100 years [5]. Therefore, understanding how P deficiency shapes root systems and how plants can overcome P deficiency is critical for future breeding of P efficient crops to ensure nutrition of the ever-growing world population in the future.

carboxylates required to mobilize P in incubation studies are much higher than those thought to occur in the rhizosphere of most plants [11]. As an example, a recent study using nearisogenic wheat (*Triticum aestivum*) lines that differed in citrate efflux failed to find evidence that higher citrate efflux led to any improvements in P uptake or crop yields [12]. The capacity for enhanced root exudation in rice to mobilize soil P is discussed in detail below (Section 2.3).

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 131

Arbuscular mycorrhizal (AM) fungi are recognized for enhancing nutrient availability, notably P, to most plants [13]. AM fungi are obligate biotrophs that establish mutualistic associations with the roots of over 90% of all plant species via a complex system of intraradical and extraradical hyphae, in which the external mycelium of AM fungi acts as an extension of host plant roots, thus increasing the effective surface area to absorb nutrients and water [13–15]. The transfer of nutrients from AM fungi to plant roots is typically facilitated by highly branched fungal structures within the root cortex, known as arbuscules [15]. The transfer of nutrients from AM fungi to roots occurs in exchange for sugars generated from photosynthesis and is typically facilitated by specific transporters expressed at the interface

To what extent the AM symbiosis is beneficial to crop species depends on environmental factors and varies between different crops and among varieties of the same crop. It is generally assumed that high rates of P fertilization diminish the potential benefits from AM symbiosis [17]. Further, one may assume that crops with a rather fine and dense root system would benefit less from the increase in the effective surface area provided by external hyphae compared to crops with less fine roots. Within crop species, varietal differences may be of importance and should ideally be explored in crop improvement programs. However, the multiple interactions between AM fungi, other soil microorganisms and plant roots, each potentially affected by soil properties, climatic factors, and crop management, have made a selection for

Application of free-living soil bacteria and fungi to plants can increase plant growth and nutrition status. As early as 1948 it was reported that isolated soil bacteria could enhance plant P solubilized from calcium phosphate [10]. Today efforts continue toward identifying microbiotaroot interactions beyond AM that enhance crop phosphorus acquisition. These are driven by commercial and regulatory pressures to supplement mined P reserves and optimize the recycling of P from soil and biomass pools [18], and by the increasingly comprehensive genomebased methods to characterize microbiomes [19] to explore to increase agricultural fertility [20]. Beneficial microbiota includes those contributing directly to plant or soil P fertility processes, and those indirectly contributing via control of plant host diseases, soil toxicities, and weeds that compete with crop resource uptake. P fertility mechanisms include (1) promoting greater root surface area, (2) increasing inorganic P availability in the soil solution, and (3) altering organic P pools in soil (e.g. rates of turnover). The evidence is mounting that microbiota can change molecular events within the plant (e.g. induce proton release for rhizosphere acidifica-

a stable increase of AM-variety symbiotic efficiency a rather challenging task.

**1.3. Association with beneficial bacteria and fungi other than AM**

tion, gene regulation involved in ion uptake) [21].

**1.2. Association with mycorrhizal fungi**

of plant root and arbuscule [15, 16].

Due to the immobile nature of P in soil, increasing root foraging for P is theoretically one of the best means by which to improve the efficiency of P acquisition by plants. Research in other crops and model plants have provided indications as to what the ideal root phenotype to cope with P deficiency should look like (reviewed in [3, 6]). Overall, it is suggested that a larger root system, both in length as well as in area, is beneficial for better yield performance in low P conditions. This higher soil exploration includes a large number of roots in the top soil (soil surface until 25 cm depth), longer roots in general, with more cortical aerenchyma, smaller diameter, more and longer root hairs, more mycorrhiza, altered bacterial colonization, and higher exudation of P-solubilizing chemicals.

In this chapter, we will (1) give a general literature overview of strategies for P uptake and its optimization and (2) review the proposed ideotype for rice P efficiency and present an experiment for its optimization using transgenic rice.

### **1.1. Exudation of P-solubilizing chemicals**

Mobilization of soil-bound P via the release of P-solubilizing compounds from roots is widely proposed to be a key mechanism by which many plant species enhance P acquisition in soils. These compounds include phosphatase enzymes capable of mobilizing organic P, protons that acidify the soil to solubilize calcium (Ca)-bound P and organic compounds including phytosiderophores and carboxylates that compete with P in ligand exchange reactions [7]. The release of protons and carboxylates is a strategy that is particularly important for species from the family Proteaceae, which have often evolved in extremely P deficient soils [8]. Other species that form proteiod—or cluster—roots under P deprivation such as the model legume species *Lupinus albus*, are also highly efficient at mobilizing P from aluminum (Al)- and iron (Fe)-P complexes via efflux of a targeted surge of carboxylates (predominantly citrate and malate) and protons from the cluster roots under P deficiency [9].

Beyond species that form cluster roots, however, the role of compounds exuded from roots is less clear. Experimental evidence that non-cluster root forming species can mobilize significant amounts of P through exudation of carboxylate is lacking [10] and the concentrations of carboxylates required to mobilize P in incubation studies are much higher than those thought to occur in the rhizosphere of most plants [11]. As an example, a recent study using nearisogenic wheat (*Triticum aestivum*) lines that differed in citrate efflux failed to find evidence that higher citrate efflux led to any improvements in P uptake or crop yields [12]. The capacity for enhanced root exudation in rice to mobilize soil P is discussed in detail below (Section 2.3).

### **1.2. Association with mycorrhizal fungi**

is an essential major nutrient necessary for biosynthesis of nucleotides, proteins, bio membranes, and energy metabolism such as the provision of ATP. In contrast to most N forms, P is highly sorbed to soil particles and is therefore considered to be an immobile nutrient in soils [2]. Estimates suggest that more than half of all agricultural soils are P deficient [3] which substantially affects plant growth, both below and above ground, to the extent that P is often the most yield-limiting nutrient in resource-poor farming systems in south-east Asia and Africa. In developed nations, P and N deficiencies are generally rectified by the application of mineral fertilizers to the soil, often in excess of that taken up by the crop. This leads to rapid leaching of N, more slowly also of P, into the ground water, eventually leading to eutrophication of water systems which leads to algal blooms, anaerobic water, and death of fish and other fauna [4]. In addition, mineral phosphate fertilizers are usually produced by mining rock phosphate and, although several predictions with variable numbers are present, these resources are limited and might be depleted in 50–100 years [5]. Therefore, understanding how P deficiency shapes root systems and how plants can overcome P deficiency is critical for future breeding of P effi-

cient crops to ensure nutrition of the ever-growing world population in the future.

higher exudation of P-solubilizing chemicals.

130 Rice Crop - Current Developments

ment for its optimization using transgenic rice.

malate) and protons from the cluster roots under P deficiency [9].

**1.1. Exudation of P-solubilizing chemicals**

Due to the immobile nature of P in soil, increasing root foraging for P is theoretically one of the best means by which to improve the efficiency of P acquisition by plants. Research in other crops and model plants have provided indications as to what the ideal root phenotype to cope with P deficiency should look like (reviewed in [3, 6]). Overall, it is suggested that a larger root system, both in length as well as in area, is beneficial for better yield performance in low P conditions. This higher soil exploration includes a large number of roots in the top soil (soil surface until 25 cm depth), longer roots in general, with more cortical aerenchyma, smaller diameter, more and longer root hairs, more mycorrhiza, altered bacterial colonization, and

In this chapter, we will (1) give a general literature overview of strategies for P uptake and its optimization and (2) review the proposed ideotype for rice P efficiency and present an experi-

Mobilization of soil-bound P via the release of P-solubilizing compounds from roots is widely proposed to be a key mechanism by which many plant species enhance P acquisition in soils. These compounds include phosphatase enzymes capable of mobilizing organic P, protons that acidify the soil to solubilize calcium (Ca)-bound P and organic compounds including phytosiderophores and carboxylates that compete with P in ligand exchange reactions [7]. The release of protons and carboxylates is a strategy that is particularly important for species from the family Proteaceae, which have often evolved in extremely P deficient soils [8]. Other species that form proteiod—or cluster—roots under P deprivation such as the model legume species *Lupinus albus*, are also highly efficient at mobilizing P from aluminum (Al)- and iron (Fe)-P complexes via efflux of a targeted surge of carboxylates (predominantly citrate and

Beyond species that form cluster roots, however, the role of compounds exuded from roots is less clear. Experimental evidence that non-cluster root forming species can mobilize significant amounts of P through exudation of carboxylate is lacking [10] and the concentrations of Arbuscular mycorrhizal (AM) fungi are recognized for enhancing nutrient availability, notably P, to most plants [13]. AM fungi are obligate biotrophs that establish mutualistic associations with the roots of over 90% of all plant species via a complex system of intraradical and extraradical hyphae, in which the external mycelium of AM fungi acts as an extension of host plant roots, thus increasing the effective surface area to absorb nutrients and water [13–15]. The transfer of nutrients from AM fungi to plant roots is typically facilitated by highly branched fungal structures within the root cortex, known as arbuscules [15]. The transfer of nutrients from AM fungi to roots occurs in exchange for sugars generated from photosynthesis and is typically facilitated by specific transporters expressed at the interface of plant root and arbuscule [15, 16].

To what extent the AM symbiosis is beneficial to crop species depends on environmental factors and varies between different crops and among varieties of the same crop. It is generally assumed that high rates of P fertilization diminish the potential benefits from AM symbiosis [17]. Further, one may assume that crops with a rather fine and dense root system would benefit less from the increase in the effective surface area provided by external hyphae compared to crops with less fine roots. Within crop species, varietal differences may be of importance and should ideally be explored in crop improvement programs. However, the multiple interactions between AM fungi, other soil microorganisms and plant roots, each potentially affected by soil properties, climatic factors, and crop management, have made a selection for a stable increase of AM-variety symbiotic efficiency a rather challenging task.

### **1.3. Association with beneficial bacteria and fungi other than AM**

Application of free-living soil bacteria and fungi to plants can increase plant growth and nutrition status. As early as 1948 it was reported that isolated soil bacteria could enhance plant P solubilized from calcium phosphate [10]. Today efforts continue toward identifying microbiotaroot interactions beyond AM that enhance crop phosphorus acquisition. These are driven by commercial and regulatory pressures to supplement mined P reserves and optimize the recycling of P from soil and biomass pools [18], and by the increasingly comprehensive genomebased methods to characterize microbiomes [19] to explore to increase agricultural fertility [20].

Beneficial microbiota includes those contributing directly to plant or soil P fertility processes, and those indirectly contributing via control of plant host diseases, soil toxicities, and weeds that compete with crop resource uptake. P fertility mechanisms include (1) promoting greater root surface area, (2) increasing inorganic P availability in the soil solution, and (3) altering organic P pools in soil (e.g. rates of turnover). The evidence is mounting that microbiota can change molecular events within the plant (e.g. induce proton release for rhizosphere acidification, gene regulation involved in ion uptake) [21].

Management of beneficial microbiota is mainly done through inoculation onto the plant (seed or shoots) of microbes that are isolated from a given environment. However, management of the naturally-associating rhizosphere microbiota through the plant (e.g. plant genetics and breeding) or soil (e.g. rotations, tillage) is also performed. Molecular characterization of the whole root and soil microbiomes will likely lead to a merging of inoculant approaches with the direct engineering of rhizosphere microbiota [22], as plant hosts are intimately connected with microbiota whose genes can be transferred with that of the host genome [23]. Although many examples report beneficial responses to bacteria or fungal strains on plants in pots in glasshouses, there are few translations to farmers' fields with formulated, scaled production [18].

**2. P deficiency responses found in rice**

**2.3. Exudation of P-solubilizing chemicals by rice**

higher P acquisition efficiency for any rice genotype.

but also occur in upland systems.

**2.1. The rice root system**

**2.2. Root efficiency in rice**

Rice roots face highly dynamic soil conditions; possibly the most complex of all cereal grains. Soils have repeated flooding, irrigation and drying events, and tilling and compacting results in very soft soils, hardpans, and furrows, with dramatic variation in aeration, pH and nutrient conditions from the surface to depth [45]. These transitions are greatest in lowland systems,

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 133

The rice root system consists of the first emerging embryonic seminal roots as well as postembryonic crown roots emerging from shoot-borne nodes. In addition, all of these main root axiles can branch, forming lateral roots (LRs) of first and higher orders. A unique feature of rice is the formation of very distinct classes of these LRs—S- and L-type [46]. The most abundant LRs are the short and thin S-type LRs. L-type LRs are much longer, have a larger

In order to analyze the natural variation of P deficiency tolerance, a large panel of diverse rice genotypes (>200 genotypes) was grown in upland fields, both in a sufficient fertilized and P deficient fields, and their root systems were analyzed and correlated to yield performance and P uptake. Interestingly, while some of the high yielding genotypes did show the estimated, large root system phenotype under P deficiency, some genotypes had a surprisingly small root system—as little as a quarter of the large root system varieties—but achieved the same yield and P uptake under P deficiency [47]. This effect has been observed in a number of field trials and recently been termed '**root efficiency**'. What could be the basis of this root efficiency? Which traits could enable a smaller root system to take up P as efficiently as or even more efficiently than a bigger one? The efficiency could be based on any or all of the aforementioned P fertility traits for plants (Section 1), and these will be elaborated on for rice in the following sections.

Rice plants exude a range of compounds from their roots, and the amount and composition of these exudates change in response to P deficiency [48–50]. A number of lines of enquiry suggest that increased efflux of carboxylates, particularly citrate, enhances the capacity of rice genotypes to acquire P. Ref. [48] reported an increase in citrate efflux under P deficiency in seven rice genotypes, and citrate efflux rates were correlated with the tolerance of these rice genotypes to P deficiency (defined as ratio of biomass grown with sparingly soluble P source compared to a soluble P source). Also found was increased citrate efflux from rice roots under P deficiency. Subsequent modeling studies indicated the importance of citrate efflux in P solubilization and uptake by rice growing under aerobic conditions [51]. However, as pointed out in a review of root traits associated with the efficient acquisition of soil P by rice [6], there is no direct evidence that increased efflux of citrate, or any other carboxylate, is responsible for

diameter and form branch roots, thus producing higher order LRs (**Figure 1**).

### **1.4. Aerenchyma formation**

Aerenchyma refers to plant tissue containing enlarged gas spaces, formed in roots and shoots of wetland and dryland species through cell death "lysigenous" or cell separation "schizogenous" [24, 25]. Formation of aerenchyma can be constitutive or induced by abiotic stresses such as waterlogging [26], drought, and nutrient deficiency including phosphorus, nitrogen and sulfur deficiency [27–32]. Therefore, the presence of aerenchyma can differ even within root segments when heterogeneous abiotic conditions, such as those in field soils, are present [30, 33, 34].

Several reports from maize (*Zea mays*), bean (*Phaseolus vulgaris*), and barley (*Hordeum vulgare*) results suggested that aerenchyma formation reduces the metabolic costs of soil exploration [35–37] through the decrease of root nutrient content and respiration [31]. Nutrients such as P, released during the aerenchyma formation (cell death), could be reutilized by the plant, for example, in the continued growth of apical cells. Increased soil exploration for more P resources, coupled with less P required for root functional maintenance, would be an advantageous trait under P-limitation.

#### **1.5. Root hair production**

Root hairs are unicellular extensions of epidermal cells that constitute most of the root's surface area [38]. Root hairs have been under investigation for more than a century [39], and it was shown decades ago that root hairs are involved in P uptake from soil. P uptake by root hairs was indirectly demonstrated for wheat and barley [40], and directly for rye (*Secale cereale*) root hairs [41]. Enhanced root hair development is consistently listed as one of the cheapest and most general adaptations to P deficiency [3]. And yet, how and if their presence is beneficial for P uptake remains under discussion. Within the molecular model plant *Arabidopsis thaliana*, a study investigating phenotypic reactions of >150 ecotypes to local P levels revealed unexpected results. Half of the tested genotypes did not show any root hair reaction to local low P, while one quarter responded with a production of shorter and less, and the other quarter with longer and more root hairs [42]. Another study showed that maize root hairs were more responsive to soil moisture than to soil P level [43]. While lower water content correlated with the production of more and longer root hairs, no correlation was found with soil P concentration. A recent study on barley seedlings developing normal or very short root hairs came to the conclusion that root hairs are instrumental for water uptake from drying soil when plant transpiration rates are high [44].

### **2. P deficiency responses found in rice**

Rice roots face highly dynamic soil conditions; possibly the most complex of all cereal grains. Soils have repeated flooding, irrigation and drying events, and tilling and compacting results in very soft soils, hardpans, and furrows, with dramatic variation in aeration, pH and nutrient conditions from the surface to depth [45]. These transitions are greatest in lowland systems, but also occur in upland systems.

### **2.1. The rice root system**

Management of beneficial microbiota is mainly done through inoculation onto the plant (seed or shoots) of microbes that are isolated from a given environment. However, management of the naturally-associating rhizosphere microbiota through the plant (e.g. plant genetics and breeding) or soil (e.g. rotations, tillage) is also performed. Molecular characterization of the whole root and soil microbiomes will likely lead to a merging of inoculant approaches with the direct engineering of rhizosphere microbiota [22], as plant hosts are intimately connected with microbiota whose genes can be transferred with that of the host genome [23]. Although many examples report beneficial responses to bacteria or fungal strains on plants in pots in glasshouses, there are few translations to farmers' fields with formulated, scaled production [18].

Aerenchyma refers to plant tissue containing enlarged gas spaces, formed in roots and shoots of wetland and dryland species through cell death "lysigenous" or cell separation "schizogenous" [24, 25]. Formation of aerenchyma can be constitutive or induced by abiotic stresses such as waterlogging [26], drought, and nutrient deficiency including phosphorus, nitrogen and sulfur deficiency [27–32]. Therefore, the presence of aerenchyma can differ even within root segments when heterogeneous abiotic conditions, such as those in field soils, are present

Several reports from maize (*Zea mays*), bean (*Phaseolus vulgaris*), and barley (*Hordeum vulgare*) results suggested that aerenchyma formation reduces the metabolic costs of soil exploration [35–37] through the decrease of root nutrient content and respiration [31]. Nutrients such as P, released during the aerenchyma formation (cell death), could be reutilized by the plant, for example, in the continued growth of apical cells. Increased soil exploration for more P resources, coupled with less P required for root functional maintenance, would be an advan-

Root hairs are unicellular extensions of epidermal cells that constitute most of the root's surface area [38]. Root hairs have been under investigation for more than a century [39], and it was shown decades ago that root hairs are involved in P uptake from soil. P uptake by root hairs was indirectly demonstrated for wheat and barley [40], and directly for rye (*Secale cereale*) root hairs [41]. Enhanced root hair development is consistently listed as one of the cheapest and most general adaptations to P deficiency [3]. And yet, how and if their presence is beneficial for P uptake remains under discussion. Within the molecular model plant *Arabidopsis thaliana*, a study investigating phenotypic reactions of >150 ecotypes to local P levels revealed unexpected results. Half of the tested genotypes did not show any root hair reaction to local low P, while one quarter responded with a production of shorter and less, and the other quarter with longer and more root hairs [42]. Another study showed that maize root hairs were more responsive to soil moisture than to soil P level [43]. While lower water content correlated with the production of more and longer root hairs, no correlation was found with soil P concentration. A recent study on barley seedlings developing normal or very short root hairs came to the conclusion that root hairs are instrumental for water uptake

from drying soil when plant transpiration rates are high [44].

**1.4. Aerenchyma formation**

132 Rice Crop - Current Developments

tageous trait under P-limitation.

**1.5. Root hair production**

[30, 33, 34].

The rice root system consists of the first emerging embryonic seminal roots as well as postembryonic crown roots emerging from shoot-borne nodes. In addition, all of these main root axiles can branch, forming lateral roots (LRs) of first and higher orders. A unique feature of rice is the formation of very distinct classes of these LRs—S- and L-type [46]. The most abundant LRs are the short and thin S-type LRs. L-type LRs are much longer, have a larger diameter and form branch roots, thus producing higher order LRs (**Figure 1**).

### **2.2. Root efficiency in rice**

In order to analyze the natural variation of P deficiency tolerance, a large panel of diverse rice genotypes (>200 genotypes) was grown in upland fields, both in a sufficient fertilized and P deficient fields, and their root systems were analyzed and correlated to yield performance and P uptake. Interestingly, while some of the high yielding genotypes did show the estimated, large root system phenotype under P deficiency, some genotypes had a surprisingly small root system—as little as a quarter of the large root system varieties—but achieved the same yield and P uptake under P deficiency [47]. This effect has been observed in a number of field trials and recently been termed '**root efficiency**'. What could be the basis of this root efficiency? Which traits could enable a smaller root system to take up P as efficiently as or even more efficiently than a bigger one? The efficiency could be based on any or all of the aforementioned P fertility traits for plants (Section 1), and these will be elaborated on for rice in the following sections.

#### **2.3. Exudation of P-solubilizing chemicals by rice**

Rice plants exude a range of compounds from their roots, and the amount and composition of these exudates change in response to P deficiency [48–50]. A number of lines of enquiry suggest that increased efflux of carboxylates, particularly citrate, enhances the capacity of rice genotypes to acquire P. Ref. [48] reported an increase in citrate efflux under P deficiency in seven rice genotypes, and citrate efflux rates were correlated with the tolerance of these rice genotypes to P deficiency (defined as ratio of biomass grown with sparingly soluble P source compared to a soluble P source). Also found was increased citrate efflux from rice roots under P deficiency. Subsequent modeling studies indicated the importance of citrate efflux in P solubilization and uptake by rice growing under aerobic conditions [51]. However, as pointed out in a review of root traits associated with the efficient acquisition of soil P by rice [6], there is no direct evidence that increased efflux of citrate, or any other carboxylate, is responsible for higher P acquisition efficiency for any rice genotype.

**2.4. Rice association with mycorrhizal fungi**

efficacy in the field [62].

Rice is a host for AM fungi [15, 16] and although root colonization has been observed under irrigated lowland and upland conditions, it is generally assumed that the AM symbiosis is more important in the aerobic upland rice [54]. It has been shown that P transporters exist in rice that are specifically induced in roots colonized by AM fungi and that these P transporters (*OsPT11* and *OsPT13*) facilitate the transfer of Pi from AM fungus to plant [16]. Compared to some other crop species, however, our knowledge regarding the AM fungi community colonizing rice roots in the field remains limited, and the extent to which AM symbiosis may be exploited to benefit rice yield directly in the field is unknown. Studies comparing AM colonization in the field in a set of diverse rice genotypes indicated that considerable variation in colonization rates exists (Wissuwa, unpublished). Further, all root samples taken showed gene expression of the *OsPT11* transporter, suggesting P transfer from AM fungus to rice roots commonly occurred. Yet neither colonization rates nor gene expression levels were correlated to the large genotypic differences observed for P uptake in that set of rice genotypes (Wissuwa, unpublished), whereas P uptake correlated strongly to root size [47]. Earlier studies in one set of near-isogenic rice lines differing in root size and P uptake showed these differences remained unchanged in sterilized soil [55]. Based on the limited evidence available to date, one may tentatively conclude that the AM symbiosis contributes less to rice genotypic differences in P uptake compared to root attributes such as size, fineness or root hair length and density [56].

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 135

**2.5. Rice association with beneficial bacteria and fungi other than AM**

There are intense research efforts to apply rhizobacteria to rice to boost its productivity and increase environmental sustainability because of the enormous value of this crop and the resources used globally. Beneficial microbiota are tested to (1) reduce methane emissions [57]; (2) increase nitrogen uptake [58]; (3) fix atmospheric nitrogen within the root [59]; (4) reduce diseases [60], and (5) increase P nutrition, and to a lesser extent, that of Fe and other micronutrients [61]. In field experiments with farmer participation, beneficial microbiota is being combined with fertilizer applications. This mode of application and level of participation appears to be required for repeatedly validating inoculants that demonstrate high

The types of microorganisms previously tested for improved P uptake by rice include bacteria of the genera: *Rhizobium* [63], *Pseudomonas*, *Azotobacter*, *Azospirillum* [61, 59], and *Enterobacter* [64], as well as bacteria within a consortium of mixed genera [65]. Applied organisms may reside at the root-soil interface, or within the root (endosphere). The endosphere aerenchyma may have more stable gaseous conditions than the root-soil and outer rhizosphere zones and is a target zone for beneficial isolates, referred to as endophytes [66]. Possible functions of microbiota for P fertility are the same as those proposed for other crops (reviewed in [10, 67–69]), with the exception of much greater emphasis on endophytic diazotrophic (nitrogen fixing) bacteria, perhaps driven by the unique aerenchyma environment in rice, and/or rice-specific responsiveness to effectors associated with endophytic colonization [70]. Given the niche opportunity for endophytes within rice axile root aerenchyma, it may be beneficial to look for consortia of microorganisms or native rhizosphere (including endo-

sphere) profiles that promote these developmental features within the roots.

**Figure 1.** The rice root system structure. Top soil washed off to display the root system of a field-grown rice plant ca. 50 DAS (A). Seven DAS seedlings with many seminal roots (left) or a high proportion of lateral roots (right) (B). Stereomicroscopic image demonstrating the rice root characteristics (C). Light microscopic images of rice roots grown in nutrient solution (D) or soil (E). MR: main root (seminal or crown root), LR: lateral root, L: L-type LR, S: S-type LR, 2O: second order LR, RH: root hair.

Protons and phosphatase enzymes are also released into the rhizosphere from rice roots. The release of protons typically occurs due to an imbalance in cation/anion uptake by roots [52]. Genes encoding phosphatases are up-regulated in roots under P deficiency [53], but we are not aware of any published studies that have demonstrated that increased phosphatase efflux from the roots of rice is linked to enhanced P uptake, or that such a trait confers greater P uptake in any given rice genotype.

#### **2.4. Rice association with mycorrhizal fungi**

Protons and phosphatase enzymes are also released into the rhizosphere from rice roots. The release of protons typically occurs due to an imbalance in cation/anion uptake by roots [52]. Genes encoding phosphatases are up-regulated in roots under P deficiency [53], but we are not aware of any published studies that have demonstrated that increased phosphatase efflux from the roots of rice is linked to enhanced P uptake, or that such a trait confers greater P

**Figure 1.** The rice root system structure. Top soil washed off to display the root system of a field-grown rice plant ca. 50 DAS (A). Seven DAS seedlings with many seminal roots (left) or a high proportion of lateral roots (right) (B). Stereomicroscopic image demonstrating the rice root characteristics (C). Light microscopic images of rice roots grown in nutrient solution (D) or soil (E). MR: main root (seminal or crown root), LR: lateral root, L: L-type LR, S: S-type LR, 2O:

uptake in any given rice genotype.

second order LR, RH: root hair.

134 Rice Crop - Current Developments

Rice is a host for AM fungi [15, 16] and although root colonization has been observed under irrigated lowland and upland conditions, it is generally assumed that the AM symbiosis is more important in the aerobic upland rice [54]. It has been shown that P transporters exist in rice that are specifically induced in roots colonized by AM fungi and that these P transporters (*OsPT11* and *OsPT13*) facilitate the transfer of Pi from AM fungus to plant [16]. Compared to some other crop species, however, our knowledge regarding the AM fungi community colonizing rice roots in the field remains limited, and the extent to which AM symbiosis may be exploited to benefit rice yield directly in the field is unknown. Studies comparing AM colonization in the field in a set of diverse rice genotypes indicated that considerable variation in colonization rates exists (Wissuwa, unpublished). Further, all root samples taken showed gene expression of the *OsPT11* transporter, suggesting P transfer from AM fungus to rice roots commonly occurred. Yet neither colonization rates nor gene expression levels were correlated to the large genotypic differences observed for P uptake in that set of rice genotypes (Wissuwa, unpublished), whereas P uptake correlated strongly to root size [47]. Earlier studies in one set of near-isogenic rice lines differing in root size and P uptake showed these differences remained unchanged in sterilized soil [55]. Based on the limited evidence available to date, one may tentatively conclude that the AM symbiosis contributes less to rice genotypic differences in P uptake compared to root attributes such as size, fineness or root hair length and density [56].

#### **2.5. Rice association with beneficial bacteria and fungi other than AM**

There are intense research efforts to apply rhizobacteria to rice to boost its productivity and increase environmental sustainability because of the enormous value of this crop and the resources used globally. Beneficial microbiota are tested to (1) reduce methane emissions [57]; (2) increase nitrogen uptake [58]; (3) fix atmospheric nitrogen within the root [59]; (4) reduce diseases [60], and (5) increase P nutrition, and to a lesser extent, that of Fe and other micronutrients [61]. In field experiments with farmer participation, beneficial microbiota is being combined with fertilizer applications. This mode of application and level of participation appears to be required for repeatedly validating inoculants that demonstrate high efficacy in the field [62].

The types of microorganisms previously tested for improved P uptake by rice include bacteria of the genera: *Rhizobium* [63], *Pseudomonas*, *Azotobacter*, *Azospirillum* [61, 59], and *Enterobacter* [64], as well as bacteria within a consortium of mixed genera [65]. Applied organisms may reside at the root-soil interface, or within the root (endosphere). The endosphere aerenchyma may have more stable gaseous conditions than the root-soil and outer rhizosphere zones and is a target zone for beneficial isolates, referred to as endophytes [66]. Possible functions of microbiota for P fertility are the same as those proposed for other crops (reviewed in [10, 67–69]), with the exception of much greater emphasis on endophytic diazotrophic (nitrogen fixing) bacteria, perhaps driven by the unique aerenchyma environment in rice, and/or rice-specific responsiveness to effectors associated with endophytic colonization [70]. Given the niche opportunity for endophytes within rice axile root aerenchyma, it may be beneficial to look for consortia of microorganisms or native rhizosphere (including endosphere) profiles that promote these developmental features within the roots.

### **2.6. Aerenchyma formation in rice**

Rice roots can form lysigenous (cell death) aerenchyma in both forms: constitutive, developed from the apical parts of the roots toward the base; and inducible, promoted in all parts of the roots under anaerobic conditions such as waterlogging, drought or nutrient deficiency.

*2.7.2. Influence of root hair longevity*

*2.7.3. Root hairs impacted by experimental conditions*

length of those restricted by soil particles [75].

*2.7.4. Genotype-dependent root hair formation*

Very few studies have addressed the question of root hair longevity and how long root hairs contribute to plant water and nutrient uptake. In [56] a higher proportion of living rice root hair cells was found in the low P compared to a P-fertilized field, over five tested genotypes. It can be concluded that longer living root hairs might be an adaptation to low P conditions to prolong root hair contribution to P uptake. Nevertheless, in the top soil layer fewer living root hairs were found compared to the subsoil [56]. This can most likely be attributed to the presence of younger root segments including root tips in the deeper soil layer compared to the oldest root segments in the top soil. In future experiments, more emphasis should be laid on longevity not just of root hairs, but also of lateral and main axile roots as harnessing natural variation in this

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 137

trait could lead to improvements in breeding for more P deficiency tolerant varieties.

root hairs of comparable length and density as those found in nutrient solution.

Our recent findings highlighted the vast influence that growth medium has on root hair formation [75]. Nutrient solution led rice seedlings to produce many, long root hairs on their main roots, while the discrepancy between root types was much smaller in soil-grown plants. We were able to reproduce these results in small plastic containers (50 ml volume) already 7 days after germination (DAG) (**Figure 2**). In addition to soil and nutrient solution, we grew rice seedlings in an artificial soil (vermiculite) and an agar solution without nutrient addition. Vermiculite led to very similar root hair growth to the soil, while roots grown in agar formed

One possible explanation for the observed differences among growth conditions is the absence or presence of physical restriction which can be found in soil particles, but not in a nutrient solution. To test this, another experiment was conducted to investigate the effect of growth medium strength on root hair length in rice seedlings. The experiment comprised six concentrations of agar with two replicate boxes per concentration. Root hair length after 3 weeks of growth significantly decreased with increasing agar concentration (**Figure 2**), proving that soil strength/presence of physical restrictions is one-factor influencing root hair growth. Detection of rice root hairs in undisturbed soil using a synchrotron approach also substantiated this assumption as root hairs growing in pores were found to grow to three times the

The commonly-stated response to P deficiency of production of longer and more root hairs may depend on genotype. For example, a wide selection of genotypes of the sup-populations indica, aus, temperate japonica, aromatic, and tropical japonica were tested in buffered, diffusion-limited solid-phase solution, and genotype-dependent variable responses to the low P condition was found [71]. We recently showed that in the soil, under controlled conditions in a greenhouse as well as in the field, some genotypes even form shorter and/or fewer root hairs in low P compared to sufficiently P-fertilized soil [56]. This highlights that increased root hair production is not a general, inevitable event upon P deficiency, but a specialized adaptation of some genotypes. Similarly, **root efficiency** (P uptake per unit root length) mentioned above

The advantage of aerenchyma formation for more soil exploration with fewer nutrients required may be greater in rice than maize, for example, due to its greater tendency for aerenchyma formation and enhanced root length under low nutrient treatments. For example, phosphorus deficiency causes a 20% increase in the percent cortical area converted to aerenchyma in rice [71]. Likewise, aerenchyma formation is enhanced by both nitrogen and oxygen-deficient conditions [72]. In addition to aerenchyma, rice possesses a barrier to radial O<sup>2</sup> loss (ROL) to the external environment, which promotes diffusion of O<sup>2</sup> toward the root apex. The O<sup>2</sup> increases the redox potential in the rhizosphere and causes the oxidation of Mn4+ and Fe2+ forming the plaque on the surface of rice roots [73] (which may reduce the uptake of phytotoxic elements into plant tissue).

Although the benefits of aerenchyma and ROL barrier formation are widely reported, the molecular mechanisms that regulate their formation are not completely understood. Since the rice genome has been fully sequenced and many tools are available, further insights into the molecular determinants of aerenchyma and other rice-specific tissues are expected, in order to improve rice cultivars by using modern breeding techniques.

#### **2.7. Root hair formation in rice**

An often heard assumption is that root hairs are adaptations specific to limiting conditions and are not needed for growth in optimal conditions. Consequently, elite varieties adapted to high input, optimized soil conditions should produce short and few root hairs and be less responsive to stress conditions compared to landraces.

In the next sections, we will review recent findings regarding natural variation in rice root hair formation in response to P deficiency, growth conditions, and in respect to the rice root system structure. Finally, we will also give an example of transgenic plant generation in the attempt of increasing rice P uptake under deficient conditions.

#### *2.7.1. Root hair variation within root types*

Lateral roots were recently proposed to have different, specialized functions depending on their developmental type [74]. Supporting this proposal, we showed that the thinner lateral roots of rice produced shorter and fewer root hairs [75], and found a positive linear relationship between lateral root diameter and root hair length [56]. These results were reproduced in different growth conditions, with main roots (seminal and crown axile roots) consistently producing the longest and most root hairs, followed by L-type and S-type lateral roots (**Figure 2**) (see Section 4 for experimental details). On the other hand, all root types within a genotype had the same tendencies regarding root hair length and density, and differences between genotypes were stable per root type [56]. This indicates that the phenotypic potential per genotype is first determined by its genome and then by the environment.

#### *2.7.2. Influence of root hair longevity*

**2.6. Aerenchyma formation in rice**

136 Rice Crop - Current Developments

totoxic elements into plant tissue).

**2.7. Root hair formation in rice**

*2.7.1. Root hair variation within root types*

The O<sup>2</sup>

Rice roots can form lysigenous (cell death) aerenchyma in both forms: constitutive, developed from the apical parts of the roots toward the base; and inducible, promoted in all parts of the roots under anaerobic conditions such as waterlogging, drought or nutrient deficiency.

The advantage of aerenchyma formation for more soil exploration with fewer nutrients required may be greater in rice than maize, for example, due to its greater tendency for aerenchyma formation and enhanced root length under low nutrient treatments. For example, phosphorus deficiency causes a 20% increase in the percent cortical area converted to aerenchyma in rice [71]. Likewise, aerenchyma formation is enhanced by both nitrogen and oxygen-deficient conditions [72]. In addition to aerenchyma, rice possesses a barrier to radial O<sup>2</sup>

increases the redox potential in the rhizosphere and causes the oxidation of Mn4+ and

Fe2+ forming the plaque on the surface of rice roots [73] (which may reduce the uptake of phy-

Although the benefits of aerenchyma and ROL barrier formation are widely reported, the molecular mechanisms that regulate their formation are not completely understood. Since the rice genome has been fully sequenced and many tools are available, further insights into the molecular determinants of aerenchyma and other rice-specific tissues are expected, in

An often heard assumption is that root hairs are adaptations specific to limiting conditions and are not needed for growth in optimal conditions. Consequently, elite varieties adapted to high input, optimized soil conditions should produce short and few root hairs and be less

In the next sections, we will review recent findings regarding natural variation in rice root hair formation in response to P deficiency, growth conditions, and in respect to the rice root system structure. Finally, we will also give an example of transgenic plant generation in the

Lateral roots were recently proposed to have different, specialized functions depending on their developmental type [74]. Supporting this proposal, we showed that the thinner lateral roots of rice produced shorter and fewer root hairs [75], and found a positive linear relationship between lateral root diameter and root hair length [56]. These results were reproduced in different growth conditions, with main roots (seminal and crown axile roots) consistently producing the longest and most root hairs, followed by L-type and S-type lateral roots (**Figure 2**) (see Section 4 for experimental details). On the other hand, all root types within a genotype had the same tendencies regarding root hair length and density, and differences between genotypes were stable per root type [56]. This indicates that the phenotypic potential per

toward the root apex.

loss (ROL) to the external environment, which promotes diffusion of O<sup>2</sup>

order to improve rice cultivars by using modern breeding techniques.

responsive to stress conditions compared to landraces.

attempt of increasing rice P uptake under deficient conditions.

genotype is first determined by its genome and then by the environment.

Very few studies have addressed the question of root hair longevity and how long root hairs contribute to plant water and nutrient uptake. In [56] a higher proportion of living rice root hair cells was found in the low P compared to a P-fertilized field, over five tested genotypes. It can be concluded that longer living root hairs might be an adaptation to low P conditions to prolong root hair contribution to P uptake. Nevertheless, in the top soil layer fewer living root hairs were found compared to the subsoil [56]. This can most likely be attributed to the presence of younger root segments including root tips in the deeper soil layer compared to the oldest root segments in the top soil. In future experiments, more emphasis should be laid on longevity not just of root hairs, but also of lateral and main axile roots as harnessing natural variation in this trait could lead to improvements in breeding for more P deficiency tolerant varieties.

#### *2.7.3. Root hairs impacted by experimental conditions*

Our recent findings highlighted the vast influence that growth medium has on root hair formation [75]. Nutrient solution led rice seedlings to produce many, long root hairs on their main roots, while the discrepancy between root types was much smaller in soil-grown plants. We were able to reproduce these results in small plastic containers (50 ml volume) already 7 days after germination (DAG) (**Figure 2**). In addition to soil and nutrient solution, we grew rice seedlings in an artificial soil (vermiculite) and an agar solution without nutrient addition. Vermiculite led to very similar root hair growth to the soil, while roots grown in agar formed root hairs of comparable length and density as those found in nutrient solution.

One possible explanation for the observed differences among growth conditions is the absence or presence of physical restriction which can be found in soil particles, but not in a nutrient solution. To test this, another experiment was conducted to investigate the effect of growth medium strength on root hair length in rice seedlings. The experiment comprised six concentrations of agar with two replicate boxes per concentration. Root hair length after 3 weeks of growth significantly decreased with increasing agar concentration (**Figure 2**), proving that soil strength/presence of physical restrictions is one-factor influencing root hair growth. Detection of rice root hairs in undisturbed soil using a synchrotron approach also substantiated this assumption as root hairs growing in pores were found to grow to three times the length of those restricted by soil particles [75].

#### *2.7.4. Genotype-dependent root hair formation*

The commonly-stated response to P deficiency of production of longer and more root hairs may depend on genotype. For example, a wide selection of genotypes of the sup-populations indica, aus, temperate japonica, aromatic, and tropical japonica were tested in buffered, diffusion-limited solid-phase solution, and genotype-dependent variable responses to the low P condition was found [71]. We recently showed that in the soil, under controlled conditions in a greenhouse as well as in the field, some genotypes even form shorter and/or fewer root hairs in low P compared to sufficiently P-fertilized soil [56]. This highlights that increased root hair production is not a general, inevitable event upon P deficiency, but a specialized adaptation of some genotypes. Similarly, **root efficiency** (P uptake per unit root length) mentioned above

on a previous study of transcriptomes of a P deficiency intolerant and a tolerant rice genotype. Several genes putatively associated with root cell wall loosening and root hair extension were found to have higher expression in the roots of the tolerant genotype [53]. We chose one gene to study that encodes a putative cell wall modifying enzyme, a Xyloglycantransferase (Os11g33270.1), designated XTH2, and produced transgenic plants (see Sections 4.4 and 4.5).

moderately increased *XTH2* expression, were selected for further phenotypic characterization (**Figure 3**). Cross and longitudinal sections were prepared to compare the root anatomies of control and *XTH2* overexpressing lines and no apparent differences were found. After 4 weeks of growth either in P deficient or sufficient nutrient solution, several root parameters

**Figure 3.** Phenotypic analysis of *Ubi::XTH2* lines*.* Total *XTH2* expression in a dozen T<sup>2</sup>

of selected T<sup>2</sup>

(LSD) values are indicated per graph.

(A). Cross and longitudinal vibratome sections (B), and root hair density on S-type lateral roots (LRs) of selected T<sup>2</sup>

grown in low or high phosphorus containing nutrient solution (C). Shoot and root dry weight (D), and root number (E)

lines grown in extreme low, low (example image in F) or high phosphorus soil. Least significant difference

lines was tested, and three with very high, high, and

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 139

lines was determined by qRT-PCR

lines

The ectopic overexpression of a dozen T<sup>2</sup>

**Figure 2.** Dependence of root hair production on growth conditions. Determination of root hair density (A, B\*) and length (C, D\*) on main roots, L-type and S-type LRs. Shown are mean values over four genotypes of 4 (A, C) or 5 (B, D) replicates; +/− standard error determined at 7 DAS (A, C) or 35 DAS (B, D). Soil and nutrient solution represent low P conditions while vermiculite and agar did not receive any nutrients. Half-strength Yoshida solution with increasing amount of agar was used to measure Nipponbare root hair length (E). Least significant difference (LSD) values are indicated per graph. Please note \*: data shown in (B, D) are a sub-set of data published in [56].

may not be coupled with increased root hair production, although the assumption could be that smaller root length and weight could be associated with more hairs for a given unit of P uptake. For example, one root efficient genotype had substantially increased root hair length and density upon P deficiency (DJ123). Yet another root efficient genotype (Santhi Sufaid) exhibited shorter and fewer root hairs in P deficient soil, while the inefficient genotype (Sadri Tor Misri) that took up the most P and had the greatest root weight, produced the most root hairs of all tested genotypes [56]. These results lead us to the conclusion that root hairs may respond to P deficiency for some genotypes, and may contribute to root efficiency in others, but their development is not a predictable, universal response to P.

#### **2.8. Transgenic plants for increased P uptake: An example study**

Here we present an example experiment to optimize or increase P uptake by rice roots using a transgenic approach (see Section 4 for experimental setup). Our candidate genes are based on a previous study of transcriptomes of a P deficiency intolerant and a tolerant rice genotype. Several genes putatively associated with root cell wall loosening and root hair extension were found to have higher expression in the roots of the tolerant genotype [53]. We chose one gene to study that encodes a putative cell wall modifying enzyme, a Xyloglycantransferase (Os11g33270.1), designated XTH2, and produced transgenic plants (see Sections 4.4 and 4.5).

The ectopic overexpression of a dozen T<sup>2</sup> lines was tested, and three with very high, high, and moderately increased *XTH2* expression, were selected for further phenotypic characterization (**Figure 3**). Cross and longitudinal sections were prepared to compare the root anatomies of control and *XTH2* overexpressing lines and no apparent differences were found. After 4 weeks of growth either in P deficient or sufficient nutrient solution, several root parameters

may not be coupled with increased root hair production, although the assumption could be that smaller root length and weight could be associated with more hairs for a given unit of P uptake. For example, one root efficient genotype had substantially increased root hair length and density upon P deficiency (DJ123). Yet another root efficient genotype (Santhi Sufaid) exhibited shorter and fewer root hairs in P deficient soil, while the inefficient genotype (Sadri Tor Misri) that took up the most P and had the greatest root weight, produced the most root hairs of all tested genotypes [56]. These results lead us to the conclusion that root hairs may respond to P deficiency for some genotypes, and may contribute to root efficiency in others,

**Figure 2.** Dependence of root hair production on growth conditions. Determination of root hair density (A, B\*) and length (C, D\*) on main roots, L-type and S-type LRs. Shown are mean values over four genotypes of 4 (A, C) or 5 (B, D) replicates; +/− standard error determined at 7 DAS (A, C) or 35 DAS (B, D). Soil and nutrient solution represent low P conditions while vermiculite and agar did not receive any nutrients. Half-strength Yoshida solution with increasing amount of agar was used to measure Nipponbare root hair length (E). Least significant difference (LSD) values are indicated per graph.

Here we present an example experiment to optimize or increase P uptake by rice roots using a transgenic approach (see Section 4 for experimental setup). Our candidate genes are based

but their development is not a predictable, universal response to P.

Please note \*: data shown in (B, D) are a sub-set of data published in [56].

138 Rice Crop - Current Developments

**2.8. Transgenic plants for increased P uptake: An example study**

**Figure 3.** Phenotypic analysis of *Ubi::XTH2* lines*.* Total *XTH2* expression in a dozen T<sup>2</sup> lines was determined by qRT-PCR (A). Cross and longitudinal vibratome sections (B), and root hair density on S-type lateral roots (LRs) of selected T<sup>2</sup> lines grown in low or high phosphorus containing nutrient solution (C). Shoot and root dry weight (D), and root number (E) of selected T<sup>2</sup> lines grown in extreme low, low (example image in F) or high phosphorus soil. Least significant difference (LSD) values are indicated per graph.

were analyzed (root length and number, lateral root densities, root hair density and length on all root types); root hair density on S-type lateral roots is shown (**Figure 3**). None of the analyzed traits displayed a clear, significant difference between control and all of the *XTH2* lines, but a slightly higher number of root hairs was formed on S-type lateral roots grown in the high P nutrient solution. Also evaluated were shoot and root development after 6 weeks of growth in low or high P soil. Some of the overexpressor lines did produce more roots and a slightly increased shoot biomass in low P soil, but no consistent effect of the overexpression could be detected. In contrast, in high P soil, the overexpressor lines produced lower root biomass than the control lines (**Figure 3**).

and condition were grown in a growth cabinet with 16 h light (30°C) and 8 h dark (25°C) for 7 days. Root hair formation was analyzed using a light microscope as described earlier [75].

Half-strength Yoshida nutrient solution [77] was prepared without P. Agar (Sigma Aldrich) was added at concentrations of 1, 1.5, 2, 2.5, 3 and 3.5% to the nutrient solution. Preliminary experiments suggested that beyond 4% agar, lateral root as well as root hair growth becomes impaired (Rose, unpublished). The nutrient solution-agar mixture was poured into clear plastic boxes (200 mm high × 100 mm wide × 25 mm deep, wrapped with aluminum foil) with a 15-mm-diameter hole in the top. A duplicate set of boxes were cut open to determine the

Two germinated seeds per box were sown 5 mm deep in the agar and boxes transferred to a growth cabinet set to 14 h light (27°C) and 10 h dark (22°C). From week two, the boxes were

To amplify the *Xyloglucantransferase2* (Os11g33270) sequence, RNA from genotype DJ123 was isolated, transcribed into cDNA and used as PCR matrix with the oligonucleotides (5′–3′) CAACCCCGGGATGGCGACGACGACGG and GATCGAGCTCTCAGGCGTCGCGGTCG, which introduced the restriction endonuclease recognition sites for *Sma*I and *Sac*I, respectively. According to manufacturer's protocols the PCR product and the target vector pBIH [78] were treated with *Sma*I and *Sac*I (Fermentas, Fast Digest enzymes), the resulting fragments purified (Promega, Wizard PCR clean-up kit), ligated (Roche, Rapid DNA ligation kit), and transformed into DH5α (Promega, library efficient DH5α). The resulting plasmid contains *XTH2* under the control of the *Ubiquitin* promoter. After *pBIHubi::XTH2* sequence confirmation (using the oligonucleotides, 5′–3′: GATGGTGGTGGCAATGTCG and CGGTCGTCGCAGTAGTTGTA) one

Genetic transformation of rice varieties NERICA4 and Nipponbare were conducted by

 **lines in nutrient solution**

cium (0.1 mM) for 7 days followed by an additional 7 days in 1/3 strength Yoshida solution [77]. At 16 DAS roots were harvested and used for RNA extraction, cDNA production, and qRT-PCR analysis as described earlier [56] and for vibratome sectioning. For detection of the *XTH2* transcript, the oligonucleotides (sequences in 5′–3′) TACCACTCCTACTCCGTCCT and TGGAGTAGAGCTTCATCGGC were used. Cross and longitudinal sections of 75 μm were prepared with a vibratome (Microslicer DTK-1000, DSK) by embedding 5 mm root segments in 4% agarose followed by slicing with a frequency of 8 and cutting speed of 5–7. The

seedlings were grown in water supplemented with iron (12 μM) and cal-

plants possessing a single copy of

Phosphorus Efficient Phenotype of Rice http://dx.doi.org/10.5772/intechopen.75642 141

**4.3. Growth in agar-nutrient solution**

resistance of each agar concentration using a penetrometer.

clone (termed *Ubi::XTH2*) was selected for rice transformation.

*Agrobacterium*-methods using immature embryos [79]. T<sup>2</sup>

 **selection**

transgene as homozygote were selected [80] and subjected to further experiments.

**4.4. Plasmid construction for** *pBIHubi::XTH2*

**4.5. Rice transformation and T2**

**4.6. Phenotyping of** *Ubi::XTH2* **T2**

Pre-germinated T<sup>2</sup>

watered with deionized water to weight every 3 d until harvest after 23 d.

Although so *XTH2* had been shown to have higher expression in roots of a P deficiency tolerant genotype in a previous study [53], overexpression in our experiments did not lead to better root and shoot growth or increased root hair production. In another study, tolerance to P deficiency was conferred by overexpression of *PSTOL1*, encoding a protein kinase [76], indicating that transformation can lead to tolerance to P deficiency depending on the specific gene.

### **3. Conclusion**

Overall, it can be concluded that a number of responses to P deficiency exist in rice, yet none of these is a general mechanism found in every rice genotype. Also, often direct evidence for a beneficial effect is lacking for rice. To construct and test a rice genotype optimized for P uptake in P deficient conditions it will be necessary to harness superior traits from many sources and integrate them via marker-assisted breeding or transgenic approaches. For a future sustainable food production, it will also be necessary to overcome the dependence on mining rock phosphate as a major source of P fertilizer. This will include an increase in recycling of biomass and wastewater.

## **4. Experimental details**

### **4.1. Germplasm and germination**

Rice varieties Nerica4, DJ123, Taichung native, and Sadri Tor Misri were used for the phenotyping experiments (Sections 4.2 and 4.3), and Nerica4 and Nipponbare were used for transgenic plant generation (Section 4.5). Dormancy break, sterilization, and pre-germination were performed as described earlier [75].

### **4.2. Growth in the soil, artificial clay, and agar without the addition of nutrients**

Pre-germinated seeds were subjected to different conditions in 50 ml incubation tubes. Plants were grown in low P soil (for details see [53]), vermiculite without nutrient supplementation, and water with the addition of 1% agar (Sigma Aldrich). To exclude light, all tubes were aluminum foil-wrapped and one pre-germinated seed added per tube. Five plants per genotype and condition were grown in a growth cabinet with 16 h light (30°C) and 8 h dark (25°C) for 7 days. Root hair formation was analyzed using a light microscope as described earlier [75].

### **4.3. Growth in agar-nutrient solution**

were analyzed (root length and number, lateral root densities, root hair density and length on all root types); root hair density on S-type lateral roots is shown (**Figure 3**). None of the analyzed traits displayed a clear, significant difference between control and all of the *XTH2* lines, but a slightly higher number of root hairs was formed on S-type lateral roots grown in the high P nutrient solution. Also evaluated were shoot and root development after 6 weeks of growth in low or high P soil. Some of the overexpressor lines did produce more roots and a slightly increased shoot biomass in low P soil, but no consistent effect of the overexpression could be detected. In contrast, in high P soil, the overexpressor lines produced lower root

Although so *XTH2* had been shown to have higher expression in roots of a P deficiency tolerant genotype in a previous study [53], overexpression in our experiments did not lead to better root and shoot growth or increased root hair production. In another study, tolerance to P deficiency was conferred by overexpression of *PSTOL1*, encoding a protein kinase [76], indicating that transformation can lead to tolerance to P deficiency depending on the

Overall, it can be concluded that a number of responses to P deficiency exist in rice, yet none of these is a general mechanism found in every rice genotype. Also, often direct evidence for a beneficial effect is lacking for rice. To construct and test a rice genotype optimized for P uptake in P deficient conditions it will be necessary to harness superior traits from many sources and integrate them via marker-assisted breeding or transgenic approaches. For a future sustainable food production, it will also be necessary to overcome the dependence on mining rock phosphate as a major source of P fertilizer. This will include an increase in recy-

Rice varieties Nerica4, DJ123, Taichung native, and Sadri Tor Misri were used for the phenotyping experiments (Sections 4.2 and 4.3), and Nerica4 and Nipponbare were used for transgenic plant generation (Section 4.5). Dormancy break, sterilization, and pre-germination were

Pre-germinated seeds were subjected to different conditions in 50 ml incubation tubes. Plants were grown in low P soil (for details see [53]), vermiculite without nutrient supplementation, and water with the addition of 1% agar (Sigma Aldrich). To exclude light, all tubes were aluminum foil-wrapped and one pre-germinated seed added per tube. Five plants per genotype

**4.2. Growth in the soil, artificial clay, and agar without the addition of nutrients**

biomass than the control lines (**Figure 3**).

specific gene.

140 Rice Crop - Current Developments

**3. Conclusion**

cling of biomass and wastewater.

**4.1. Germplasm and germination**

performed as described earlier [75].

**4. Experimental details**

Half-strength Yoshida nutrient solution [77] was prepared without P. Agar (Sigma Aldrich) was added at concentrations of 1, 1.5, 2, 2.5, 3 and 3.5% to the nutrient solution. Preliminary experiments suggested that beyond 4% agar, lateral root as well as root hair growth becomes impaired (Rose, unpublished). The nutrient solution-agar mixture was poured into clear plastic boxes (200 mm high × 100 mm wide × 25 mm deep, wrapped with aluminum foil) with a 15-mm-diameter hole in the top. A duplicate set of boxes were cut open to determine the resistance of each agar concentration using a penetrometer.

Two germinated seeds per box were sown 5 mm deep in the agar and boxes transferred to a growth cabinet set to 14 h light (27°C) and 10 h dark (22°C). From week two, the boxes were watered with deionized water to weight every 3 d until harvest after 23 d.

### **4.4. Plasmid construction for** *pBIHubi::XTH2*

To amplify the *Xyloglucantransferase2* (Os11g33270) sequence, RNA from genotype DJ123 was isolated, transcribed into cDNA and used as PCR matrix with the oligonucleotides (5′–3′) CAACCCCGGGATGGCGACGACGACGG and GATCGAGCTCTCAGGCGTCGCGGTCG, which introduced the restriction endonuclease recognition sites for *Sma*I and *Sac*I, respectively. According to manufacturer's protocols the PCR product and the target vector pBIH [78] were treated with *Sma*I and *Sac*I (Fermentas, Fast Digest enzymes), the resulting fragments purified (Promega, Wizard PCR clean-up kit), ligated (Roche, Rapid DNA ligation kit), and transformed into DH5α (Promega, library efficient DH5α). The resulting plasmid contains *XTH2* under the control of the *Ubiquitin* promoter. After *pBIHubi::XTH2* sequence confirmation (using the oligonucleotides, 5′–3′: GATGGTGGTGGCAATGTCG and CGGTCGTCGCAGTAGTTGTA) one clone (termed *Ubi::XTH2*) was selected for rice transformation.

#### **4.5. Rice transformation and T2 selection**

Genetic transformation of rice varieties NERICA4 and Nipponbare were conducted by *Agrobacterium*-methods using immature embryos [79]. T<sup>2</sup> plants possessing a single copy of transgene as homozygote were selected [80] and subjected to further experiments.

#### **4.6. Phenotyping of** *Ubi::XTH2* **T2 lines in nutrient solution**

Pre-germinated T<sup>2</sup> seedlings were grown in water supplemented with iron (12 μM) and calcium (0.1 mM) for 7 days followed by an additional 7 days in 1/3 strength Yoshida solution [77]. At 16 DAS roots were harvested and used for RNA extraction, cDNA production, and qRT-PCR analysis as described earlier [56] and for vibratome sectioning. For detection of the *XTH2* transcript, the oligonucleotides (sequences in 5′–3′) TACCACTCCTACTCCGTCCT and TGGAGTAGAGCTTCATCGGC were used. Cross and longitudinal sections of 75 μm were prepared with a vibratome (Microslicer DTK-1000, DSK) by embedding 5 mm root segments in 4% agarose followed by slicing with a frequency of 8 and cutting speed of 5–7. The sections were then stained for 1 min with 0.05% toluidine blue, briefly washed with water and mounted with 50% glycerol for light microscope (Olympus BX50, Olympus) imaging.

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Selected *Ubi::XTH2* lines with very high, high, and moderate ectopic overexpression as well as control (untransformed Nerica4 and Mock transformed) lines were grown in nutrient solution with low (1 μM) or sufficient (100 μM) P nutrition (NaH<sup>2</sup> PO<sup>4</sup> 2H<sup>2</sup> O). The nutrient solution was changed and increased from 1/3 to 1/2 and finally to full-strength Yoshida solution [77] weekly, while pH was adjusted regularly to 5.7. At 35 DAS root hair parameters were determined as described previously [75].

#### **4.7. Phenotyping of** *Ubi::XTH2* **T2 lines in the soil**

Soil containing three levels of P: extreme low P (80% low P soil mixed with 20% subsoil), low P soil, and P-replete soil (fertilized) were used. The soil was sieved and filled into boxes while softly compacted to simulate field conditions. Fertilizer was supplied in the equivalent amount to 30-0-30 or 30-30-30 kg ha−1 (N, P<sup>2</sup> O5 , K<sup>2</sup> O, respectively).

Four germinated seeds (with similar size) were sown directly in soil and thinned to two plants after 1 week of germination. Water was supplied regularly to field capacity to simulate the wet/dry cycle in upland condition. Plants were grown in a greenhouse with temperature and relative humidity varying between 25 and 32°C and 30–50%. At 40 DAS plants were harvested to evaluate plant height, number of leaves, number of tillers, main root length, root number, shoot and root dry matter.

### **Acknowledgements**

Parts of the presented research were supported by post-doctoral scholarships from the Japanese Society for the Promotion of Science (JSPS) awarded to JK (nèe Nestler) and TR.

### **Author details**

Josefine Kant<sup>1</sup> \*, Takuma Ishizaki<sup>2</sup> , Juan Pariasca-Tanaka<sup>3</sup> , Terry Rose<sup>4</sup> , Matthias Wissuwa<sup>3</sup> and Michelle Watt<sup>1</sup>

\*Address all correspondence to: j.kant@juelich.de

1 Institute for Bio- and Geosciences, Plant Sciences (IBG-2), Forschungszentrum Juelich, Juelich, Germany

2 Tropical Agricultural Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan

3 Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Japan

4 Southern Cross Plant Science, Southern Cross University, Lismore, Australia

### **References**

sections were then stained for 1 min with 0.05% toluidine blue, briefly washed with water and mounted with 50% glycerol for light microscope (Olympus BX50, Olympus) imaging.

Selected *Ubi::XTH2* lines with very high, high, and moderate ectopic overexpression as well as control (untransformed Nerica4 and Mock transformed) lines were grown in nutrient solu-

was changed and increased from 1/3 to 1/2 and finally to full-strength Yoshida solution [77] weekly, while pH was adjusted regularly to 5.7. At 35 DAS root hair parameters were deter-

Soil containing three levels of P: extreme low P (80% low P soil mixed with 20% subsoil), low P soil, and P-replete soil (fertilized) were used. The soil was sieved and filled into boxes while softly compacted to simulate field conditions. Fertilizer was supplied in the equivalent

Four germinated seeds (with similar size) were sown directly in soil and thinned to two plants after 1 week of germination. Water was supplied regularly to field capacity to simulate the wet/dry cycle in upland condition. Plants were grown in a greenhouse with temperature and relative humidity varying between 25 and 32°C and 30–50%. At 40 DAS plants were harvested to evaluate plant height, number of leaves, number of tillers, main root length, root number,

Parts of the presented research were supported by post-doctoral scholarships from the Japanese Society for the Promotion of Science (JSPS) awarded to JK (nèe Nestler) and TR.

, Juan Pariasca-Tanaka<sup>3</sup>

1 Institute for Bio- and Geosciences, Plant Sciences (IBG-2), Forschungszentrum Juelich,

3 Crop, Livestock and Environment Division, Japan International Research Center for

4 Southern Cross Plant Science, Southern Cross University, Lismore, Australia

2 Tropical Agricultural Research Front, Japan International Research Center for Agricultural

O5 , K<sup>2</sup>

 **lines in the soil**

PO<sup>4</sup> 2H<sup>2</sup>

O, respectively).

, Terry Rose<sup>4</sup>

, Matthias Wissuwa<sup>3</sup>

O). The nutrient solution

tion with low (1 μM) or sufficient (100 μM) P nutrition (NaH<sup>2</sup>

mined as described previously [75].

142 Rice Crop - Current Developments

**4.7. Phenotyping of** *Ubi::XTH2* **T2**

shoot and root dry matter.

**Acknowledgements**

**Author details**

and Michelle Watt<sup>1</sup>

Juelich, Germany

Sciences (JIRCAS), Tsukuba, Japan

Josefine Kant<sup>1</sup>

amount to 30-0-30 or 30-30-30 kg ha−1 (N, P<sup>2</sup>

\*, Takuma Ishizaki<sup>2</sup>

\*Address all correspondence to: j.kant@juelich.de

Agricultural Sciences (JIRCAS), Tsukuba, Japan


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**Chapter 9**

**Provisional chapter**

**Heavy Metal and Mineral Element-Induced Abiotic**

**Heavy Metal and Mineral Element-Induced Abiotic** 

The adverse effect of nonliving factors on living organisms is described as abiotic stress. It includes drought, excessive watering, extreme temperatures, salinity, and mineral toxicity. Rice is an important cereal crop, grown under diverse ecological and agricultural conditions. Heavy metal contamination of agricultural land causes abiotic stress to the crop plant as well as has a drastic effect on humans. Increased metal concentration in plants leads to the production of reactive oxygen species which results in cell death and thus affects the crop production in plants. In addition, increased heavy metal concentration in the plant has deleterious effects on its consumers. Like other organisms, plants have also designed ways to deal with such stress situations. In this chapter, abiotic stress due to metal toxicity in rice plant, which includes uptake and sequestration mechanisms, biochemical changes taking place in the plant and variation in their gene expression is elucidated. Based on several molecular and biochemical studies in various reviews and research papers, the role of different transporters like zinc-regulated transporter (ZIP), natural resistance-associated macrophage protein (NRAMP), copper transporter (COPT), yellow stripe like (YSL), heavy metal ATPase (HMA), metal tolerance protein (MTP) and other vascular transporters involved in the above processes in rice plant will be discussed

**Keywords:** transporters, reactive oxygen species, vacuolar sequestration capacity,

Any negative impact of nonliving factors on living organisms in a particular environment can be described as abiotic stress. There are different types of abiotic stress like those due to

DOI: 10.5772/intechopen.76080

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Stress in Rice Plant**

**Abstract**

in this chapter.

**1. Introduction**

antioxidant system, gene regulation

**Stress in Rice Plant**

Anitha Mani and Kavitha Sankaranarayanan

Anitha Mani and Kavitha Sankaranarayanan

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76080


#### **Heavy Metal and Mineral Element-Induced Abiotic Stress in Rice Plant Heavy Metal and Mineral Element-Induced Abiotic Stress in Rice Plant**

DOI: 10.5772/intechopen.76080

Anitha Mani and Kavitha Sankaranarayanan Anitha Mani and Kavitha Sankaranarayanan

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76080

#### **Abstract**

[74] McCormack ML, Dickie IA, Eissenstat DM, et al. Redefining fine roots improves understanding of below-ground contributions to terrestrial biosphere processes. New

[75] Nestler J, Keyes SD, Wissuwa M. Root hair formation in rice (*Oryza sativa* L.) differs between root types and is altered in artificial growth conditions. Journal of Experimental

[76] Gamuyao R, Chin JH, Pariasca-Tanaka J, Pesaresi P, Catausan S, Dalid C, Slamet-Loedin I, Tecson-Mendoza EM, Wissuwa M, Heuer S. The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency. Nature. 2012;**488**:535-541. DOI: 10.1038/

[77] Yoshida S, Forno DA, Cock JH, Gomez KA. Laboratory Manual for Physiological Studies

[78] Becker D. Binary vectors which allow the exchange of plant selectable markers and

[79] Ishizaki T, Kumashiro T. Genetic transformation of NERICA, interspecific hybrid rice between *Oryza glaberrima* and *O. sativa*, mediated by *Agrobacterium tumefaciens*. Plant

[80] Ishizaki T, Kumashiro T. Investigations of copy number of transgene, fertility and expression level of an introduced GUS gene in transgenic NERICA produced by *Agrobacterium*mediated methods. In Vitro Cellular & Developmental Biology- Plant. 2011;**47**:339-347.

of Rice. 2nd ed. The International Rice Research Institute; 1972. pp. 1-70

Phytologist. 2015;**205**:505-518. DOI: 10.1111/nph.13363

Botany. 2016;**67**:3699-3708. DOI: 10.1093/jxb/erw115

reporter genes. Nucleic Acids Research. 1990;**18**:203

DOI: 10.1007/s11627-011-9341-z

Cell Reports. 2008;**27**:319-327. DOI: 10.1007/s00299-007-0465-x

nature11346

148 Rice Crop - Current Developments

The adverse effect of nonliving factors on living organisms is described as abiotic stress. It includes drought, excessive watering, extreme temperatures, salinity, and mineral toxicity. Rice is an important cereal crop, grown under diverse ecological and agricultural conditions. Heavy metal contamination of agricultural land causes abiotic stress to the crop plant as well as has a drastic effect on humans. Increased metal concentration in plants leads to the production of reactive oxygen species which results in cell death and thus affects the crop production in plants. In addition, increased heavy metal concentration in the plant has deleterious effects on its consumers. Like other organisms, plants have also designed ways to deal with such stress situations. In this chapter, abiotic stress due to metal toxicity in rice plant, which includes uptake and sequestration mechanisms, biochemical changes taking place in the plant and variation in their gene expression is elucidated. Based on several molecular and biochemical studies in various reviews and research papers, the role of different transporters like zinc-regulated transporter (ZIP), natural resistance-associated macrophage protein (NRAMP), copper transporter (COPT), yellow stripe like (YSL), heavy metal ATPase (HMA), metal tolerance protein (MTP) and other vascular transporters involved in the above processes in rice plant will be discussed in this chapter.

**Keywords:** transporters, reactive oxygen species, vacuolar sequestration capacity, antioxidant system, gene regulation

### **1. Introduction**

Any negative impact of nonliving factors on living organisms in a particular environment can be described as abiotic stress. There are different types of abiotic stress like those due to

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

drought, excessive watering, that is, water-logging/flooding, extreme temperatures (cold, frost and heat), salinity and mineral (metal and metalloid) toxicity. These have a negative impact on seed germination, plant growth, development, yield, and seed quality of crops. Changes in environmental conditions affect the biological and physiological response of plants. This chapter deals with abiotic stress due to metal toxicity in rice plants, which includes uptake and sequestration mechanisms and biochemical changes taking place in the plant.

with 10 genome types [5, 6] *O. sativa* is composed of two subspecies: *japonica* and *indica*. It is also a model monocotyledon plant as it has a small genome, which has been sequenced (for

Heavy Metal and Mineral Element-Induced Abiotic Stress in Rice Plant

http://dx.doi.org/10.5772/intechopen.76080

151

Plants are sessile and its roots are exposed to various stresses like deficiency and an excess of mineral elements. Heavy metal contamination and accumulation in water, soil, and air due to various reasons has become a serious environmental problem and has greatly affected rice growth and quality. Heavy metals accumulated in rice are toxic to growth, metabolism, and development of plants. Thus, the transfer of heavy metals from soil to plants of commercial agricultural value, such as rice, is of great concern as it may lead to biomagnification via food chain causing several deleterious effects to the consumer. Heavy metals can enter the human body through the food chain, leading to an increased prevalence of chronic diseases, deformities and cancer. The uptake of heavy metal ions by rice plants poses a threat to the consumer's health. Thus, it is important to understand the uptake and sequestration of heavy metal in the

Availability of metals in soil to plants is controlled by three steps: (1) soil conditions (upland or flooded soil and soil solution pH); (2) mineralization (ionization and complex formation)

In addition to genetic variation, metal uptake is sometimes limited by its bioavailability in the soil. The availability of metal ions like Fe, Zn, and Cd for plant uptake varies mainly depending on soil redox potential. Generally, rice is cultivated under flooded conditions. The practice of flooding paddy field increases Fe availability while decreasing Zn and Cd availability while moderate soil drying improves Zn and Cd uptake and but decreases Fe uptake [9]. In both drained and flooded soils, Zn mainly exists as Zn2+, while some of it binds to organic substances, and is immobilized as Zn-sulfide (ZnS) in the anaerobic layer of soil [10]. In drained acidic soils, cadmium exists in its ionized state as Cd2+ ion, whereas Cd in alkaline

soil immobilizes Cd as Cd-sulfide (CdS) and colloidal-bound Cd [12]. Drying of soil converts CdS to Cd2+ and increases its availability to plants. In acidic soil, Fe is ionized as Fe2+/Fe3+, and

Both Cd and Pb are nonessential elements for plants and are toxic even at a very low concentrations, but are readily transported within the plants. The uptake and sequestration of Cd and Pb by crops is of great concern, due to their accumulation in the edible parts of the plant. The uptake of Cd and Pb, their transport, and accumulation by plants are strongly influenced by soil properties and vary with plant species [13, 14]. Cd is freely taken up by the plants and its uptake increases with increased external concentration. The amount of Cd accumulated after its uptake and its translocation to different organs varies with species and with cultivars within the species. The ability for uptake and sequestration of metals in different parts of the plants varies between different plants. There exists a huge difference in metal uptake and translocation between plant species and even between different cultivars of the same species [15]. Roots of most cereal crop plants are present at a depth of 25 cm from the soil surface from

.

and humic acid-bound Cd [11]. Flooding of

rice plant in order to understand the stress caused by it to the plant.

both the cultivars *japonica* [7] and *indica* [8]).

and (3) uptake and efflux transporters.

paddy field soils is present in the forms of CdCO<sup>3</sup>

Fe in aerobic alkaline soils is immobilized as Fe(OH)<sup>3</sup>

where the heavy metals are absorbed by the plant [16].

For their overall growth and development, plants require 14 different mineral elements [1]. These elements are present in the soil and are taken up by the roots, translocated to the shoots, and then distributed to different organs and tissues of the plant depending on their needs [1].

Minerals comprise of both metals and metalloids that are toxic to both plants and animals even at a very low concentration. Some of these heavy metals such as arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) or selenium (Se), do not perform any known physiological function in plants, and are called nonessential metals. Others, such as cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn), are essential elements as they are required for the normal growth and metabolism of plants. Essential elements can lead to poisoning when their concentration rises beyond their optimal levels.

Plants growing on metal-contaminated soils are categorized as resistant varieties, which have adapted to this stressed environment. Heavy metal resistance is attained by the plants either by avoidance/tolerance or by both. Plants, which can prevent the entry of metal ions into their cell cytoplasm, are categorized as avoiders, while the plants which can detoxify the metal ions can cross the plasma membrane or organellar membranes are grouped as tolerant varieties. Plants were thus classified into three groups as: metal excluders, indicators and accumulators/ hyperaccumulators by Baker and Walker [2] based on the approach used by the plants to grow on metal-contaminated soils. Excluder group of plants restricts the uptake and translocation of metal ions to their shoots thus maintaining low levels of heavy metals in the shoots even when grown over a varied range of metal concentrations in soil. Most of the plants belong to the excluder group. Plants, which are classified as metal indicators, can accumulate metals in their aerial shoot system. The level of metal ions in their aerial biomass generally indicates the concentration of metal in the soil. Plants, which are classified as metal accumulators/hyperaccumulators, can take up, transport and accumulate metals generously in their aerial parts to levels higher than the metal concentration found in the soil [2]. The antioxidant defense system in plants helps to accumulate and tolerate the side effects of high levels of internal metal concentrations. Antioxidant system is activated in order to combat the deleterious effects caused by reactive oxygen species (ROS) generated due to stress [3]. Metals can interfere with mineral nutrition and change the concentration and composition of plant nutrients. Metals can also alter the conformation of proteins, including transporters, or other regulatory proteins [4].

Rice (*Oryza sativa L*.) is one of the most important cereal crops in most part of the world and is cultivated in tropical and temperate regions of the world. Rice is the staple food for half of the world's population majorly for many South East Asian countries. It ranks second next to wheat among the most cultivated cereals in the world to feed the ever-growing population. The genus *Oryza* contains 21 wild and 2 cultivated species (*Oryza sativa* and *O. glaberrima*) with 10 genome types [5, 6] *O. sativa* is composed of two subspecies: *japonica* and *indica*. It is also a model monocotyledon plant as it has a small genome, which has been sequenced (for both the cultivars *japonica* [7] and *indica* [8]).

drought, excessive watering, that is, water-logging/flooding, extreme temperatures (cold, frost and heat), salinity and mineral (metal and metalloid) toxicity. These have a negative impact on seed germination, plant growth, development, yield, and seed quality of crops. Changes in environmental conditions affect the biological and physiological response of plants. This chapter deals with abiotic stress due to metal toxicity in rice plants, which includes uptake

For their overall growth and development, plants require 14 different mineral elements [1]. These elements are present in the soil and are taken up by the roots, translocated to the shoots, and then distributed to different organs and tissues of the plant depending on their needs [1]. Minerals comprise of both metals and metalloids that are toxic to both plants and animals even at a very low concentration. Some of these heavy metals such as arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) or selenium (Se), do not perform any known physiological function in plants, and are called nonessential metals. Others, such as cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn), are essential elements as they are required for the normal growth and metabolism of plants. Essential elements can lead to poisoning when their concentration rises beyond their optimal levels.

Plants growing on metal-contaminated soils are categorized as resistant varieties, which have adapted to this stressed environment. Heavy metal resistance is attained by the plants either by avoidance/tolerance or by both. Plants, which can prevent the entry of metal ions into their cell cytoplasm, are categorized as avoiders, while the plants which can detoxify the metal ions can cross the plasma membrane or organellar membranes are grouped as tolerant varieties. Plants were thus classified into three groups as: metal excluders, indicators and accumulators/ hyperaccumulators by Baker and Walker [2] based on the approach used by the plants to grow on metal-contaminated soils. Excluder group of plants restricts the uptake and translocation of metal ions to their shoots thus maintaining low levels of heavy metals in the shoots even when grown over a varied range of metal concentrations in soil. Most of the plants belong to the excluder group. Plants, which are classified as metal indicators, can accumulate metals in their aerial shoot system. The level of metal ions in their aerial biomass generally indicates the concentration of metal in the soil. Plants, which are classified as metal accumulators/hyperaccumulators, can take up, transport and accumulate metals generously in their aerial parts to levels higher than the metal concentration found in the soil [2]. The antioxidant defense system in plants helps to accumulate and tolerate the side effects of high levels of internal metal concentrations. Antioxidant system is activated in order to combat the deleterious effects caused by reactive oxygen species (ROS) generated due to stress [3]. Metals can interfere with mineral nutrition and change the concentration and composition of plant nutrients. Metals can also alter the conformation of proteins, including transporters, or other regulatory proteins [4].

Rice (*Oryza sativa L*.) is one of the most important cereal crops in most part of the world and is cultivated in tropical and temperate regions of the world. Rice is the staple food for half of the world's population majorly for many South East Asian countries. It ranks second next to wheat among the most cultivated cereals in the world to feed the ever-growing population. The genus *Oryza* contains 21 wild and 2 cultivated species (*Oryza sativa* and *O. glaberrima*)

and sequestration mechanisms and biochemical changes taking place in the plant.

150 Rice Crop - Current Developments

Plants are sessile and its roots are exposed to various stresses like deficiency and an excess of mineral elements. Heavy metal contamination and accumulation in water, soil, and air due to various reasons has become a serious environmental problem and has greatly affected rice growth and quality. Heavy metals accumulated in rice are toxic to growth, metabolism, and development of plants. Thus, the transfer of heavy metals from soil to plants of commercial agricultural value, such as rice, is of great concern as it may lead to biomagnification via food chain causing several deleterious effects to the consumer. Heavy metals can enter the human body through the food chain, leading to an increased prevalence of chronic diseases, deformities and cancer. The uptake of heavy metal ions by rice plants poses a threat to the consumer's health. Thus, it is important to understand the uptake and sequestration of heavy metal in the rice plant in order to understand the stress caused by it to the plant.

Availability of metals in soil to plants is controlled by three steps: (1) soil conditions (upland or flooded soil and soil solution pH); (2) mineralization (ionization and complex formation) and (3) uptake and efflux transporters.

In addition to genetic variation, metal uptake is sometimes limited by its bioavailability in the soil. The availability of metal ions like Fe, Zn, and Cd for plant uptake varies mainly depending on soil redox potential. Generally, rice is cultivated under flooded conditions. The practice of flooding paddy field increases Fe availability while decreasing Zn and Cd availability while moderate soil drying improves Zn and Cd uptake and but decreases Fe uptake [9]. In both drained and flooded soils, Zn mainly exists as Zn2+, while some of it binds to organic substances, and is immobilized as Zn-sulfide (ZnS) in the anaerobic layer of soil [10]. In drained acidic soils, cadmium exists in its ionized state as Cd2+ ion, whereas Cd in alkaline paddy field soils is present in the forms of CdCO<sup>3</sup> and humic acid-bound Cd [11]. Flooding of soil immobilizes Cd as Cd-sulfide (CdS) and colloidal-bound Cd [12]. Drying of soil converts CdS to Cd2+ and increases its availability to plants. In acidic soil, Fe is ionized as Fe2+/Fe3+, and Fe in aerobic alkaline soils is immobilized as Fe(OH)<sup>3</sup> .

Both Cd and Pb are nonessential elements for plants and are toxic even at a very low concentrations, but are readily transported within the plants. The uptake and sequestration of Cd and Pb by crops is of great concern, due to their accumulation in the edible parts of the plant. The uptake of Cd and Pb, their transport, and accumulation by plants are strongly influenced by soil properties and vary with plant species [13, 14]. Cd is freely taken up by the plants and its uptake increases with increased external concentration. The amount of Cd accumulated after its uptake and its translocation to different organs varies with species and with cultivars within the species. The ability for uptake and sequestration of metals in different parts of the plants varies between different plants. There exists a huge difference in metal uptake and translocation between plant species and even between different cultivars of the same species [15]. Roots of most cereal crop plants are present at a depth of 25 cm from the soil surface from where the heavy metals are absorbed by the plant [16].

Roots are the primary target for the accumulation of metals, and metals like Cd and Pb are accumulated mainly in roots [14]. Plants have developed several strategies to decrease metal ion toxicity, one of which is the cellular transport system. There are several groups of metal transporter proteins identified in plants.

**2. ZIP family**

oxidized nor reduced [20].

OsIRT1 can only uptake Fe but not Cu.

ZIP family of transporters is named after the first proteins identified ZRT, iron-regulated transporter (IRT) like protein [20]. They are present in bacteria, fungus, plants, and humans. ZIP proteins can transport divalent cations like Fe2+, Zn+2, Mn+2 and Cd+2 [22]. Zn as such cannot be transported across the cell membrane, it requires specific zinc transporters for its transport into the cytoplasm. Generally, ZIP family of transporter proteins transport cations into the cell cytoplasm and play a role in cellular metal ion homeostasis. ZIP proteins generally localized at the plasma membrane and are involved in either moving metals or to remobilize them from intracellular compartments into the cytoplasm [21]. In rice, 14 putative ZIP family of transporter proteins have been identified. It is divided into two subfamilies based on their amino acid sequence similarities. Zinc is taken up by these transporters as a divalent cation and plays role in cellular activities in the form of tetrahedral complexes as it is neither

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IRT1 was the first member of ZIP family to be identified. It is a Fe(II) transporter and is involved in the uptake of iron from the soil. Studies have proved that in addition to Fe, it can transport Mn and Zn. IRT1 mediates Cd accumulation in iron-deficient plants. Depending on the plant species, there are two mechanisms for the uptake of Fe by IRT transporter from soil. The first strategy is used by all dicot and non-gramineous monocot plants. In this process, iron uptake by IRT1 transporter happens after ferric Fe is reduced to ferrous Fe by ferric chelate reductase (FRO2) on the plasma membrane [23, 24]. The second strategy is used by graminaceous plants. It is characterized by the secretion of mugineic acid (MA) and forms mugineic acid-ferric complex followed by its uptake by IRT1 transporter [25]. Though the rice root does not have Fe reductase enzyme activity [26], ferrous Fe is abundantly present in paddy fields due to reductive status in soil. Both OsIRT1 and OsIRT2 are involved in Fe uptake in rice plant. They are expressed mostly in roots and induced by Fe deficiency [27].

OsZIP1 and OsZIP3 are involved in the transport of Zn but not Fe/Mn. OsZIP4, OsZIP5, and OsZIP8 are functional rice zinc transporters expressed on the plasma membrane [28]. OsZIP1 and OsZIP3 are upregulated in roots and shoots upon Zn deprivation. Zn deficiency upregulates OsZIP2 only in roots [27], whereas Zn deficiency upregulates OsZIP5 and OsZIP7 in rice shoots. In the mature rice plant, OsZIP7a and OsZIP8 are expressed constitutively and weakly in roots, culms, leaves and flowering spikes [29]. Expression of OsZIP9 is also induced by Zn deficiency and it complements the absence of OsZIP5. OsZIP4 is expressed in Zn deficient shoots and roots especially in phloem cells and meristems. Zn deficiency induces the expression of OsZIP8 in both roots and shoots. Expression of OsZIP5 is relatively higher in roots than in shoots and is specific to zinc [27]. OsZIP4 is localized to the plasma membrane. It is a Zn-regulated Zn transporter involved in the transport of Zn. It controls the supply of Zn to developing young leaves and is involved in remobilization of Zn from old to young leaves [28]. In rice Zn transporters OsZIP1, OsZIP3, OsZIP4, and OsZIP5 are induced by Zn deficiency [26, 27, 30]. OsZIP1, OsZIP3, and OsZIP4 are expressed in the vascular bundles in both rice

Rice has a distinct root system. Anatomically, rice root is characterized by the two casparian strips present on the exodermis and endodermis. The apoplastic flow of water and movement of mineral elements is prevented in between the cell layers by casparian strips, which act as a barrier [17]. Also, the mature rice root has a well developed aerenchyma, which has well developed vascular bundles for the upstream translocation of metals and other mineral elements from the roots to the shoots. Apoptosis of cortical cells creates the aerenchyma, which has an apoplastic space and no symplastic connections. The apoplastic space is connected by remaining spoke-like connections between the exodermis and the endodermis [18]. Thus to reach the stele, the mineral nutrients in rice roots have to be transported via the symplast of both the exodermis and the endodermis and also through the apoplast of the aerenchyma. The root tips, which lack aerenchyma and casparian strips, can also accumulate mineral elements but in small percentage due to their highly undeveloped vascular system [19]. For efficient translocation of mineral nutrition from the soil solution to the stele in rice, roots require cooperation between both the influx and efflux transporters. Transporters involved in this uptake process have been identified for some mineral elements, but most of them remain unknown. Thus plants take up heavy metals through their roots majorly via various transporters from the soil and accumulate them in their aerial parts.

Metal ions are transported from the soil into the root and then distributed throughout the plant, after crossing both cellular and organellar membranes [20]. All the characterized plant transporter proteins, which are responsible for metal homeostasis, are membrane proteins that mediate heavy metal movement through membranes. Transporters in plants either act at the plasma membrane to move metals into the cytoplasm or at the intracellular organellar membrane to re-circulate metals from intracellular compartment into the cytoplasm. They are classified into different families such as natural resistance-associated macrophage protein (NRAMP), zinc-regulated transporter (ZIP), yellow stripe 1-like family (YSL), and Ctr/copper transporter (COPT) family of high-affinity Cu uptake proteins. Plant transporters have been identified in *Arabidopsis thaliana* to be involved in metal efflux from the cytoplasm either across the plasma membrane or into the organelles. They are classified into two families namely P1B-ATPase family/CPX- ATPase and cation diffusion facilitator (CDF) family/metal tolerance protein1 (MTP1) [21]. Similar metal transporter families are also present in rice.

Various such transporters have been identified in different plant species, which are involved in the metal uptake and sequestration process. This chapter focuses on rice plant transporters that are involved in the uptake of mineral elements in roots and its sequestration into the vacuoles and also the biochemical changes taking place in the rice plant during abiotic stress due to heavy metals.

## **2. ZIP family**

Roots are the primary target for the accumulation of metals, and metals like Cd and Pb are accumulated mainly in roots [14]. Plants have developed several strategies to decrease metal ion toxicity, one of which is the cellular transport system. There are several groups of metal

Rice has a distinct root system. Anatomically, rice root is characterized by the two casparian strips present on the exodermis and endodermis. The apoplastic flow of water and movement of mineral elements is prevented in between the cell layers by casparian strips, which act as a barrier [17]. Also, the mature rice root has a well developed aerenchyma, which has well developed vascular bundles for the upstream translocation of metals and other mineral elements from the roots to the shoots. Apoptosis of cortical cells creates the aerenchyma, which has an apoplastic space and no symplastic connections. The apoplastic space is connected by remaining spoke-like connections between the exodermis and the endodermis [18]. Thus to reach the stele, the mineral nutrients in rice roots have to be transported via the symplast of both the exodermis and the endodermis and also through the apoplast of the aerenchyma. The root tips, which lack aerenchyma and casparian strips, can also accumulate mineral elements but in small percentage due to their highly undeveloped vascular system [19]. For efficient translocation of mineral nutrition from the soil solution to the stele in rice, roots require cooperation between both the influx and efflux transporters. Transporters involved in this uptake process have been identified for some mineral elements, but most of them remain unknown. Thus plants take up heavy metals through their roots majorly via various transporters from the soil and accumulate them in

Metal ions are transported from the soil into the root and then distributed throughout the plant, after crossing both cellular and organellar membranes [20]. All the characterized plant transporter proteins, which are responsible for metal homeostasis, are membrane proteins that mediate heavy metal movement through membranes. Transporters in plants either act at the plasma membrane to move metals into the cytoplasm or at the intracellular organellar membrane to re-circulate metals from intracellular compartment into the cytoplasm. They are classified into different families such as natural resistance-associated macrophage protein (NRAMP), zinc-regulated transporter (ZIP), yellow stripe 1-like family (YSL), and Ctr/copper transporter (COPT) family of high-affinity Cu uptake proteins. Plant transporters have been identified in *Arabidopsis thaliana* to be involved in metal efflux from the cytoplasm either across the plasma membrane or into the organelles. They are classified into two families namely P1B-ATPase family/CPX- ATPase and cation diffusion facilitator (CDF) family/metal tolerance protein1 (MTP1) [21]. Similar metal transporter families are

Various such transporters have been identified in different plant species, which are involved in the metal uptake and sequestration process. This chapter focuses on rice plant transporters that are involved in the uptake of mineral elements in roots and its sequestration into the vacuoles and also the biochemical changes taking place in the rice plant during abiotic stress

transporter proteins identified in plants.

152 Rice Crop - Current Developments

their aerial parts.

also present in rice.

due to heavy metals.

ZIP family of transporters is named after the first proteins identified ZRT, iron-regulated transporter (IRT) like protein [20]. They are present in bacteria, fungus, plants, and humans. ZIP proteins can transport divalent cations like Fe2+, Zn+2, Mn+2 and Cd+2 [22]. Zn as such cannot be transported across the cell membrane, it requires specific zinc transporters for its transport into the cytoplasm. Generally, ZIP family of transporter proteins transport cations into the cell cytoplasm and play a role in cellular metal ion homeostasis. ZIP proteins generally localized at the plasma membrane and are involved in either moving metals or to remobilize them from intracellular compartments into the cytoplasm [21]. In rice, 14 putative ZIP family of transporter proteins have been identified. It is divided into two subfamilies based on their amino acid sequence similarities. Zinc is taken up by these transporters as a divalent cation and plays role in cellular activities in the form of tetrahedral complexes as it is neither oxidized nor reduced [20].

IRT1 was the first member of ZIP family to be identified. It is a Fe(II) transporter and is involved in the uptake of iron from the soil. Studies have proved that in addition to Fe, it can transport Mn and Zn. IRT1 mediates Cd accumulation in iron-deficient plants. Depending on the plant species, there are two mechanisms for the uptake of Fe by IRT transporter from soil. The first strategy is used by all dicot and non-gramineous monocot plants. In this process, iron uptake by IRT1 transporter happens after ferric Fe is reduced to ferrous Fe by ferric chelate reductase (FRO2) on the plasma membrane [23, 24]. The second strategy is used by graminaceous plants. It is characterized by the secretion of mugineic acid (MA) and forms mugineic acid-ferric complex followed by its uptake by IRT1 transporter [25]. Though the rice root does not have Fe reductase enzyme activity [26], ferrous Fe is abundantly present in paddy fields due to reductive status in soil. Both OsIRT1 and OsIRT2 are involved in Fe uptake in rice plant. They are expressed mostly in roots and induced by Fe deficiency [27]. OsIRT1 can only uptake Fe but not Cu.

OsZIP1 and OsZIP3 are involved in the transport of Zn but not Fe/Mn. OsZIP4, OsZIP5, and OsZIP8 are functional rice zinc transporters expressed on the plasma membrane [28]. OsZIP1 and OsZIP3 are upregulated in roots and shoots upon Zn deprivation. Zn deficiency upregulates OsZIP2 only in roots [27], whereas Zn deficiency upregulates OsZIP5 and OsZIP7 in rice shoots. In the mature rice plant, OsZIP7a and OsZIP8 are expressed constitutively and weakly in roots, culms, leaves and flowering spikes [29]. Expression of OsZIP9 is also induced by Zn deficiency and it complements the absence of OsZIP5. OsZIP4 is expressed in Zn deficient shoots and roots especially in phloem cells and meristems. Zn deficiency induces the expression of OsZIP8 in both roots and shoots. Expression of OsZIP5 is relatively higher in roots than in shoots and is specific to zinc [27]. OsZIP4 is localized to the plasma membrane. It is a Zn-regulated Zn transporter involved in the transport of Zn. It controls the supply of Zn to developing young leaves and is involved in remobilization of Zn from old to young leaves [28].

In rice Zn transporters OsZIP1, OsZIP3, OsZIP4, and OsZIP5 are induced by Zn deficiency [26, 27, 30]. OsZIP1, OsZIP3, and OsZIP4 are expressed in the vascular bundles in both rice shoot and root and only in the epidermal cells in rice root [26, 30]. OsIRT1, OsZIP5, and OsZIP4 are upregulated in Zn-deficient roots and OsZIP4, OsZIP5 and OsZIP7 are upregulated in Zn deficient shoots [28].

that YS1 is the Fe(III)-PS transporter. The YS1 protein is upregulated by Fe deficiencies in roots and shoots, and functions as a proton-coupled symporter to transport Fe(III)-PS [40].

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Similar to maize YS1 gene sequence, 18 such genes have been putatively identified in rice and named as yellow stripe-like (YSL) genes [41]. Though YSL is part of the larger oligopeptide transporter (OPT) family, which is also present in fungi [42], but YSL transporter family members can only be found in plants [43]. YSL family of transporters cannot transport free metals as such but can transport only metals along with nicotianamine (NA) or its derivatives [42]. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe [41]. NA is a non-proteogenic amino acid, synthesized from *S*-adenosyl-methionine by the enzyme NA synthase (NAS) [44]. It is a structural analog of 2′-deoxymugineic acid (DMA), which is formed by NA aminotransferase (NAAT) and DMA synthase (DMAS) [45]. In monocot plants, YSL transporters are associated with metal uptake from the soil, while in both monocots and dicots plants they are involved in long-distance

In rice, OsYSL2 transports Fe(II)-NA complex and Mn(II)-NA complex and is mainly expressed in the phloem cells of the vascular bundles, especially in the companion cells of Fe-deficient leaves [47]. It also mediates long-distance transport of manganese, especially to the grain [47, 48]. The expression of OsYSL2 is induced by Fe deficiency in the leaves but not in the roots. OsYSL2 is important for Fe translocation at the early stage of growth [35] also important for long-distance transport during grain filling, particularly for Fe translocation to

OsYSL15 is localized to the plasma membrane and mediates the uptake of MA-Fe complex [49]. Fe deficiency induces *OsYSL15* in roots but is unaffected by Zn, Mn or Cu deficiency [49]. Under Fe deficiency, *OsYSL15* is expressed in the epidermis, endodermis, cortex, and vascular bundles of the roots and leaves [49]. The OsYSL15 transporter contribution in paddy soil is little as the secreted MA diffuses out of the rhizosphere. In neutral and alkaline soils, Fe3+ binds to mineral and organic substances strongly such that ions are hardly available to plants and the iron is solubilized by forming a complex with a phytosiderophore, DMA [50], which is excreted by rice roots by OsTOM1 (rice DMA effluxer) [51]. Fe(III)-DMA complexes in the soil solution is then taken up by OsYSL15 [49]. Under flooded conditions, rice plants

OsYSL16 is a Cu-NA transporter which delivers Cu to the developing tissues and seeds through phloem transport. During vegetative growth, OsYSL16 is expressed in the roots, leaves, and unelongated nodes and during the reproductive stage, it is highly expressed in

Among the 18 OsYSL genes in rice, OsYSL15 transports Fe(III)-DMA and Fe(II)-NA [49]. OsYSL15 expression is strongly induced in the roots and shoots by Fe deficiency. OsYSL2, induced by Fe deficiency, is localized to the plasma membrane and transports Fe(II)-NA and Mn(II)-NA, but not Fe(III)-DMA [47]. OsYSL18 is a transporter of Fe(III)-DMA but not of

metal translocation [46].

the endosperm [48].

the upper nodes [52].

may absorb both Fe(III)-DMA and Fe2+ [26].

OsZIP4 is expressed in the vascular bundles in rice root and shoot and also root and shoot meristem during Zn deficiency. Basically, OsZIP1 and OsZIP3 are involved in Zn uptake in roots and Zn homeostasis in shoots [30]. OsZIP4 transports Zn, specifically into vascular bundles and meristem [28]. OsZIP6 is transcriptionally activated in the shoot and root tissues in response to the deficiency in Fe2+, Zn2+, and Mn2+. Ion transport by OsZIP6 is pH dependent and enhanced transport is observed at acidic pH. OsZIP6 is involved in iron uptake in roots and transport of Fe, Zn, and Mn in the shoots of rice [31]. OsZIP1 and OsZIP3 transport only Zn2+ and not Fe2+ or Mn2+ [30], OsZIP4 transports Zn2+ and not Fe2+ [28] and OsIRT1 similarly transports Fe2+ and not Cu2+ [32]. OsZIP6 transports at least three transition metal ions, namely, Fe2+, Co2+, and Cd2+. Substrate affinity for OsZIP6 is in the order Co2+ > Cd2+ > Fe2+. ZIP transporters show both high and low affinity transport [31]. In rice, zinc transporter OsZIP1 exhibits enhanced transport at pH 4.7, in contrast to OsZIP3, where maximum activity is observed at pH 6.0 [30]. OsZIP6 is expressed in both roots and shoots at maximum tillering and mid-grain filling stages [33]. OsZIP8 is a plasma membrane zinc transporter in rice that functions in Zn uptake and distribution. During Zn deficiency, it is highly upregulated in shoots and roots [27].

In rice seed, Zn is present in the embryo, endosperm, and the aleurone layer. The Zn content is specifically high in the embryo [34]. During germination, Zn content in the endosperm decreases, while Zn content increases in the radicle and leaf primordium. Zn content increases in the scutellum and its vascular bundle after 24 hrs of sowing [34]. During germination, expression of ZIP family of transporter members decreases [35]. In the embryo meristematic tissues, Zn accumulation is limited. For such a partial localization of Zn, a decrease in OsZIP family transcripts is required. Different rice genotypes vary mainly in their efficiency to utilize Zn and in their grain Zn contents [36]. Thus, the wrong expression of ZIPs could lead to the irregular distribution of the essential micronutrients in the plant.

### **3. Yellow stripe-like (YSL) proteins**

Though iron is abundant in the earth's crust, it is mostly unavailable to plants because, at a neutral pH, it forms insoluble ferric oxide complexes in aerobic environment [37]. Graminaceous plants use the chelation-based strategy II. In response to Fe deficiency, these plant cells release phytosiderophores (PSs), which belong to the mugineic acid (MA) family and are derived from the precursor nicotianamine (NA). These molecules bind to Fe(III) and specific plasma membrane transporter proteins to import the Fe(III)-PS complexes [38]. The molecular mechanism controlling Fe(III)-uptake was elucidated by cloning the membrane transporter from the maize yellow stripe 1 (ys1) mutant, which showed characteristic interveinal chlorosis or yellow patches [39]. Because that mutant is deficient in Fe(III)-PS uptake, it has been suggested that YS1 is the Fe(III)-PS transporter. The YS1 protein is upregulated by Fe deficiencies in roots and shoots, and functions as a proton-coupled symporter to transport Fe(III)-PS [40].

shoot and root and only in the epidermal cells in rice root [26, 30]. OsIRT1, OsZIP5, and OsZIP4 are upregulated in Zn-deficient roots and OsZIP4, OsZIP5 and OsZIP7 are upregu-

OsZIP4 is expressed in the vascular bundles in rice root and shoot and also root and shoot meristem during Zn deficiency. Basically, OsZIP1 and OsZIP3 are involved in Zn uptake in roots and Zn homeostasis in shoots [30]. OsZIP4 transports Zn, specifically into vascular bundles and meristem [28]. OsZIP6 is transcriptionally activated in the shoot and root tissues in response to the deficiency in Fe2+, Zn2+, and Mn2+. Ion transport by OsZIP6 is pH dependent and enhanced transport is observed at acidic pH. OsZIP6 is involved in iron uptake in roots and transport of Fe, Zn, and Mn in the shoots of rice [31]. OsZIP1 and OsZIP3 transport only Zn2+ and not Fe2+ or Mn2+ [30], OsZIP4 transports Zn2+ and not Fe2+ [28] and OsIRT1 similarly transports Fe2+ and not Cu2+ [32]. OsZIP6 transports at least three transition metal ions, namely, Fe2+, Co2+, and Cd2+. Substrate affinity for OsZIP6 is in the order Co2+ > Cd2+ > Fe2+. ZIP transporters show both high and low affinity transport [31]. In rice, zinc transporter OsZIP1 exhibits enhanced transport at pH 4.7, in contrast to OsZIP3, where maximum activity is observed at pH 6.0 [30]. OsZIP6 is expressed in both roots and shoots at maximum tillering and mid-grain filling stages [33]. OsZIP8 is a plasma membrane zinc transporter in rice that functions in Zn uptake and distribution. During Zn deficiency, it is highly upregulated in

In rice seed, Zn is present in the embryo, endosperm, and the aleurone layer. The Zn content is specifically high in the embryo [34]. During germination, Zn content in the endosperm decreases, while Zn content increases in the radicle and leaf primordium. Zn content increases in the scutellum and its vascular bundle after 24 hrs of sowing [34]. During germination, expression of ZIP family of transporter members decreases [35]. In the embryo meristematic tissues, Zn accumulation is limited. For such a partial localization of Zn, a decrease in OsZIP family transcripts is required. Different rice genotypes vary mainly in their efficiency to utilize Zn and in their grain Zn contents [36]. Thus, the wrong expression of ZIPs could lead to

Though iron is abundant in the earth's crust, it is mostly unavailable to plants because, at a neutral pH, it forms insoluble ferric oxide complexes in aerobic environment [37]. Graminaceous plants use the chelation-based strategy II. In response to Fe deficiency, these plant cells release phytosiderophores (PSs), which belong to the mugineic acid (MA) family and are derived from the precursor nicotianamine (NA). These molecules bind to Fe(III) and specific plasma membrane transporter proteins to import the Fe(III)-PS complexes [38]. The molecular mechanism controlling Fe(III)-uptake was elucidated by cloning the membrane transporter from the maize yellow stripe 1 (ys1) mutant, which showed characteristic interveinal chlorosis or yellow patches [39]. Because that mutant is deficient in Fe(III)-PS uptake, it has been suggested

the irregular distribution of the essential micronutrients in the plant.

**3. Yellow stripe-like (YSL) proteins**

lated in Zn deficient shoots [28].

154 Rice Crop - Current Developments

shoots and roots [27].

Similar to maize YS1 gene sequence, 18 such genes have been putatively identified in rice and named as yellow stripe-like (YSL) genes [41]. Though YSL is part of the larger oligopeptide transporter (OPT) family, which is also present in fungi [42], but YSL transporter family members can only be found in plants [43]. YSL family of transporters cannot transport free metals as such but can transport only metals along with nicotianamine (NA) or its derivatives [42]. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe [41]. NA is a non-proteogenic amino acid, synthesized from *S*-adenosyl-methionine by the enzyme NA synthase (NAS) [44]. It is a structural analog of 2′-deoxymugineic acid (DMA), which is formed by NA aminotransferase (NAAT) and DMA synthase (DMAS) [45]. In monocot plants, YSL transporters are associated with metal uptake from the soil, while in both monocots and dicots plants they are involved in long-distance metal translocation [46].

In rice, OsYSL2 transports Fe(II)-NA complex and Mn(II)-NA complex and is mainly expressed in the phloem cells of the vascular bundles, especially in the companion cells of Fe-deficient leaves [47]. It also mediates long-distance transport of manganese, especially to the grain [47, 48]. The expression of OsYSL2 is induced by Fe deficiency in the leaves but not in the roots. OsYSL2 is important for Fe translocation at the early stage of growth [35] also important for long-distance transport during grain filling, particularly for Fe translocation to the endosperm [48].

OsYSL15 is localized to the plasma membrane and mediates the uptake of MA-Fe complex [49]. Fe deficiency induces *OsYSL15* in roots but is unaffected by Zn, Mn or Cu deficiency [49]. Under Fe deficiency, *OsYSL15* is expressed in the epidermis, endodermis, cortex, and vascular bundles of the roots and leaves [49]. The OsYSL15 transporter contribution in paddy soil is little as the secreted MA diffuses out of the rhizosphere. In neutral and alkaline soils, Fe3+ binds to mineral and organic substances strongly such that ions are hardly available to plants and the iron is solubilized by forming a complex with a phytosiderophore, DMA [50], which is excreted by rice roots by OsTOM1 (rice DMA effluxer) [51]. Fe(III)-DMA complexes in the soil solution is then taken up by OsYSL15 [49]. Under flooded conditions, rice plants may absorb both Fe(III)-DMA and Fe2+ [26].

OsYSL16 is a Cu-NA transporter which delivers Cu to the developing tissues and seeds through phloem transport. During vegetative growth, OsYSL16 is expressed in the roots, leaves, and unelongated nodes and during the reproductive stage, it is highly expressed in the upper nodes [52].

Among the 18 OsYSL genes in rice, OsYSL15 transports Fe(III)-DMA and Fe(II)-NA [49]. OsYSL15 expression is strongly induced in the roots and shoots by Fe deficiency. OsYSL2, induced by Fe deficiency, is localized to the plasma membrane and transports Fe(II)-NA and Mn(II)-NA, but not Fe(III)-DMA [47]. OsYSL18 is a transporter of Fe(III)-DMA but not of Fe(II)-NA [53]. Its expression in flowers and the phloem of lamina joints indicates that it is involved in translocating Fe to the reproductive organs and phloem joints.

into cytosol or vacuoles or are localized on the lysosomal membrane and are involved in the

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The COPT family of transporters in rice consists of seven members: COPT1–COPT7. In rice plant, COPT proteins are specifically involved in Cu transport. It can transport only Cu(I) but not other bivalent ions such as Mn, Zn or Fe. In rice plant, COPT1–COPT7 are plasma membrane-localized proteins. As these transporters can form symmetrical homotrimer or heterotrimer structure with a diameter that is only suitable for Cu(I) transport and not other divalent ions [59] or heterocomplex with themselves or each other [60] or heterocomplex with

COPT1 and COPT5 can exist as homodimers or a heterodimer. Only COPT1 and COPT5 bind to rice XA13 protein, a protein which is susceptible to pathogenic bacterium *Xanthomonas oryzae pv. Oryzae* (Xoo) [61] and mediate Cu transport in rice plant. In rice plant, all the COPTs except COPT1 and COPT5, function independently or together and mediate Cu transport in different tissues. COPT6 acts as a cofactor and aids the efficient localization of COPT2, COPT3 or COPT4 to the plasma membrane for mediating Cu transport. COPT1 and COPT5 show similar tissue and also develop-specific expression patterns. When compared to the sheath, stem, and panicle expression levels of these two genes, they have a higher level of expression in root and leaf tissues. COPT4 has higher expression level in root in comparison to other tissues. COPT1, COPT4, and COPT5 have higher expression level in young leaves than in old leaves, particularly COPT1 and COPT4. COPT2, COPT3, and COPT7 show higher expression levels in old leaves when compared with young leaves. COPT6 is not expressed in root and is highly expressed in leaf than in other tissues. COPT6 is constitutively expressed in differentaged leaves but has a low level of expression in shoot tissue at seedling stage. The expression of rice COPT1 and COPT5 are induced by Cu deficiency and suppressed by excess Cu in both shoot and root tissues. COPT1 and COPT5 together mediate Cu transport in rice plant [61]. The expression of rice COPT2, COPT3, COPT4, COPT6, and COPT7, is also affected by the variation in Cu levels. In both root and shoot tissues, COPT7 shows a similar response as COPT1 and COPT5 to Cu deficiency and overdose. In different-aged leaves of a mature plant, COPT6 is constitutively expressed while in the seedling stage, the shoot tissue has low expression levels. In shoot, COPT6 is induced under Cu deficiency state and suppressed in Cu overdose but no COPT6 expression is detected in root either with or without Cu deficiency. Cu overdose suppresses the expression of COPT2, COPT3, and COPT4 in both root and shoot tissues but their expression is not influenced by Cu deficiency. Expression of COPTs is also influenced by other bivalent cations. Mn deficiency induces expression of COPT1 in root and COPT3 and COPT7 in shoot and slightly suppresses the expression of COPT2 and COPT4 in root. The expression of COPT1, COPT5, and COPT7 is induced by Zn deficiency and COPT4 expression is slightly suppressed in the root. Zn deficiency also induces the expression of COPT5, COPT6, and COPT7 in the shoot. In root, Fe deficiency moderately induces COPT1 and suppresses COPT2 and COPT5 while in the shoot, it induces COPT2, COPT5, COPT6, and COPT7. In root, no COPT6 expression has been found either in the presence or absence of Mn, Zn, or Fe deficiency. COPT family of transporter proteins functions uniquely in different tissues, during various developmental stages, and in different environmental conditions. In rice plant, COPT2, COPT3, COPT4, COPT6 and COPT7 mediate Cu transport either

supply of Cu from vacuoles or lysosomes to the cytosol [58].

other proteins which are involved in Cu transport [61].

Expression of OsYSL2, OsYSL9 and OsYSL15 genes increases when Fe is limited. OsYSL9 is induced by Fe deficiency in the shoots but not the roots. On the other hand, OsYSL16 is constitutively expressed in both roots and shoots at levels similar to OsYSL2, OsYSL9 and OsYSL15 genes, but the alteration of Fe concentration has not shown any effect on the expression of OsYSL16 [47].

OsYSL13 is mostly expressed in the shoots, and its expression is reduced under Fe deficient conditions. OsYSL14 is expressed in both roots and shoots irrespective of the external Fe concentration. OsYSL15 is expressed only in roots and Fe deficiency highly induces its expression. OsYSL16 is expressed in both roots and shoots, and Fe deficiency slightly increases its expression in roots.

OsYSL2 is localized in the plasma membrane. It is not a Fe(III)-phytosiderophore transporter which is involved in the uptake of Fe from the soil [47]. In rice plant, OsYSL2 is expressed in the root companion cells and leaves phloem. OsYSL2 can also transport manganese-NA complex [47].

OsYSL6 is required for detoxification of excess Mn in rice thus helps in Mn tolerance. Irrespective of the Fe status, OsYSL6 is constitutively expressed in both roots and leaves. While OsYSL6 expression is slightly reduced under Fe-deficiency condition. The expression level increases with leaf age. This pattern is similar for Mn concentration in the different leaves [48].

YSL16 is a plasma membrane-localized transporter and is directly involved in distribution and remobilization of Cu as Cu-NA complex in the developing tissues and rice seed. It loads Cu-NA complex into the phloem, which is required for remobilization of Cu from older leaves to developing tissues like young leaves and seeds [52]. OsYSL16 is expressed in many cell types and is more preferentially expressed in the vascular tissues of roots and leaves. It has been studied that enhanced tolerance to a low-Fe environment can be achieved through over expression of OsYSL16.

OsYSL18 is a Fe(III)-DMA transporter which is involved in Fe distribution mediated by DMA in the reproductive organs, lamina joints, and phloem cells at the leaf sheath base. It is localized in the plasma membrane [54]. The other remaining putative OsYSL transporters in rice need to be functionally characterized in future.

### **4. Ctr/COPT family: copper transporter**

This family of transporters is found only in eukaryotes. In plants, it is known as COPT transporters [55] and in animals and fungi as Ctr [56]. COPT family of proteins are important for copper uptake from soil and its transport to pollen in plants [57]. COPT proteins are localized on the plasma membrane and are involved in the transport of Cu from extracellular spaces into cytosol or vacuoles or are localized on the lysosomal membrane and are involved in the supply of Cu from vacuoles or lysosomes to the cytosol [58].

Fe(II)-NA [53]. Its expression in flowers and the phloem of lamina joints indicates that it is

Expression of OsYSL2, OsYSL9 and OsYSL15 genes increases when Fe is limited. OsYSL9 is induced by Fe deficiency in the shoots but not the roots. On the other hand, OsYSL16 is constitutively expressed in both roots and shoots at levels similar to OsYSL2, OsYSL9 and OsYSL15 genes, but the alteration of Fe concentration has not shown any effect on the expres-

OsYSL13 is mostly expressed in the shoots, and its expression is reduced under Fe deficient conditions. OsYSL14 is expressed in both roots and shoots irrespective of the external Fe concentration. OsYSL15 is expressed only in roots and Fe deficiency highly induces its expression. OsYSL16 is expressed in both roots and shoots, and Fe deficiency slightly increases its

OsYSL2 is localized in the plasma membrane. It is not a Fe(III)-phytosiderophore transporter which is involved in the uptake of Fe from the soil [47]. In rice plant, OsYSL2 is expressed in the root companion cells and leaves phloem. OsYSL2 can also transport manganese-NA

OsYSL6 is required for detoxification of excess Mn in rice thus helps in Mn tolerance. Irrespective of the Fe status, OsYSL6 is constitutively expressed in both roots and leaves. While OsYSL6 expression is slightly reduced under Fe-deficiency condition. The expression level increases with leaf age. This pattern is similar for Mn concentration in the different

YSL16 is a plasma membrane-localized transporter and is directly involved in distribution and remobilization of Cu as Cu-NA complex in the developing tissues and rice seed. It loads Cu-NA complex into the phloem, which is required for remobilization of Cu from older leaves to developing tissues like young leaves and seeds [52]. OsYSL16 is expressed in many cell types and is more preferentially expressed in the vascular tissues of roots and leaves. It has been studied that enhanced tolerance to a low-Fe environment can be achieved through

OsYSL18 is a Fe(III)-DMA transporter which is involved in Fe distribution mediated by DMA in the reproductive organs, lamina joints, and phloem cells at the leaf sheath base. It is localized in the plasma membrane [54]. The other remaining putative OsYSL transporters in rice

This family of transporters is found only in eukaryotes. In plants, it is known as COPT transporters [55] and in animals and fungi as Ctr [56]. COPT family of proteins are important for copper uptake from soil and its transport to pollen in plants [57]. COPT proteins are localized on the plasma membrane and are involved in the transport of Cu from extracellular spaces

involved in translocating Fe to the reproductive organs and phloem joints.

sion of OsYSL16 [47].

156 Rice Crop - Current Developments

expression in roots.

complex [47].

leaves [48].

over expression of OsYSL16.

need to be functionally characterized in future.

**4. Ctr/COPT family: copper transporter**

The COPT family of transporters in rice consists of seven members: COPT1–COPT7. In rice plant, COPT proteins are specifically involved in Cu transport. It can transport only Cu(I) but not other bivalent ions such as Mn, Zn or Fe. In rice plant, COPT1–COPT7 are plasma membrane-localized proteins. As these transporters can form symmetrical homotrimer or heterotrimer structure with a diameter that is only suitable for Cu(I) transport and not other divalent ions [59] or heterocomplex with themselves or each other [60] or heterocomplex with other proteins which are involved in Cu transport [61].

COPT1 and COPT5 can exist as homodimers or a heterodimer. Only COPT1 and COPT5 bind to rice XA13 protein, a protein which is susceptible to pathogenic bacterium *Xanthomonas oryzae pv. Oryzae* (Xoo) [61] and mediate Cu transport in rice plant. In rice plant, all the COPTs except COPT1 and COPT5, function independently or together and mediate Cu transport in different tissues. COPT6 acts as a cofactor and aids the efficient localization of COPT2, COPT3 or COPT4 to the plasma membrane for mediating Cu transport. COPT1 and COPT5 show similar tissue and also develop-specific expression patterns. When compared to the sheath, stem, and panicle expression levels of these two genes, they have a higher level of expression in root and leaf tissues. COPT4 has higher expression level in root in comparison to other tissues. COPT1, COPT4, and COPT5 have higher expression level in young leaves than in old leaves, particularly COPT1 and COPT4. COPT2, COPT3, and COPT7 show higher expression levels in old leaves when compared with young leaves. COPT6 is not expressed in root and is highly expressed in leaf than in other tissues. COPT6 is constitutively expressed in differentaged leaves but has a low level of expression in shoot tissue at seedling stage. The expression of rice COPT1 and COPT5 are induced by Cu deficiency and suppressed by excess Cu in both shoot and root tissues. COPT1 and COPT5 together mediate Cu transport in rice plant [61]. The expression of rice COPT2, COPT3, COPT4, COPT6, and COPT7, is also affected by the variation in Cu levels. In both root and shoot tissues, COPT7 shows a similar response as COPT1 and COPT5 to Cu deficiency and overdose. In different-aged leaves of a mature plant, COPT6 is constitutively expressed while in the seedling stage, the shoot tissue has low expression levels. In shoot, COPT6 is induced under Cu deficiency state and suppressed in Cu overdose but no COPT6 expression is detected in root either with or without Cu deficiency. Cu overdose suppresses the expression of COPT2, COPT3, and COPT4 in both root and shoot tissues but their expression is not influenced by Cu deficiency. Expression of COPTs is also influenced by other bivalent cations. Mn deficiency induces expression of COPT1 in root and COPT3 and COPT7 in shoot and slightly suppresses the expression of COPT2 and COPT4 in root. The expression of COPT1, COPT5, and COPT7 is induced by Zn deficiency and COPT4 expression is slightly suppressed in the root. Zn deficiency also induces the expression of COPT5, COPT6, and COPT7 in the shoot. In root, Fe deficiency moderately induces COPT1 and suppresses COPT2 and COPT5 while in the shoot, it induces COPT2, COPT5, COPT6, and COPT7. In root, no COPT6 expression has been found either in the presence or absence of Mn, Zn, or Fe deficiency. COPT family of transporter proteins functions uniquely in different tissues, during various developmental stages, and in different environmental conditions. In rice plant, COPT2, COPT3, COPT4, COPT6 and COPT7 mediate Cu transport either solely or cooperatively with each other. In different tissues of rice plant COPT2, COPT3, or COPT4 function along with COPT6 for Cu transport except in root. Expression of COPT2, COPT3, COPT4, and COPT6 has been observed in stem, sheath, leaf, and panicle tissues. Root shows relatively high expression levels of COPT3 and COPT4 but no expression of COPT6. In leaves, the expression of COPT2, COPT3, and COPT4 is developmentally regulated but not that of COPT6. In rice shoot, Cu deficiency strongly induces the expression of COPT6 but not COPT2, COPT3, and COPT4. COPT7 mediates Cu transport in rice all by itself. Based on its expression pattern, it has been suggested that COPT7 functions in different tissues and is unaffected by Cu deficiency [62].

members [68]. NRAT1 plays an important role in rice Al tolerance by reducing the level of toxic Al in the root cell wall and transporting Al into the root cell vacuole for sequestration. Rice is the most Al tolerant of all the cereal crops and OsNRAMP4 plays an important role in

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OsNRAMP5 is a plasma membrane protein involved in Mn and Fe transport [70]. OsNRAMP5 gene expression increases slightly in the roots when plants are under Fe or Zn deficiency but varying levels of Mn in the surrounding does not affect it [71]. It is expressed in the mature root zone at the PM of the exodermal and endodermal layers [70, 71]. OsNRAMP5 in rice plant is essential for the uptake of Mn from the soil. In rice plant, OsNRAMP5 is constitutively involved in Fe and Mn uptake, it also plays a role in Fe and Mn transport during flowering

OsNRAMP5 is highly expressed in hulls. It is also expressed in leaves but the expression level decreases with leaf age. In rice plant, OsNRAMP5 transporter is present in the vascular bundles of roots and shoots particularly the parenchyma cells surrounding the xylem. *OsNRAMP5* is also highly expressed in stele cells especially in the xylem region, thus plays an important role in the xylem-mediated root-to-shoot transport. Thus OsNRAMP5 plays an important role in the uptake, translocation, and distribution of Mn in rice plants. *OsNRAMP5* is highly expressed in stele cells especially in the xylem region, thus plays an important role in the xylem-mediated root-to-shoot transport [72]. OsNRAMP5 is a major transporter for Cd

Recently, OsNRAMP6 has been identified to be involved in uptake of Fe and Mn. It is a plasma membrane-localized protein. It negatively regulates the rice plant immunity as loss of

The P1B-type ATPases, known as heavy metal ATPases (HMAs) in plants, play an important role in metal transport. HMAs vary in their tissue distribution, subcellular localization, and metal specificity. HMA transporters can be divided into two subgroups based on their metalsubstrate specificity, they are Cu/Ag group and Zn/Co/Cd/Pb group. Rice plant has nine such *HMA* genes. OsHMA1–OsHMA3 are members of the Zn/Co/Cd/Pb subgroup in rice. Unlike dicots, only a few reports on HMAs from monocots are available. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd and participates in their transport to developing seeds in rice. OsHMA9 phylogenetically belongs to the Cu/Ag subgroup but also plays a role in Zn, Cd, and Pb transport [74]. OsHMA3 transports only Cd and in root cells is involved in the sequestration of Cd into vacuoles [75, 76]. *OsHMA3* has been identified as a responsive gene for quantitative trait loci of Cd concentration in the rice cultivars Anjana Dhan and Cho-kokoku, and loss of function of this protein leads to high Cd accumulation in the shoots [75–77]. There is little information available on the role of OsHMA1 and is thought to be involved in Zn transport. *OsHMA1* expression in shoot tissue is highly upregulated by Zn deficiency [78]. OsHMA1 is suggested to play a role in Zn transport in the

its function results in increased resistance against *M. oryzae* [73].

**6. Heavy metal ATPases (HMAs)**

this [69].

and seed development [70].

uptake in rice [71].

### **5. NRAMP family**

Natural resistance-associated macrophage protein (NRAMP) family of transporters are found in the three domains of life [63]. NRAMP transporters have a wide range of metal substrates, typically transport Fe+2, Mn+2, Co+2, and Zn+2 [63].

The first plant NRAMP genes cloned were from rice [64]. In rice, there are seven Nramp transporters, OsNRAMP1-OsNRAMP7. Though, not all have been functionally characterized [63]. Many of the NRAMP family proteins function as Fe transporters. *OsNRAMP1* is highly upregulated by Fe deficiency. OsNRAMP1 is a plasma membrane-localized transporter and is involved in the transport of Cd and Fe. *OsNRAMP1* expression is observed mainly in roots at the vegetative state and is involved in cellular uptake of Cd and is responsible for high Cd accumulation in rice [65]. The differences observed in Cd accumulation among different rice cultivars are because of differences in *OsNRAMP1* expression levels in roots [65]. *OsNRAMP1* expression is higher during the reproductive stage in leaf blade and stem.

OsNRAMP3 is localized to the plasma membrane and is specifically expressed in vascular bundles, particularly in companion cells of phloem. *OsNRAMP3* is constitutively expressed in the rice node [66]. OsNRAMP3 is a Mn-influx transporter involved in Mn distribution and redistribution to young leaves from old leaf via phloem cells. With leaf aging, the expression of OsNRAMP3 in leaves increases slightly in rice plants. OsNRAMP3 transports Mn from the enlarged vascular bundles to the younger tissues and panicles during Mn deficiency in order to meet its minimal growth requirement. On the other hand, when Mn is in excess, OsNRAMP3 is internalized in vesicles and rapidly degraded. Then, Mn is preferentially loaded into the older leaves, which are directly connected to the enlarged vascular bundles, thereby protecting the developing tissues from Mn toxicity. This indicates the role of posttranslational regulation of OsNRAMP3 in response to environmental nutrient availability. Rice plant utilizes OsNRAMP3 to respond to environmental changes to Mn availability. OsNRAMP3 is involved in Mn translocation but not Mn uptake [67].

OsNRAMP4 is also known as Nramp aluminum transporter1 (Nrat1) is the first transporter in this family to be identified as the trivalent Al ion transporter [68]. In contrast to other rice NRAMP members, OsNRAMP4 does not show transport activity for other divalent metal ions, like Zn, Mn, and Fe. It also shares relatively low similarity with the other OsNRAMP members [68]. NRAT1 plays an important role in rice Al tolerance by reducing the level of toxic Al in the root cell wall and transporting Al into the root cell vacuole for sequestration. Rice is the most Al tolerant of all the cereal crops and OsNRAMP4 plays an important role in this [69].

OsNRAMP5 is a plasma membrane protein involved in Mn and Fe transport [70]. OsNRAMP5 gene expression increases slightly in the roots when plants are under Fe or Zn deficiency but varying levels of Mn in the surrounding does not affect it [71]. It is expressed in the mature root zone at the PM of the exodermal and endodermal layers [70, 71]. OsNRAMP5 in rice plant is essential for the uptake of Mn from the soil. In rice plant, OsNRAMP5 is constitutively involved in Fe and Mn uptake, it also plays a role in Fe and Mn transport during flowering and seed development [70].

OsNRAMP5 is highly expressed in hulls. It is also expressed in leaves but the expression level decreases with leaf age. In rice plant, OsNRAMP5 transporter is present in the vascular bundles of roots and shoots particularly the parenchyma cells surrounding the xylem. *OsNRAMP5* is also highly expressed in stele cells especially in the xylem region, thus plays an important role in the xylem-mediated root-to-shoot transport. Thus OsNRAMP5 plays an important role in the uptake, translocation, and distribution of Mn in rice plants. *OsNRAMP5* is highly expressed in stele cells especially in the xylem region, thus plays an important role in the xylem-mediated root-to-shoot transport [72]. OsNRAMP5 is a major transporter for Cd uptake in rice [71].

Recently, OsNRAMP6 has been identified to be involved in uptake of Fe and Mn. It is a plasma membrane-localized protein. It negatively regulates the rice plant immunity as loss of its function results in increased resistance against *M. oryzae* [73].

### **6. Heavy metal ATPases (HMAs)**

solely or cooperatively with each other. In different tissues of rice plant COPT2, COPT3, or COPT4 function along with COPT6 for Cu transport except in root. Expression of COPT2, COPT3, COPT4, and COPT6 has been observed in stem, sheath, leaf, and panicle tissues. Root shows relatively high expression levels of COPT3 and COPT4 but no expression of COPT6. In leaves, the expression of COPT2, COPT3, and COPT4 is developmentally regulated but not that of COPT6. In rice shoot, Cu deficiency strongly induces the expression of COPT6 but not COPT2, COPT3, and COPT4. COPT7 mediates Cu transport in rice all by itself. Based on its expression pattern, it has been suggested that COPT7 functions in different tissues and is

Natural resistance-associated macrophage protein (NRAMP) family of transporters are found in the three domains of life [63]. NRAMP transporters have a wide range of metal substrates,

The first plant NRAMP genes cloned were from rice [64]. In rice, there are seven Nramp transporters, OsNRAMP1-OsNRAMP7. Though, not all have been functionally characterized [63]. Many of the NRAMP family proteins function as Fe transporters. *OsNRAMP1* is highly upregulated by Fe deficiency. OsNRAMP1 is a plasma membrane-localized transporter and is involved in the transport of Cd and Fe. *OsNRAMP1* expression is observed mainly in roots at the vegetative state and is involved in cellular uptake of Cd and is responsible for high Cd accumulation in rice [65]. The differences observed in Cd accumulation among different rice cultivars are because of differences in *OsNRAMP1* expression levels in roots [65]. *OsNRAMP1*

OsNRAMP3 is localized to the plasma membrane and is specifically expressed in vascular bundles, particularly in companion cells of phloem. *OsNRAMP3* is constitutively expressed in the rice node [66]. OsNRAMP3 is a Mn-influx transporter involved in Mn distribution and redistribution to young leaves from old leaf via phloem cells. With leaf aging, the expression of OsNRAMP3 in leaves increases slightly in rice plants. OsNRAMP3 transports Mn from the enlarged vascular bundles to the younger tissues and panicles during Mn deficiency in order to meet its minimal growth requirement. On the other hand, when Mn is in excess, OsNRAMP3 is internalized in vesicles and rapidly degraded. Then, Mn is preferentially loaded into the older leaves, which are directly connected to the enlarged vascular bundles, thereby protecting the developing tissues from Mn toxicity. This indicates the role of posttranslational regulation of OsNRAMP3 in response to environmental nutrient availability. Rice plant utilizes OsNRAMP3 to respond to environmental changes to Mn availability.

OsNRAMP4 is also known as Nramp aluminum transporter1 (Nrat1) is the first transporter in this family to be identified as the trivalent Al ion transporter [68]. In contrast to other rice NRAMP members, OsNRAMP4 does not show transport activity for other divalent metal ions, like Zn, Mn, and Fe. It also shares relatively low similarity with the other OsNRAMP

expression is higher during the reproductive stage in leaf blade and stem.

OsNRAMP3 is involved in Mn translocation but not Mn uptake [67].

unaffected by Cu deficiency [62].

typically transport Fe+2, Mn+2, Co+2, and Zn+2 [63].

**5. NRAMP family**

158 Rice Crop - Current Developments

The P1B-type ATPases, known as heavy metal ATPases (HMAs) in plants, play an important role in metal transport. HMAs vary in their tissue distribution, subcellular localization, and metal specificity. HMA transporters can be divided into two subgroups based on their metalsubstrate specificity, they are Cu/Ag group and Zn/Co/Cd/Pb group. Rice plant has nine such *HMA* genes. OsHMA1–OsHMA3 are members of the Zn/Co/Cd/Pb subgroup in rice. Unlike dicots, only a few reports on HMAs from monocots are available. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd and participates in their transport to developing seeds in rice. OsHMA9 phylogenetically belongs to the Cu/Ag subgroup but also plays a role in Zn, Cd, and Pb transport [74]. OsHMA3 transports only Cd and in root cells is involved in the sequestration of Cd into vacuoles [75, 76]. *OsHMA3* has been identified as a responsive gene for quantitative trait loci of Cd concentration in the rice cultivars Anjana Dhan and Cho-kokoku, and loss of function of this protein leads to high Cd accumulation in the shoots [75–77]. There is little information available on the role of OsHMA1 and is thought to be involved in Zn transport. *OsHMA1* expression in shoot tissue is highly upregulated by Zn deficiency [78]. OsHMA1 is suggested to play a role in Zn transport in the plant throughout its growth and developmental stages [74]. Enhanced activity of OsHMA3 is related to increased storage of Cd in roots and its decreased transport to the shoot and the final accumulation in rice grains [74]. OsHMA2 is localized at the root pericycle and plays a major role in of transport of Zn and Cd during xylem loading [74, 75].

*OsMTP1* was characterized recently [86, 87]. In mature leaves and stem, it is highly expressed [86]. Generally, OsMTP1 transports Zn but can also transport Co, Fe, and Cd. Earlier OsMTP1 has been shown to transport Ni [86]. The vacuolar localization of OsMTP1 in the tonoplast, compartmentalizes primarily Zn, but also Co, Fe, and Cd, and serves as a detoxification system when these metals are available in excess. OsMTP1 is expressed constitutively and upregulated by Cd [86]. OsMTP1, OsMTP5, and OsMTP12 belong to the Zn-CDF subgroup [82, 84]. Expression of OsMTP1 in leaves, stems, roots, and flowers is relatively low and spatially and temporally regulated during development of rice. Also, it shows differential response to Cd stress. Transgenic assays in rice have shown that OsMTP1 expression levels can change plant

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In rice, OsMTP8.1 is the first Mn-CDF member to be identified. It is localized on to the tonoplast, and its over expression in rice enhances Mn accumulation and tolerance [88]. OsMTP9 is also a Mn-CDF member, which is involved in the uptake and translocation of Mn in rice plant [89]. Mn and other heavy metals induce the expression of *OsMTP11* in rice. In rice plant, *OsMTP1* is involved in Zn and Cd homeostasis/stress and mediates their translocation from roots to the aerial parts [86]. In most rice plant tissues *OsMTP11* is constitutively

OsMTP8.1 is localized to the tonoplast and involved in the detoxification of manganese by sequestering excess manganese to the vacuoles [90]. In rice root, OsMTP9 is polarly localized at the proximal side of both exodermis and endodermis opposite to Nramp5. The cooperative transport by Nramp5 and MTP9 efficiently transport Mn leading to its high accumulation in

The Mn-CDF group in plants is further clustered into two subgroups, Groups 8 and 9 [82]. In the rice genome, there are three members (MTP9/11/11.1) of Group 9. MTP9 shows much higher expression in the roots than in the basal region and shoots. The expression is unaffected by the deficiency of iron, zinc, copper, and manganese. The expression of MTP9 in roots is eightfold higher in the basal parts than that in apical parts. At the reproductive growth stage, MTP9 is also expressed in other organs such as nodes and leaf sheath in addition to the roots. MTP9 is polarly located at the proximal side of the exodermis and the endodermis, which is in opposition to Nramp5 [71]. Therefore, MTP9 at the proximal side of the exodermis releases manganese taken up by Nramp5 to the apoplast of a spoke-like structure in the aerenchyma, whereas MTP9 at the proximal side of endodermis further releases manganese toward the apoplast of stele including xylem vessels. Thus polar localization of transporters plays an important role in the directional transport of minerals. Recently, a number of transporters have been found to show polar localization. However, our understanding of the molecular mechanism underlying polar localization is still very poor. Also, MTP9 is different from other members of the Mn-CDF group as it shows a distinct expression pattern in tissue and subcellular localization. MTP9 is mainly expressed in the roots, but MTP8.1 from rice [90] in the same group is mainly expressed in shoots rather than roots. Different from other members, rice MTP9 is localized to the plasma membrane. These differences are associated with the role of MTP9 in manganese uptake in rice roots. In conclusion, MTP9 is a plasma membrane-localized efflux transporter for manganese uptake and translocation in rice roots.

cation absorption and in turn has affect on Zn, Ni, and Cd contents [86].

expressed.

rice [91].

*OsHMA3* gene selectively sequesters Cd into the vacuoles thus limits the root-to-shoot translocation of Cd [75, 76]. In rice plant, *OsHMA2* gene has also been shown to be involved in the translocation of Cd through xylem from root to shoot [79, 80].

In root cells, OsHMA4 is a vacuolar membrane-localized transporter and is involved in sequestering Cu into the vacuoles. OsHMA4 specifically transports Cu. Increased Cu accumulation in rice grain due to increased root-to-shoot translocation of Cu has been observed when OsHMA4 function is lost. In rice OsHMA4–OsHMA9 are members of the Cu/Ag subgroup of HMAs. OsHMA5 is a Cu transporter, localized to the plasma membrane [81]. In rice, OsHMA5 is involved in transferring Cu into the xylem for its root-to-shoot translocation and/ or Cu detoxification in roots [81]. OsHMA4 is induced under long-term exposure of excess Cu and its expression is suppressed by Cu deficiency. In mature root zone, OsHMA4 is localized at the pericycle [81]. OsHMA4 regulates the cellular Cu concentration before loading to the xylem depending on its environmental concentration. OsHMA3 is localized in all root cells [75]. In future, the mechanism responsible for the transporter substrate specificity of the HMAs needs to be studied.
