**4. Achievements made through molecular mapping approaches with respect to salinity tolerance in rice**

By using linkage and association mapping approaches, a number of QTLs linked to salinity tolerance in rice, have been identified. Detail of identified QTLs is given below.

### **4.1. Linkage mapping**

Linkage mapping has been very successful in the identification of QTLs linked to salinity tolerance in rice. A number of significant QTLs associated with salinity tolerance in rice were identified through linkage mapping approach (**Table 1**). In these studies, the mapping populations used were F2 population, F<sup>3</sup> population, F2:4 population, near-isogenic lines, recombinant inbred lines, doubled haploid population, backcross-inbred lines, BC<sup>3</sup> F5 lines, BC2 F8 advanced backcross introgression lines, and reciprocal introgression lines. Marker systems used in these studies included SSR, RFLP, SSLP, SSR AFLP, and SNPs.

Morphological parameters are supposed to be indicators of salt tolerance. There were various reports in which QTLs related to morphological traits under salt stress were identified [12, 32, 34–43]. In these mapping studies, the plant material was phenotyped at the seedling, tillering, or the maturity stage. Data for different morphological traits were recorded in these studies. These traits included seed germination (%), seedling survival days, seedling vigor, seedling root length, shoot length, fresh shoot weight, dry shoot weight, dry root weight, reduction rate of dry weight, reduction rate of fresh weight, reduction rate of leaf area, reduction rate of seedling height, tiller number, salt tolerance rating, score of salt toxicity of leaves, plant height, and grain yield-related traits. A number of significant QTLs were identified in these studies. These identified QTLs included a QTL for seedling survival days [32]; a QTL for root length flanked by restriction fragment length polymorphism (RFLP) markers RG162-RG653 [36]; QTLs with heritability values up to 53.3% [34]; two significant QTLs, *qST1* and *qST3*, for salt tolerance at seedling stage with 35.5–36.9% phenotypic variance explained values, respectively [38]; same QTLs conferring salt tolerance at both seedling and tillering stages [40], SSR marker RM223 associated with salt tolerance in rice [39], and a major QTL for straw yield, *qSY-3* [12]. These studies also suggested that it is possible to combine favorable alleles associated with salt tolerance in a single cultivar through marker-assisted selection (MAS) of main effect QTLs (M-QTLs) [42]. Similarly, pleiotropic effects were found for some QTLs which were found associated with both drought and salt tolerance [43].

There are also a number of reports of QTLs identified for different physio-biochemical traits through linkage mapping [11, 33, 44–52]. Traits which were studied in these reports were shoot Na+ concentration; shoot K<sup>+</sup> concentration; leaf Na+ concentration; leaf K<sup>+</sup> concentration; Na+ uptake; K<sup>+</sup> uptake; Na<sup>+</sup> absorption; K<sup>+</sup> adsorption; Na+ /K<sup>+</sup> absorption ratio; K<sup>+</sup> /Na+ ratio;


**Table 1.** QTLs identified through linkage mapping studies.

polymorphism (SSLP), and single nucleotide polymorphism (SNP) [12, 32–35] are different types of DNA markers which are employed for genotyping in molecular mapping studies.

By using linkage and association mapping approaches, a number of QTLs linked to salinity

Linkage mapping has been very successful in the identification of QTLs linked to salinity tolerance in rice. A number of significant QTLs associated with salinity tolerance in rice were identified through linkage mapping approach (**Table 1**). In these studies, the mapping popu-

advanced backcross introgression lines, and reciprocal introgression lines. Marker systems

Morphological parameters are supposed to be indicators of salt tolerance. There were various reports in which QTLs related to morphological traits under salt stress were identified [12, 32, 34–43]. In these mapping studies, the plant material was phenotyped at the seedling, tillering, or the maturity stage. Data for different morphological traits were recorded in these studies. These traits included seed germination (%), seedling survival days, seedling vigor, seedling root length, shoot length, fresh shoot weight, dry shoot weight, dry root weight, reduction rate of dry weight, reduction rate of fresh weight, reduction rate of leaf area, reduction rate of seedling height, tiller number, salt tolerance rating, score of salt toxicity of leaves, plant height, and grain yield-related traits. A number of significant QTLs were identified in these studies. These identified QTLs included a QTL for seedling survival days [32]; a QTL for root length flanked by restriction fragment length polymorphism (RFLP) markers RG162-RG653 [36]; QTLs with heritability values up to 53.3% [34]; two significant QTLs, *qST1* and *qST3*, for salt tolerance at seedling stage with 35.5–36.9% phenotypic variance explained values, respectively [38]; same QTLs conferring salt tolerance at both seedling and tillering stages [40], SSR marker RM223 associated with salt tolerance in rice [39], and a major QTL for straw yield, *qSY-3* [12]. These studies also suggested that it is possible to combine favorable alleles associated with salt tolerance in a single cultivar through marker-assisted selection (MAS) of main effect QTLs (M-QTLs) [42]. Similarly, pleiotropic effects were found for some QTLs

population, F2:4 population, near-isogenic lines, recom-

concentration; leaf K<sup>+</sup>

absorption ratio; K<sup>+</sup>

/K<sup>+</sup>

F5

lines, BC2

concentration;

ratio;

/Na+

F8

**4. Achievements made through molecular mapping approaches with** 

tolerance in rice, have been identified. Detail of identified QTLs is given below.

binant inbred lines, doubled haploid population, backcross-inbred lines, BC<sup>3</sup>

used in these studies included SSR, RFLP, SSLP, SSR AFLP, and SNPs.

which were found associated with both drought and salt tolerance [43].

absorption; K<sup>+</sup>

concentration; shoot K<sup>+</sup>

uptake; Na<sup>+</sup>

There are also a number of reports of QTLs identified for different physio-biochemical traits through linkage mapping [11, 33, 44–52]. Traits which were studied in these reports were

adsorption; Na+

concentration; leaf Na+

**respect to salinity tolerance in rice**

population, F<sup>3</sup>

**4.1. Linkage mapping**

184 Rice Crop - Current Developments

lations used were F2

shoot Na+

uptake; K<sup>+</sup>

Na+

ratio of leaf Na+ to sheath Na+ concentrations; sodium (Na+ ) and potassium (K<sup>+</sup> ) in roots; Na+ concentration and Na/K ratio in the flag leaf; and sodium (Na<sup>+</sup> ), potassium (K<sup>+</sup> ), and calcium (Ca++) accumulation traits. Major discoveries in these studies included a major QTL (*QKr1.2*) identified for K<sup>+</sup> content in the root on chromosome 1 explaining 30% of the total variation [48]; pollen fertility, Na<sup>+</sup> concentration and Na/K ratio in the flag leaf were found as the most important attributes for salt tolerance at the reproductive stage in rice [52], QTLs for sodium and potassium uptake were identified on different linkage groups (chromosomes) [33] suggesting that different pathways are involved in Na<sup>+</sup> and K<sup>+</sup> uptake; and a major locus controlling Na+ uptake (*QTLsur-7*) was identified on chromosome 7, with *R*<sup>2</sup> value of 72.57% [11].

of information in the present rice genetics knowledge pool. Random distribution in the rice germplasm of favorable alleles associated with salt tolerance is a worthwhile finding which should be considered while exploring and selecting crossing parents in breeding programmes.

Abiotic Stress Tolerance in Rice (*Oryza sativa* L.): A Genomics Perspective of Salinity Tolerance

http://dx.doi.org/10.5772/intechopen.73571

187

Climate change has affected world agriculture a lot. The most pronounced effects of climate change are the heat stress and periodic drought conditions in major rice producing countries of the world. Due to periodic drought conditions, the already existing problem of high amounts of salts in the upper surface soil has intensified. So, there is a dire need to opt for a coordinated approach to address the problem of salinity stress for rice production. Genomics has great potential to assist in this coordinated programme. With the help of molecular mapping approaches, a number of major and minor QTLs associated with salinity tolerance in rice have been identified in recent years and there are further accelerated research efforts underway in this direction. The identified QTLs are valuable resources for marker-assisted selection (MAS) to develop elite salt tolerant rice cultivars. Great task is needed to be done in this regard so that marker-assisted breeding (MAB) approach can be implemented successfully in routine breeding programmes. In future, efforts should be directed to develop climate-smart rice cultivars which can perform stably under diverse environmental conditions. Identified QTLs and rice germplasm found tolerant to salinity stress can be exploited in three major ways: (a) to understand the molecular genetics of salt tolerance in rice; (b) salinity stress tolerant rice germplasm might be incorporated into salt-tolerant rice cultivars development molecular breeding programmes; (c) identified QTLs incorporated into MAS for screening rice germplasm against salinity stress. New genes involved in salt tolerance will be identified by this approach. Genome sequence of rice, both indica and japonica subspecies, is available now. In the next phase of annotation of the rice genome, molecular mapping results can be of

Lot of work related to molecular mapping for salinity tolerance in rice is to be performed yet. The main cautious point is the plant phenotyping for salt stress tolerance. Accuracy in the phenotyping work is the key in the authentic identification of QTLs related to salt tolerance. Hydroponics should be tried for this purpose. Under salinity stress conditions, phenotyping at germination, seedling, tillering, and reproductive phases require different strategies and care. In case of quantitative traits, such as salinity stress tolerance, there is pronounced effect of environment. Efforts should be made to design a judicious phenotyping plan which can minimize effect of environment. In case of plant genotyping work, robust marker systems with high resolution power such as SNPs should be preferred over other marker systems.

Previous research efforts have pointed out that the distribution of favorable alleles, associated with salt-tolerance, is random among the rice germplasm [53]. So, it is possible to pyramid favorable alleles of salt-tolerance in an elite rice genotype through well-planned crossing programme. This elite rice cultivar will have great potential with regard to salt tolerance. In view of inland intrusion of the seas, we have to concentrate on the coastal areas to fully exploit

**5. Future prospects and conclusions**

help in combination with the comparative genomics approach.

Genotyping-by-sequencing (GBS) is another option.

### **4.2. Association mapping**

In recent years, association mapping is widely used to identify QTLs in plants. Association mapping approach is relatively new arrival in plant genetics. There are some reports of association mapping for salt tolerance in rice [13, 53–58]. Main findings of these association studies are presented in **Table 2**. In these studies, rice mapping populations used consisted of European Rice Core collection (ERCC) containing 180 japonica accessions [53], 96 rice germplasm accessions including Nona Bokra [55], 220 rice accessions [56], 341 japonica rice accessions [57], 94 rice genotypes [58], and 24 indica rice genotypes [13]. Traits for which data were recorded in these studies included Na+ /K<sup>+</sup> ratio, survival days of seedlings, shoot K<sup>+</sup> /Na+ ratio, Na+ uptake, Ca++ uptake, total cations uptake, Ca++ uptake ratio, K<sup>+</sup> uptake ratio, Na<sup>+</sup> /K<sup>+</sup> uptake and salinity tolerance scoring. Major findings made in these studies included an observation that distribution of favorable alleles associated with salt tolerance was random in ERCC [53]; 40 new allelic variants found in coding sequences of five salt-related genes [54]; STS marker, RM22418, for *SKC1*, on Chr. 8 was found associated with salinity tolerance [55]; region containing *Saltol* was found associated with Na+ /K<sup>+</sup> ratio [56]; marker RM3412 was found associated to salinity tolerance at seedling stage due to its close linkage to *SKC* gene [58]; and the report that other QTLs, in addition to *Saltol*, might be involved in salinity tolerance [58]. These reports highlighted that in rice germplasm there might be other genomic regions involved in salt tolerance. These genomic regions need to be characterized in future to add a wealth


**Table 2.** QTLs identified through association mapping studies.

of information in the present rice genetics knowledge pool. Random distribution in the rice germplasm of favorable alleles associated with salt tolerance is a worthwhile finding which should be considered while exploring and selecting crossing parents in breeding programmes.

### **5. Future prospects and conclusions**

ratio of leaf Na+

186 Rice Crop - Current Developments

identified for K<sup>+</sup>

ling Na+

Na+

Na+ /K<sup>+</sup>

protection

[48]; pollen fertility, Na<sup>+</sup>

**4.2. Association mapping**

recorded in these studies included Na+

taining *Saltol* was found associated with Na+

ratio equilibrium; signaling cascade; stress

**Table 2.** QTLs identified through association mapping studies.

Survival days of seedlings and shoot K<sup>+</sup>

to sheath Na+

gesting that different pathways are involved in Na<sup>+</sup>

concentration and Na/K ratio in the flag leaf; and sodium (Na<sup>+</sup>

concentrations; sodium (Na+

(Ca++) accumulation traits. Major discoveries in these studies included a major QTL (*QKr1.2*)

important attributes for salt tolerance at the reproductive stage in rice [52], QTLs for sodium and potassium uptake were identified on different linkage groups (chromosomes) [33] sug-

In recent years, association mapping is widely used to identify QTLs in plants. Association mapping approach is relatively new arrival in plant genetics. There are some reports of association mapping for salt tolerance in rice [13, 53–58]. Main findings of these association studies are presented in **Table 2**. In these studies, rice mapping populations used consisted of European Rice Core collection (ERCC) containing 180 japonica accessions [53], 96 rice germplasm accessions including Nona Bokra [55], 220 rice accessions [56], 341 japonica rice accessions [57], 94 rice genotypes [58], and 24 indica rice genotypes [13]. Traits for which data were

and salinity tolerance scoring. Major findings made in these studies included an observation that distribution of favorable alleles associated with salt tolerance was random in ERCC [53]; 40 new allelic variants found in coding sequences of five salt-related genes [54]; STS marker, RM22418, for *SKC1*, on Chr. 8 was found associated with salinity tolerance [55]; region con-

/K<sup>+</sup>

ated to salinity tolerance at seedling stage due to its close linkage to *SKC* gene [58]; and the report that other QTLs, in addition to *Saltol*, might be involved in salinity tolerance [58]. These reports highlighted that in rice germplasm there might be other genomic regions involved in salt tolerance. These genomic regions need to be characterized in future to add a wealth

uptake (*QTLsur-7*) was identified on chromosome 7, with *R*<sup>2</sup>

/K<sup>+</sup>

**Trait Plant material used Marker system** 

Salinity tolerance 180 japonica accessions SNPs, SSR [53]

Salinity tolerance 96 germplasm accessions SSR [55] Stress-responsive genes 220 rice accessions SNPs [56]

Seedling stage salt tolerance 94 rice genotypes SSR [58]

/Na+

uptake, Ca++ uptake, total cations uptake, Ca++ uptake ratio, K<sup>+</sup>

content in the root on chromosome 1 explaining 30% of the total variation

and K<sup>+</sup>

concentration and Na/K ratio in the flag leaf were found as the most

ratio, survival days of seedlings, shoot K<sup>+</sup>

) and potassium (K<sup>+</sup>

), potassium (K<sup>+</sup>

uptake; and a major locus control-

uptake ratio, Na<sup>+</sup>

ratio [56]; marker RM3412 was found associ-

**used**

392 rice accessions SNPs [54]

ratio 341 japonica rice accessions SSR [57]

value of 72.57% [11].

) in roots; Na+

), and calcium

/Na+

**Reference**

/K<sup>+</sup>

ratio,

uptake

Climate change has affected world agriculture a lot. The most pronounced effects of climate change are the heat stress and periodic drought conditions in major rice producing countries of the world. Due to periodic drought conditions, the already existing problem of high amounts of salts in the upper surface soil has intensified. So, there is a dire need to opt for a coordinated approach to address the problem of salinity stress for rice production. Genomics has great potential to assist in this coordinated programme. With the help of molecular mapping approaches, a number of major and minor QTLs associated with salinity tolerance in rice have been identified in recent years and there are further accelerated research efforts underway in this direction. The identified QTLs are valuable resources for marker-assisted selection (MAS) to develop elite salt tolerant rice cultivars. Great task is needed to be done in this regard so that marker-assisted breeding (MAB) approach can be implemented successfully in routine breeding programmes. In future, efforts should be directed to develop climate-smart rice cultivars which can perform stably under diverse environmental conditions. Identified QTLs and rice germplasm found tolerant to salinity stress can be exploited in three major ways: (a) to understand the molecular genetics of salt tolerance in rice; (b) salinity stress tolerant rice germplasm might be incorporated into salt-tolerant rice cultivars development molecular breeding programmes; (c) identified QTLs incorporated into MAS for screening rice germplasm against salinity stress. New genes involved in salt tolerance will be identified by this approach. Genome sequence of rice, both indica and japonica subspecies, is available now. In the next phase of annotation of the rice genome, molecular mapping results can be of help in combination with the comparative genomics approach.

Lot of work related to molecular mapping for salinity tolerance in rice is to be performed yet. The main cautious point is the plant phenotyping for salt stress tolerance. Accuracy in the phenotyping work is the key in the authentic identification of QTLs related to salt tolerance. Hydroponics should be tried for this purpose. Under salinity stress conditions, phenotyping at germination, seedling, tillering, and reproductive phases require different strategies and care. In case of quantitative traits, such as salinity stress tolerance, there is pronounced effect of environment. Efforts should be made to design a judicious phenotyping plan which can minimize effect of environment. In case of plant genotyping work, robust marker systems with high resolution power such as SNPs should be preferred over other marker systems. Genotyping-by-sequencing (GBS) is another option.

Previous research efforts have pointed out that the distribution of favorable alleles, associated with salt-tolerance, is random among the rice germplasm [53]. So, it is possible to pyramid favorable alleles of salt-tolerance in an elite rice genotype through well-planned crossing programme. This elite rice cultivar will have great potential with regard to salt tolerance. In view of inland intrusion of the seas, we have to concentrate on the coastal areas to fully exploit their agricultural production potential. This is also imperative in view of alarming increase in human population and to feed this population we have to exploit every available land for agricultural production. It is hoped that genomics approaches will play a greater part in this exploitation of land by providing salt tolerant crop cultivars.

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Abiotic Stress Tolerance in Rice (*Oryza sativa* L.): A Genomics Perspective of Salinity Tolerance

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### **Author details**

Muhammad Saeed

Address all correspondence to: saeed\_pbg@gcuf.edu.pk

Department of Botany, Faculty of Life Sciences, Government College University, Faisalabad, Pakistan

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Department of Botany, Faculty of Life Sciences, Government College University, Faisalabad,

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Genetic Linkage Maps. Wageningen, The Netherlands: CPRO-DLO; 2001

experimental and natural populations. Genomics. 1987;**1**(2):174-181

of Statistics, North Carolina State University; 2001

informatics, Zhejiang University; 2005

samples. PLoS Genetics. 2007;**3**:e4

ance in rice. Rice Science. 2005;**12**:25-32

Science. 2000;**78**:162-164

SNP, marker sets. Molecular Plant Breeding. 2013;**5**:47-63

Physiologia Plantarum. 1983;**59**:189-195

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190 Rice Crop - Current Developments

of Chicago Press; 2004

2007;**23**:2633-2635

1998;**12**:72-78

**125**:406-422


[50] Wang Z, Chen Z, Cheng J, Lai Y, Wang J, Bao Y, Huang J, Zhang H. QTL analysis of Na<sup>+</sup> and K<sup>+</sup> concentrations in roots and shoots under different levels of NaCl stress in rice (*Oryza sativa* L.). PLoS One. 2012;**7**:e51202

**Chapter 11**

**Provisional chapter**

**Natural Resistance of Sri Lankan Rice (***Oryza sativa* **L.)**

**Natural Resistance of Sri Lankan Rice (***Oryza sativa* **L.)** 

DOI: 10.5772/intechopen.76991

**Varieties to Broad-Spectrum Herbicides (Glyphosate**

**Varieties to Broad-Spectrum Herbicides (Glyphosate** 

Since studies on herbicide-resistant rice (HRR) are limited in Sri Lanka, the present study conducted to screen the naturally existing glyphosate and glufosinate resistance in traditional and inbred rice varieties. Six traditional varieties and nineteen inbred lines were selected for the study. Complete randomized design with three pots with 10 replicates for each herbicide concentration was employed. Optimal concentrations of glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) were applied at 3–4 leaf stages. Varieties ≥50% survival percentage was considered as resistant to respective herbicides. Twelve varieties showed resistance (≥50%) at 0.5 gl−1 glyphosate concentration. Survived plants were monitored and agro-morphological and yield characters/parameters were measured. Fifteen varieties were to glufosinate at 0.05 gl−1. Even though no significant differences (p > 0.05) were observed in growth parameters across control and treated plants, there was a yield penalty. Nine varieties (At362, Bg352, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454, and Pachchaperumal) indicated moderate resistance to both glyphosate and glufosinate. The emerged HRRs indicated varying responses of agro-morphological and yield characters across the type of herbicide and the variety. Glyphosate reduced the growth parameters and yield penalty compared to glufosinate treated varieties. These HRR varieties have a higher potential in rice breeding programs and in develop-

> © 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.

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,

Shyama R. Weerakoon, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and Sachithri Munasighe

Shyama R. Weerakoon, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and Sachithri Munasighe

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.76991

ing HR rice varieties in future.

**Keywords:** glyphosate, glufosinate, herbicide resistance, *Oryza sativa*

**and Glufosinate)**

**and Glufosinate)**

**Abstract**


**Provisional chapter**

### **Natural Resistance of Sri Lankan Rice (***Oryza sativa* **L.) Varieties to Broad-Spectrum Herbicides (Glyphosate and Glufosinate) Natural Resistance of Sri Lankan Rice (***Oryza sativa* **L.) Varieties to Broad-Spectrum Herbicides (Glyphosate and Glufosinate)**

DOI: 10.5772/intechopen.76991

Shyama R. Weerakoon, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and Sachithri Munasighe Shyama R. Weerakoon, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and Sachithri Munasighe

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.76991

#### **Abstract**

[50] Wang Z, Chen Z, Cheng J, Lai Y, Wang J, Bao Y, Huang J, Zhang H. QTL analysis of Na<sup>+</sup>

[51] Ghomi K, Rabiei B, Sabouri H, Sabouri A. Mapping QTLs for traits related to salinity tolerance at seedling stage of rice (*Oryza sativa* L.): An agrigenomics study of an Iranian rice population. OMICS: A Journal of Integrative Biology. 2013;**17**(5):242-251. DOI: 10.1089/

[52] Hossain H, Rahman MA, Aslam MS, Singh RK. Mapping of quantitative trait loci associated with reproductive stage salt tolerance in rice. Journal of Agronomy and Crop

[53] Ahmadi N, Negrão S, Katsantonis D, Frouin J, Ploux J, Letourmy P, Droc G, Babo P, Trindade H, Bruschi G, Greco R, Oliveira MM, Piffanelli P, Courtois B. Targeted associa-

[54] Negrão S, Almadanim MC, Pires IS, Abreu IA, Maroco J, Courtois B, Gregorio GB, McNally KL, Oliveira MM. New allelic variants found in key rice salt-tolerance genes:

[55] Emon RM, Islam MM, Halder J, Fan Y. Genetic diversity and association mapping for salinity tolerance in Bangladeshi rice landraces. Crop Journal. 2015;**3**:440-444

[56] Kumar V, Singh A, Mithra SVA, Krishnamurthy SL, Parida SK, Jain S, Tiwari KK, Kumar P, Rao AR, Sharma SK, Khurana JP, Singh NK, Mohapatra T. Genome-wide association mapping of salinity tolerance in rice (*Oryza sativa*). DNA Research. 2015;**22**:133-145 [57] Zheng H, Wang J, Zhao H, Liu H, Sun J, Guo L, Zou D. Genetic structure, linkage disequilibrium and association mapping of salt tolerance in japonica rice germplasm at the

[58] Krishnamurthy SL, Sharma SK, Kumar V, Tiwari S, Singh NK.Analysis of genomic region spanning *Saltol* using SSR markers in rice genotypes showing differential seedlings stage

salt tolerance. Journal of Plant Biochemistry and Biotechnology. 2016;**25**:331-336

allelic make-up of the salt tolerant indica variety Nona Bokra. Theoretical and Applied

/K<sup>+</sup>

homeostasis without the

tion analysis identified japonica rice varieties achieving Na<sup>+</sup>

An association study. Plant Biotechnology Journal. 2013;**11**:87-100

seedling stage. Molecular Breeding. 2015;**35**:152

concentrations in roots and shoots under different levels of NaCl stress in rice

and K<sup>+</sup>

192 Rice Crop - Current Developments

omi.2012.0097

Science. 2015;**201**:17-31

Genetics. 2011;**123**:881-895

(*Oryza sativa* L.). PLoS One. 2012;**7**:e51202

Since studies on herbicide-resistant rice (HRR) are limited in Sri Lanka, the present study conducted to screen the naturally existing glyphosate and glufosinate resistance in traditional and inbred rice varieties. Six traditional varieties and nineteen inbred lines were selected for the study. Complete randomized design with three pots with 10 replicates for each herbicide concentration was employed. Optimal concentrations of glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) were applied at 3–4 leaf stages. Varieties ≥50% survival percentage was considered as resistant to respective herbicides. Twelve varieties showed resistance (≥50%) at 0.5 gl−1 glyphosate concentration. Survived plants were monitored and agro-morphological and yield characters/parameters were measured. Fifteen varieties were to glufosinate at 0.05 gl−1. Even though no significant differences (p > 0.05) were observed in growth parameters across control and treated plants, there was a yield penalty. Nine varieties (At362, Bg352, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454, and Pachchaperumal) indicated moderate resistance to both glyphosate and glufosinate. The emerged HRRs indicated varying responses of agro-morphological and yield characters across the type of herbicide and the variety. Glyphosate reduced the growth parameters and yield penalty compared to glufosinate treated varieties. These HRR varieties have a higher potential in rice breeding programs and in developing HR rice varieties in future.

**Keywords:** glyphosate, glufosinate, herbicide resistance, *Oryza sativa*

© 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, © 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.

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

### **1. Introduction**

Rice, one of the most important grains, fulfills the carbohydrate requirement of people in the tropical countries and to a lesser extent in subtemperate areas. The cultivated rice belongs to the grass family Gramineae (Poaceae) under the tribe-Oryzeae of the subfamily Pooideae [1]. However, the genus *Oriza* has been divided into several sections and placed *O. sativa* under Series Sativa in Section Sativae [2]. *O. sativa,* an indigenous rice species in Asia, is a diploid species consisting 24 chromosomes. The genomic formula of *O. sativa* is AA [2]. The species *O. sativa* is an annual grass, with round, hollow, jointed culms, rather flat, sessile leaf blades, and a terminal panicle, under favorable conditions. As the other members in the tribe Oryzeae, rice is well-adapted to aquatic and swampy habitats [3].

Rice weeds adversely affected on final yield in number of ways. Weed increases the cost of production of rice. The cost of rice weed control, including herbicides, cultural and mechanical practices, and hand weeding, is estimated to be about 5% of world rice production and amount to US\$3.5 billion annually. When the10% loss of rough rice grain yield is added to this cost, the world's total estimated cost for rice weeds and their control amounts to 15% of

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

http://dx.doi.org/10.5772/intechopen.76991

195

Weeds indirectly limit production and act as a host of plant harboring pathogens and pests that adversely affect rice. Furthermore, weeds intervene rice harvesting and increase harvest costs through direct interference with the harvesting operation and by causing lodging. Contamination of rough rice by the seeds of the weeds reduces the grain quality and market value for example weed red rice (*Oryza sativa f. spontanea*) has a pigmented layer that shatters easily and readily contaminates rough rice. Removing all traces of the pigmented layer requires intense milling and results in decreased grain quality and lower milling rates [12]. The drudgery of weeding and labor shortages have made rice farming unattractive. In most tropical countries, farmers spend more time on weeding, by hand or with simple tools, than on any other farming task. Hand weeding of one (01) ha. of rice requires from 100 to 780 laborhours per crop, depending on the rice culture. Due to these adverse effects, there is a need to

Herbicides are chemical substances used to kill plants which are often placed under the group of chemicals known as pesticides that prevent, destroy, repel, or mitigate any pest [13]. Herbicides, in general, are classified using different criteria such as activity, timing of application, method of application, mechanism of action and chemical family. Based on the time of application there are three main categories of herbicides recommended for rice. "Pre-plant herbicides" are applied before the crop is planted in order to eliminate weeds that have germinated before planting or were left from following (e.g., glyphosate, glufosinate). "Pre-emergence herbicides" are applied after the crop has been planted but before weeds emerge (butachlor, pretilachloroxadiazon, pendimethalin, oxadiargyl) and "Post-emergence herbicides" are applied after weeds have emerged (bisparybacbispyribac, pentagon, 2,4-D). These herbicides are either broad-spectrum (nonselective) or narrowspectrum (selective). Some of the most common modes of actions are auxin mimics, mitosis inhibitors, photosynthesis inhibitors, amino acid synthesis inhibitors and lipid biosynthesis

The usage of pre-emergent, broad spectrum herbicide in controlling weeds in rice cultivation has become a popular method among the farmers since it minimizes cost, labor and time. Glyphosate and glufosinate are the most commonly used broad-spectrum herbicides (BSHs) in rice fields and glyphosate usage is comparatively higher. Glyphosate (*N*-(phosphonomethyl)

killing all plant types including grasses, perennials and woody plants. Glyphosate is a versatile herbicide used by farmers, land managers and gardeners to simply, safely and effectively control unwanted vegetation. Initially glyphosate was patented and sold by Monsanto

P is a broad spectrum, nonselective systemic herbicide. It is effective in

total annual production [12].

improve the present weed control practices.

inhibitors.

glycine) or C3

H8 NO5

Rice is cultivated on about 156 million hectares of land to produce about 696 million tons annually in Asian countries which account for 90% of the world's total rice production [4]. There is a growing trend of increasing rice consumptions since 2000s, which surpasses the production. On an annual basis, global rice demand keeps increasing by *ca.* 8 million tons implying that during next 10 years, the rice production need to increase to 80 million tons which is double the present production [5].

The increasing world population especially in tropical countries where rice serves as the staple food, one billion people per year demands an additional rice production (100 million MT) [6]. In future, it is apparent that rice production will continue to grow rapidly as increasing populations attempt to secure food supplies. In order to obtain a good yield in rice, farmers are required to overcome several biotic and abiotic stresses. Among biotic stresses, weeds stand out as the major threat to rice cultivation, which reduce the yield qualitatively and quantitatively. Over the past few decades, climate change has induced transformations in the weed flora of arable ecosystems and the changes in the climate have also influenced weeds indirectly by enforcing adaptations to agronomic practice [7]. Therefore, it is imperative to develop effective weed control strategies while maintaining crop yield [8]. Globally, *ca.*10% loss of rice yield is attributed to weed and specific quantity is more or less closer to 46 million tons (based on 1987 world rough rice production). Depending on the predominant weed flora and on the control methods practiced by farmers, loss of yield caused by weeds varies across countries in the world. In Sri Lanka, a country considered self-sufficient in rice, weeds are the major biotic stress in rice production and account for 30–40% of yield losses [9]. Thus, there is a need to take timely and appropriate measures to preserve the country's rice production.

Rice weeds are the major barriers to rice production because they possess the ability to compete for CO2 , space, moisture, sunlight and nutrients. Under certain conditions, crops fail to successfully compete with weeds [10]. Weed flora varies spatially due to type of rice culture, soil type, hydrology, tillage, cultural practices and irrigation pattern and so on. Approximately, 134 weed species belonging to 32 taxonomic families were identified in rice fields in Sri Lanka, and they were categorized as grasses, sedges and broad leaves [11].

Rice weeds adversely affected on final yield in number of ways. Weed increases the cost of production of rice. The cost of rice weed control, including herbicides, cultural and mechanical practices, and hand weeding, is estimated to be about 5% of world rice production and amount to US\$3.5 billion annually. When the10% loss of rough rice grain yield is added to this cost, the world's total estimated cost for rice weeds and their control amounts to 15% of total annual production [12].

**1. Introduction**

194 Rice Crop - Current Developments

rice production.

for CO2

Rice, one of the most important grains, fulfills the carbohydrate requirement of people in the tropical countries and to a lesser extent in subtemperate areas. The cultivated rice belongs to the grass family Gramineae (Poaceae) under the tribe-Oryzeae of the subfamily Pooideae [1]. However, the genus *Oriza* has been divided into several sections and placed *O. sativa* under Series Sativa in Section Sativae [2]. *O. sativa,* an indigenous rice species in Asia, is a diploid species consisting 24 chromosomes. The genomic formula of *O. sativa* is AA [2]. The species *O. sativa* is an annual grass, with round, hollow, jointed culms, rather flat, sessile leaf blades, and a terminal panicle, under favorable conditions. As the other members in the tribe Oryzeae,

Rice is cultivated on about 156 million hectares of land to produce about 696 million tons annually in Asian countries which account for 90% of the world's total rice production [4]. There is a growing trend of increasing rice consumptions since 2000s, which surpasses the production. On an annual basis, global rice demand keeps increasing by *ca.* 8 million tons implying that during next 10 years, the rice production need to increase to 80 million tons

The increasing world population especially in tropical countries where rice serves as the staple food, one billion people per year demands an additional rice production (100 million MT) [6]. In future, it is apparent that rice production will continue to grow rapidly as increasing populations attempt to secure food supplies. In order to obtain a good yield in rice, farmers are required to overcome several biotic and abiotic stresses. Among biotic stresses, weeds stand out as the major threat to rice cultivation, which reduce the yield qualitatively and quantitatively. Over the past few decades, climate change has induced transformations in the weed flora of arable ecosystems and the changes in the climate have also influenced weeds indirectly by enforcing adaptations to agronomic practice [7]. Therefore, it is imperative to develop effective weed control strategies while maintaining crop yield [8]. Globally, *ca.*10% loss of rice yield is attributed to weed and specific quantity is more or less closer to 46 million tons (based on 1987 world rough rice production). Depending on the predominant weed flora and on the control methods practiced by farmers, loss of yield caused by weeds varies across countries in the world. In Sri Lanka, a country considered self-sufficient in rice, weeds are the major biotic stress in rice production and account for 30–40% of yield losses [9]. Thus, there is a need to take timely and appropriate measures to preserve the country's

Rice weeds are the major barriers to rice production because they possess the ability to compete

and they were categorized as grasses, sedges and broad leaves [11].

, space, moisture, sunlight and nutrients. Under certain conditions, crops fail to successfully compete with weeds [10]. Weed flora varies spatially due to type of rice culture, soil type, hydrology, tillage, cultural practices and irrigation pattern and so on. Approximately, 134 weed species belonging to 32 taxonomic families were identified in rice fields in Sri Lanka,

rice is well-adapted to aquatic and swampy habitats [3].

which is double the present production [5].

Weeds indirectly limit production and act as a host of plant harboring pathogens and pests that adversely affect rice. Furthermore, weeds intervene rice harvesting and increase harvest costs through direct interference with the harvesting operation and by causing lodging. Contamination of rough rice by the seeds of the weeds reduces the grain quality and market value for example weed red rice (*Oryza sativa f. spontanea*) has a pigmented layer that shatters easily and readily contaminates rough rice. Removing all traces of the pigmented layer requires intense milling and results in decreased grain quality and lower milling rates [12]. The drudgery of weeding and labor shortages have made rice farming unattractive. In most tropical countries, farmers spend more time on weeding, by hand or with simple tools, than on any other farming task. Hand weeding of one (01) ha. of rice requires from 100 to 780 laborhours per crop, depending on the rice culture. Due to these adverse effects, there is a need to improve the present weed control practices.

Herbicides are chemical substances used to kill plants which are often placed under the group of chemicals known as pesticides that prevent, destroy, repel, or mitigate any pest [13]. Herbicides, in general, are classified using different criteria such as activity, timing of application, method of application, mechanism of action and chemical family. Based on the time of application there are three main categories of herbicides recommended for rice. "Pre-plant herbicides" are applied before the crop is planted in order to eliminate weeds that have germinated before planting or were left from following (e.g., glyphosate, glufosinate). "Pre-emergence herbicides" are applied after the crop has been planted but before weeds emerge (butachlor, pretilachloroxadiazon, pendimethalin, oxadiargyl) and "Post-emergence herbicides" are applied after weeds have emerged (bisparybacbispyribac, pentagon, 2,4-D). These herbicides are either broad-spectrum (nonselective) or narrowspectrum (selective). Some of the most common modes of actions are auxin mimics, mitosis inhibitors, photosynthesis inhibitors, amino acid synthesis inhibitors and lipid biosynthesis inhibitors.

The usage of pre-emergent, broad spectrum herbicide in controlling weeds in rice cultivation has become a popular method among the farmers since it minimizes cost, labor and time. Glyphosate and glufosinate are the most commonly used broad-spectrum herbicides (BSHs) in rice fields and glyphosate usage is comparatively higher. Glyphosate (*N*-(phosphonomethyl) glycine) or C3 H8 NO5 P is a broad spectrum, nonselective systemic herbicide. It is effective in killing all plant types including grasses, perennials and woody plants. Glyphosate is a versatile herbicide used by farmers, land managers and gardeners to simply, safely and effectively control unwanted vegetation. Initially glyphosate was patented and sold by Monsanto Company in the 1970s under the trade name Roundup and after that glyphosate-based products have become the most commonly used herbicides in the U.S. [14]. This widespread adoption is the result of glyphosate's ability to control a broad spectrum of weeds, its extensive economic and environmental benefits and its strong safety profile. Glyphosate is currently undergoing registration review by the US Environmental Protection Agency (EPA or the Agency) and it is essential that farmers, land managers and gardeners retain access to this important tool for weed control.

selection, where the herbicide is the selection pressure, susceptible plants are killed while

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

http://dx.doi.org/10.5772/intechopen.76991

197

During the last two decades, considerable effort has been made to breed HR crops and it was expected to relieve the constraints imposed by different combinations of chemicals, overcome problems associated with herbicide residues, expand the range of compounds available for selective use in-crop, simplify the crop management and extend the useful life span of the

Rice cultivars resistant to glufosinate [23], sulfonylureas, imidazolinones and glyphosate have already been developed and are being field-tested, mostly in the USA but also in South America and Japan [24–26]. The main reason for developing HR rice is to attain control of weed species that fail to control rice weeds selectively [27]. In addition, introduction of HR rice improves current cropping systems, with more efficient weed control measures and could reduce the amount of land required to satisfy the global rice needs and fulfill the increase in the future demand of rice. Particularly, HR rice provides the farmer with new efficient chemical options for weed control, for instance, glyphosate and glufosinate target both monocotyledonous and dicotyledonous weeds, which probably allow less herbicide use in terms of amount and number of applications. In relation to HR, both herbicides were post-emergence, which means that doses can be adjusted to actual weed infestation, and spraying can be performed within a wider time frame due to their high efficacy and crop tolerance. Therefore, HR could result in adequate control of hard-to-kill weeds. In addition, weed populations already resistant to currently used herbicides could be controlled with these broad-spectrum

Many studies focused on optimization of weed management in HR have been conducted with rice resistant to either glufosinate or imidazolinone. Almost complete control of weedy rice and other grasses, including *Echinochloa crus-galli* (L.) Beauv. was achieved in glufosinate-resistant rice in Arkansas (USA) by sequential applications of glufosinate. Initial studies on weed control in Imidazolinone resistant rice (IMI rice) were conducted with imazethapyr, an herbicide proven effective against weedy rice and other rice weeds when applied as a soil or foliar treatment. Imidazolinone resistant rice varieties carrying an insensitive target acetolactatesynthase (ALS) enzyme, which is the target site of these herbicides, were developed through anther culture and backcrossing without exposure to mutagens or genetic transformation [29]. Further, imidazolinone-tolerant rice variety was engineered through mutation of the rice variety AS3510 with EMS. The resulted M2 plants were sprayed with imazethapyr. A single surviving plant was identified, and the progeny of this rice plant showed tolerance to several AHAS-inhibiting herbicides [30]. This mutant line was referred to as 93AS3510, and subsequently two imidazolinone-tolerant rice varieties, CL121 and CL141 were developed with this tolerance trait and were first marketed in

Even with such achievements, inadequate weed and pest management practices led to creation of a yield gap in the rice production. Literature on the subject revealed that studies have focused on the importance of controlling weeds including hard-to-control *Echinochloa* spp.

herbicide resistant plants survive.

current nonselective herbicides [22].

herbicides [28].

the USA in 2001 [31, 32].

As an herbicide, glyphosate is activated by absorbing into the plant mainly though leaves and also through soft stalk tissue. Subsequently, glyphosate is transported throughout the plant where it acts on various enzyme systems inhibiting amino acid metabolism (shikimic acid pathway). Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate-synthase, the sixth enzyme in the shikimate biosynthetic pathway that produces the essential aromatic amino acids (tryptophan, tyrosine and phenylalanine) and subsequently phenolics, lignins, tannins and other phenylpropanoids [15]. The shikimate pathway is found in all microorganisms and crop plants. This pathway is essential for the biosynthesis of chorismate, the precursor for aromatic amino acids and aromatic secondary metabolites [16] (Priestman *et al*., 2005). Glyphosate is reported to be causing a significant damage to rice yield with a reduction of yield up to 80% by blocking the shikimate pathway of crop plant [17].

Glufosinate is converted within the plant cell into the phytotoxin named as phosphinothricin (PT). As a structural analogue of glutamic acid, PT inhibits glutamine synthatase—GS (E.C.6.3.1.2.), competitively and irreversibly [18, 19]. GS is an essential ammonia assimilation enzyme found in plants. Inhibition of GS causes a rapid, toxic accumulation of intercellular ammonia resulting in metabolic disruption and inhibition of photosystem I and photosystem II in treated plants [18, 19] (Senseman, 2007; Hensley, 2009). Over 40 monocotyledonous and more than 150 dicotyledonous species are sensitive to PT [20].

In relation to herbicide, usage of the terms "tolerance" and "resistance" are inconsistent among the general public and even weed scientists. Among the members of the weed science community, tolerance and resistance are used interchangeably. Further, herbicide manufacturers/seed companies that develop and/or market HR crop cultivars/varieties generally refer to these as herbicide-tolerant. The present study recognizes the definition of herbicide tolerance and resistance established by the Weed Science Society of America (WSSA) [21].

The official Weed Science Society of America [21] defines herbicide resistance as "the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type. In a plant, resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis". Herbicide tolerance is defined by WSSA as "tolerance is the inherent ability of a species to survive and reproduce after herbicide treatment. This implies that there was no selection or genetic manipulation to make the plant tolerant; it is naturally tolerant" [21]. Resistance may occur in plants as the result of random and frequent mutation. Through selection, where the herbicide is the selection pressure, susceptible plants are killed while herbicide resistant plants survive.

Company in the 1970s under the trade name Roundup and after that glyphosate-based products have become the most commonly used herbicides in the U.S. [14]. This widespread adoption is the result of glyphosate's ability to control a broad spectrum of weeds, its extensive economic and environmental benefits and its strong safety profile. Glyphosate is currently undergoing registration review by the US Environmental Protection Agency (EPA or the Agency) and it is essential that farmers, land managers and gardeners retain access to this

As an herbicide, glyphosate is activated by absorbing into the plant mainly though leaves and also through soft stalk tissue. Subsequently, glyphosate is transported throughout the plant where it acts on various enzyme systems inhibiting amino acid metabolism (shikimic acid pathway). Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate-synthase, the sixth enzyme in the shikimate biosynthetic pathway that produces the essential aromatic amino acids (tryptophan, tyrosine and phenylalanine) and subsequently phenolics, lignins, tannins and other phenylpropanoids [15]. The shikimate pathway is found in all microorganisms and crop plants. This pathway is essential for the biosynthesis of chorismate, the precursor for aromatic amino acids and aromatic secondary metabolites [16] (Priestman *et al*., 2005). Glyphosate is reported to be causing a significant damage to rice yield with a reduction of yield up to 80% by blocking the shikimate pathway of crop

Glufosinate is converted within the plant cell into the phytotoxin named as phosphinothricin (PT). As a structural analogue of glutamic acid, PT inhibits glutamine synthatase—GS (E.C.6.3.1.2.), competitively and irreversibly [18, 19]. GS is an essential ammonia assimilation enzyme found in plants. Inhibition of GS causes a rapid, toxic accumulation of intercellular ammonia resulting in metabolic disruption and inhibition of photosystem I and photosystem II in treated plants [18, 19] (Senseman, 2007; Hensley, 2009). Over 40 monocotyledonous and

In relation to herbicide, usage of the terms "tolerance" and "resistance" are inconsistent among the general public and even weed scientists. Among the members of the weed science community, tolerance and resistance are used interchangeably. Further, herbicide manufacturers/seed companies that develop and/or market HR crop cultivars/varieties generally refer to these as herbicide-tolerant. The present study recognizes the definition of herbicide tolerance and resistance established by the Weed Science Society of America

The official Weed Science Society of America [21] defines herbicide resistance as "the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type. In a plant, resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis". Herbicide tolerance is defined by WSSA as "tolerance is the inherent ability of a species to survive and reproduce after herbicide treatment. This implies that there was no selection or genetic manipulation to make the plant tolerant; it is naturally tolerant" [21]. Resistance may occur in plants as the result of random and frequent mutation. Through

more than 150 dicotyledonous species are sensitive to PT [20].

important tool for weed control.

196 Rice Crop - Current Developments

plant [17].

(WSSA) [21].

During the last two decades, considerable effort has been made to breed HR crops and it was expected to relieve the constraints imposed by different combinations of chemicals, overcome problems associated with herbicide residues, expand the range of compounds available for selective use in-crop, simplify the crop management and extend the useful life span of the current nonselective herbicides [22].

Rice cultivars resistant to glufosinate [23], sulfonylureas, imidazolinones and glyphosate have already been developed and are being field-tested, mostly in the USA but also in South America and Japan [24–26]. The main reason for developing HR rice is to attain control of weed species that fail to control rice weeds selectively [27]. In addition, introduction of HR rice improves current cropping systems, with more efficient weed control measures and could reduce the amount of land required to satisfy the global rice needs and fulfill the increase in the future demand of rice. Particularly, HR rice provides the farmer with new efficient chemical options for weed control, for instance, glyphosate and glufosinate target both monocotyledonous and dicotyledonous weeds, which probably allow less herbicide use in terms of amount and number of applications. In relation to HR, both herbicides were post-emergence, which means that doses can be adjusted to actual weed infestation, and spraying can be performed within a wider time frame due to their high efficacy and crop tolerance. Therefore, HR could result in adequate control of hard-to-kill weeds. In addition, weed populations already resistant to currently used herbicides could be controlled with these broad-spectrum herbicides [28].

Many studies focused on optimization of weed management in HR have been conducted with rice resistant to either glufosinate or imidazolinone. Almost complete control of weedy rice and other grasses, including *Echinochloa crus-galli* (L.) Beauv. was achieved in glufosinate-resistant rice in Arkansas (USA) by sequential applications of glufosinate. Initial studies on weed control in Imidazolinone resistant rice (IMI rice) were conducted with imazethapyr, an herbicide proven effective against weedy rice and other rice weeds when applied as a soil or foliar treatment. Imidazolinone resistant rice varieties carrying an insensitive target acetolactatesynthase (ALS) enzyme, which is the target site of these herbicides, were developed through anther culture and backcrossing without exposure to mutagens or genetic transformation [29]. Further, imidazolinone-tolerant rice variety was engineered through mutation of the rice variety AS3510 with EMS. The resulted M2 plants were sprayed with imazethapyr. A single surviving plant was identified, and the progeny of this rice plant showed tolerance to several AHAS-inhibiting herbicides [30]. This mutant line was referred to as 93AS3510, and subsequently two imidazolinone-tolerant rice varieties, CL121 and CL141 were developed with this tolerance trait and were first marketed in the USA in 2001 [31, 32].

Even with such achievements, inadequate weed and pest management practices led to creation of a yield gap in the rice production. Literature on the subject revealed that studies have focused on the importance of controlling weeds including hard-to-control *Echinochloa* spp. and *Eleusine* spp. [8]. In addition, herbicide resistant (HR) conspecific weeds such as weedy rice with varying dormancy patterns have become more abundant in rice fields in Sri Lanka throughout the cropping season. As a result, Sri Lankan farmers tend to use pre- and postemergent herbicides in land preparation specially to control weedy rice. These pre- and postemergent herbicides include selective and nonselective (broad spectrum) herbicides. As far as selective herbicide usage is considered, the number of application and their amount to control common weeds such as: *Cyperus iria* L. (family: Cyperaceae), *Echinochloa* sp. (family: Gramineae), *Monochoria vaginalis* (family: Pontederiaceae) and weedy rice (*Oryza sativa f. spontanea*; family: Poaceae) has been increased considerably leading to sever threats to the rice growing environment [33]. Thus, it is critically important to evaluate the possibility of applying commonly used broad-spectrum herbicides; glyphosate and glufosinate as postemergent herbicides along with herbicide resistant technology to eliminate hard-to-control weeds. Thus, the objective of the present study was to evaluate the herbicide resistance of Sri Lankan traditional and inbred cultivated rice varieties to pre-emergent herbicide—glyphosate and glufosinate.

### **2. Methodology**

Seeds of twenty-five rice (*Oryza sativa* L.) varieties (**Table 1**) were collected from the Rice Research and Development Institute (RRDI) of Sri Lanka. These lines were maintained in a plant house at the Open University of Sri Lanka, located in low country wet zone of the Western province, with an average temperature of 28–32°C and 65–70% relative humidity.

The selected seeds were pre-soaked overnight and allowed to germinate. One week old seedlings were planted in pots (with 23 cm diameter) filled with puddle soil (5.5 kg per pot) and excess plantlets were thinned out 1 week after planting [34] leaving 10 plants per pot. Fertilizer application and other crop management practices were performed according to the recommendations of the Department of Agriculture, Sri Lanka.

Glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) [35] were applied at 3–4 leaf stage (Department of Agriculture, Sri Lanka) of plants separately. The research design used was complete randomized design (CRD) with three pots (10 replicates in each pot) for each treatment and nontreated plants served as the control.

The total number of plants and the number of surviving plants were counted for each variety and percentage resistance (PR) was calculated as follows: plants with ≥50% resistance to herbicides were arbitrary considered as resistant varieties [36].

[bickses were arbitrary considered as resistant varieties [36].

$$\text{PR (\%)} = \left[\frac{\text{Number of survivors seeking in a variety}}{\text{Total number of seedlings grown in the same variety}}\right] \times 100$$

**3. Results and discussion**

Evaluation of natural resistance to glyphosate and glufosinate among rice varieties.

**Selection number Name Age (month) Attributes** 1 Bg94-1 3 ½ High yield WP

3 Bg300 3 Resistant to GM-1, BL, BB, Bph 4 Bg304 3 Resistant to GM 1&2, BL, BB, Bph 5 Bg305 3 Resistant to GM-1 and 11, BPH, BL

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

 Bg352 3 ½ Resistant to BL, BB & GM-1, Bph Bg357 3 ½ Resistant to GM-1& 2, BL, BB, Bph Bg359 Resistant to GM 1 & 2, BL, BB, Bph Bg360 3 ½ Resistant to GM-1, GM-2, BL, Bph

12 Ld3 65 3 ½ Resistant to iron toxicity

15 Bg379-2 4 ½ Resistant to Bph and BB 16 Bg403 4 Resistant to BB, BL and Bph

20 *Kaluheenati* 4 Moderately tolerant Gr. 2

17 Bg450 4 ½ Resistant GM-I

19 H4 4 Resistant to BL

Source: Jeyawardena *et al.*, 2010, RRDI, Batalagoda, Department of Agriculture, Sri Lanka

and BLB

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199

2 Bg250 2 ½

10 At362 3 ½ 11 Bw364 3 ½

13 Bg366 3 ½ 14 Bg369 3 ½

18 Bg454 4 ½

 *Kuruluthuda* 4 *Suwadal* 5 *Rathhal* 5 24 *Madel* 5 *Pachchaperumal* 3 ½

Bph: brown plant hopper; PS: photo period sensitivity.

The results obtained from the screening for glufosinate resistant and glyphosate resistant varieties revealed that some of the selected traditional rice varieties and inbred lines possess

BB: bacterial leaf blight; BL: rice blast disease; GM-1: biotype one of rice gall midge; GM-2: biotype two of rice gall midge;

**Table 1.** List of chosen Sri Lankan rice varieties form the results of a previous for the study on natural herbicide resistance.

Agro-morphological characters of resistant plant were measured/evaluated in 2 weeks after sawing and the yield parameters, respectively, by application of respective herbicide.

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides… http://dx.doi.org/10.5772/intechopen.76991 199


BB: bacterial leaf blight; BL: rice blast disease; GM-1: biotype one of rice gall midge; GM-2: biotype two of rice gall midge; Bph: brown plant hopper; PS: photo period sensitivity.

Source: Jeyawardena *et al.*, 2010, RRDI, Batalagoda, Department of Agriculture, Sri Lanka

**Table 1.** List of chosen Sri Lankan rice varieties form the results of a previous for the study on natural herbicide resistance.

### **3. Results and discussion**

and *Eleusine* spp. [8]. In addition, herbicide resistant (HR) conspecific weeds such as weedy rice with varying dormancy patterns have become more abundant in rice fields in Sri Lanka throughout the cropping season. As a result, Sri Lankan farmers tend to use pre- and postemergent herbicides in land preparation specially to control weedy rice. These pre- and postemergent herbicides include selective and nonselective (broad spectrum) herbicides. As far as selective herbicide usage is considered, the number of application and their amount to control common weeds such as: *Cyperus iria* L. (family: Cyperaceae), *Echinochloa* sp. (family: Gramineae), *Monochoria vaginalis* (family: Pontederiaceae) and weedy rice (*Oryza sativa f. spontanea*; family: Poaceae) has been increased considerably leading to sever threats to the rice growing environment [33]. Thus, it is critically important to evaluate the possibility of applying commonly used broad-spectrum herbicides; glyphosate and glufosinate as postemergent herbicides along with herbicide resistant technology to eliminate hard-to-control weeds. Thus, the objective of the present study was to evaluate the herbicide resistance of Sri Lankan traditional and inbred cultivated rice varieties to pre-emergent herbicide—glyphosate

Seeds of twenty-five rice (*Oryza sativa* L.) varieties (**Table 1**) were collected from the Rice Research and Development Institute (RRDI) of Sri Lanka. These lines were maintained in a plant house at the Open University of Sri Lanka, located in low country wet zone of the Western province, with an average temperature of 28–32°C and 65–70% relative

The selected seeds were pre-soaked overnight and allowed to germinate. One week old seedlings were planted in pots (with 23 cm diameter) filled with puddle soil (5.5 kg per pot) and excess plantlets were thinned out 1 week after planting [34] leaving 10 plants per pot. Fertilizer application and other crop management practices were performed according to the

Glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) [35] were applied at 3–4 leaf stage (Department of Agriculture, Sri Lanka) of plants separately. The research design used was complete randomized design (CRD) with three pots (10 replicates in each pot) for each treatment and

The total number of plants and the number of surviving plants were counted for each variety and percentage resistance (PR) was calculated as follows: plants with ≥50% resistance to her-

Agro-morphological characters of resistant plant were measured/evaluated in 2 weeks after

sawing and the yield parameters, respectively, by application of respective herbicide.

recommendations of the Department of Agriculture, Sri Lanka.

bicides were arbitrary considered as resistant varieties [36].

*PR* (%) <sup>=</sup> [ *Number of survivng seedlings in <sup>a</sup> variety* \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Total number of seedlings grown in the same variety*] <sup>×</sup> <sup>100</sup>

nontreated plants served as the control.

and glufosinate.

humidity.

**2. Methodology**

198 Rice Crop - Current Developments

Evaluation of natural resistance to glyphosate and glufosinate among rice varieties.

The results obtained from the screening for glufosinate resistant and glyphosate resistant varieties revealed that some of the selected traditional rice varieties and inbred lines possess the ability to resist the detrimental effects of those broad-spectrum herbicides (**Figure 1**). Two rice varieties (*Rathal*—2% and Bg305—1%) were found to be lethal to 0.05 gl−1 glufosinate concentration whereas no such varieties were observed under the application of 0.5 gl−1 glyphosate. Fifteen rice varieties (At362—90%, Bg250—83%, Bg300—96%, Bg352— 100%, Bg357—53%, Bg359—100%, Bg360—96%, Bg366—73%, Bg369—83%, Bg379/2—93%, Bg403—100%, Bg450—57%, Bg454—97%, Bg94/1—73%, *Pachchaperumal*—53%) showed natural resistance under glufosinate application and 12 rice varieties (At362—75%, Bg352— 50%, Bg359—55%, Bg366—65%, Bg369—60%, Bg379/2—65%, Bg403—60%, Bg454—55%, Ld365—70%, *Kaluheenati*—55%, *Kuruluthuda*—55%, *Pachchaperumal*—70%) were able to survive under glyphosate application. Results indicated that nine varieties (At362, Bg352, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454 and *Pachchaperumal*) were resistant for both glyphosate and glufosinate (**Figure 1**).

On the other hand, according to the results of the study, relatively high survival percentage toward both BSHs was reported by inbred rice varieties which possess many valuable attributes other than glyphosate- and glufosinate-resistance such as resistant to GM-1, GM-2, BL, BB, Bph and have high yield potential (**Table 1**). These rice varieties could be incorporated in

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201

Comparison table of plant height and yield parameters of glyphosate treated and untreated rice varieties are shown in **Table 2**. Though rice plant showed considerable HR in general, growth

**Rice variety Plant height (cm) 1000-grain weight (g) Yield/plant (g)**

At362 66.33 (1.20) 25.00 (0.44) 23.43 (2.69) Bg359 62.00 (2.08) 22.46 (0.32) 12.38 (0.46) Bg366 46.67 (2.73) 23.17 (0.28) 5.34 (0.60)

Bg379-2 64.00 (0.58) 25.67 (0.44) 5.22 (0.70) Bg403 67.00 (1.15) 21.39 (0.38) 15.00 (1.09)

Bw364 58.67 (2.33) 19.23 (2.25) 12.51 (2.65) Ld365 59.00 (2.08) 13.26 (0.29) 4.04 (0.77) *Kaluheenati* 73.33 (0.88) 22.89 (1.51) 4.69 (0.75)

*Pachchaperumal* 70.33 (1.20) 31.64 (0.38) 11.31 (1.02)

At362 50.33 (0.88) 16.86 (0.32) 12.61 (3.25) Bg359 52.00 (3.06) 16.37 (0.29) 5.38 (0.94) Bg366 27.67 (3.67) 18.49 (0.90) 3.19 (0.36)

Bg379-2 48.67 (3.48) 15.27 (1.41) 1.84 (0.40) Bg403 49.00 (2.65) 17.56 (0.38) 6.50 (1.45)

Ld365 48.00 (2.00) 9.85 (0.20) 1.90 (0.38) *Kaluheenati* 59.83 (3.68) 14.37 (0.59) 3.11 (0.50)

*Pachchaperumal* 61.00 (3.12) 22.94 (1.34) 3.29 (0.37)

**Table 2.** Summary of the parametric variables; plant height, 1000-grain weight and yield per plant control and treated

rice breeding programs to strengthen the sustainable cultivation.

**Control**

**Treated**

Bg369 34.33 (1.67)

Bg454 52.00 (2.08)

*Kuruluthuda* 70.00 (1.15)

Bg369 48.67 (2.33)

Bg454 45.33 (1.45)

*Kuruluthuda* 64.33 (2.33)

with glyphosate (0.5 g/l).

Very limited studies have been conducted regarding the natural or induced herbicide resistance in Sri Lankan rice varieties [36] and findings of Sri Lankan rice varieties which are able to resist broad-spectrum herbicides (BSHs) such as glufosinate and glyphosate have hardly been recorded. In this study, nine rice varieties with the ability to resistant the application of concentrations, 0.05 gl−1glufosinate and 0.5 gl−1 glyphosate have been identified. Among these varieties, only two red grain rice varieties *(Pachchaperumal* and At362) are included indicating that most of the cultivated traditional rice varieties, except *Pachchaperumal* did not possess the ability to resist both glufosinate and glyphosate as observed in inbred rice varieties. However, further studies are required to confirm such findings. Sri Lankans do admire red grain rice such as *Kuruluthud*a, *Kaluheenati* and *Rathal* due to their high nutritive qualities (**Table 1**), and it is important to note that such varieties need to be developed as BSHs resistant varieties in future.

**Figure 1.** Comparison between natural resistances in selected rice (*Oryza sativa* L.) varieties to glyphosate and glufosinate.

On the other hand, according to the results of the study, relatively high survival percentage toward both BSHs was reported by inbred rice varieties which possess many valuable attributes other than glyphosate- and glufosinate-resistance such as resistant to GM-1, GM-2, BL, BB, Bph and have high yield potential (**Table 1**). These rice varieties could be incorporated in rice breeding programs to strengthen the sustainable cultivation.

the ability to resist the detrimental effects of those broad-spectrum herbicides (**Figure 1**). Two rice varieties (*Rathal*—2% and Bg305—1%) were found to be lethal to 0.05 gl−1 glufosinate concentration whereas no such varieties were observed under the application of 0.5 gl−1 glyphosate. Fifteen rice varieties (At362—90%, Bg250—83%, Bg300—96%, Bg352— 100%, Bg357—53%, Bg359—100%, Bg360—96%, Bg366—73%, Bg369—83%, Bg379/2—93%, Bg403—100%, Bg450—57%, Bg454—97%, Bg94/1—73%, *Pachchaperumal*—53%) showed natural resistance under glufosinate application and 12 rice varieties (At362—75%, Bg352— 50%, Bg359—55%, Bg366—65%, Bg369—60%, Bg379/2—65%, Bg403—60%, Bg454—55%, Ld365—70%, *Kaluheenati*—55%, *Kuruluthuda*—55%, *Pachchaperumal*—70%) were able to survive under glyphosate application. Results indicated that nine varieties (At362, Bg352, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454 and *Pachchaperumal*) were resistant for both glyphosate

Very limited studies have been conducted regarding the natural or induced herbicide resistance in Sri Lankan rice varieties [36] and findings of Sri Lankan rice varieties which are able to resist broad-spectrum herbicides (BSHs) such as glufosinate and glyphosate have hardly been recorded. In this study, nine rice varieties with the ability to resistant the application of concentrations, 0.05 gl−1glufosinate and 0.5 gl−1 glyphosate have been identified. Among these varieties, only two red grain rice varieties *(Pachchaperumal* and At362) are included indicating that most of the cultivated traditional rice varieties, except *Pachchaperumal* did not possess the ability to resist both glufosinate and glyphosate as observed in inbred rice varieties. However, further studies are required to confirm such findings. Sri Lankans do admire red grain rice such as *Kuruluthud*a, *Kaluheenati* and *Rathal* due to their high nutritive qualities (**Table 1**), and it is important to note that such varieties need to be developed as BSHs resistant varieties in future.

**Figure 1.** Comparison between natural resistances in selected rice (*Oryza sativa* L.) varieties to glyphosate and glufosinate.

and glufosinate (**Figure 1**).

200 Rice Crop - Current Developments

Comparison table of plant height and yield parameters of glyphosate treated and untreated rice varieties are shown in **Table 2**. Though rice plant showed considerable HR in general, growth


**Table 2.** Summary of the parametric variables; plant height, 1000-grain weight and yield per plant control and treated with glyphosate (0.5 g/l).

retardation is indicated by the decrease in plant height resulting stunting of glyphosate treated plants. Similarly, the yield parameters such as 1000-grain weight yield per plant also showed apparent decrease in treated plants. However, yield per plant of the treated At362 indicated comparatively less reduction of yield. These findings led to conclude that though most of the rice varieties included in the study were resistant to the glyphosate, there was a considerable yield penalty. Similarly, the response of rice varieties included in the study to glufosinate are summarized in **Table 3** and according to the table, a general trend of decreasing plant height that is stunting growth and yield parameters specially yield per plant was observed. Comparatively, glufosinate treated At362 variety indicated low reduction in yield per plant (**Table 3**).

The nonparametric variables of treated and untreated rice varieties with glyphosate and glufosinate are shown in **Tables 4** and **5**, respectively. According to the tables, it is evident that there was no discernible different in growth parameters; however, yield parameters were

**Number of leaves/**

**Number of panicles/**

**Number of seeds/**

203

**panicle**

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**plant**

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

**plant**

At362 1 1 2 15 Bg359 0 3 1 14 Bg366 1 2 1 10

Bg379 1 2 1 10 Bg403 1 5 2 20

Bw364 1 1 1 15 Ld365 1 5 2 20 *Kaluheenati* 1 3 2 6

*Pachchaperumal* 1 3 1 15

At362 0 2 3 34 Bg359 1 4 2 10 Bg366 2 1 1 5

Bg379 1 4 1 9 Bg403 1 3 3 20

Bw364 1 1 2 22 Ld365 0 2 1 15 *Kaluheenati* 0 1 1 11

*Pachchaperumal* 0 2 2 10

panicles per plant and number of seeds per panicle of control and treated with glyphosate (0.5 gl−1).

**Table 4.** Summary of nonparametric variables; number of tillers per plant, number of leaves per plant, number of

considerably varied between the herbicide treated plants.

**Rice variety Number of tillers/**

Bg369 1 2

Bg454 1 3

*Kuruluthuda* 1 3

Bg369 1 2

Bg454 1 2

*Kuruluthuda* 1 4

**Control**

**Treated**

**plant**


**Table 3.** Summary of the parametric variables; plant height, 1000-grain weight and yield per plant of control and treated with glufosinate (0.05 gl−1).

The nonparametric variables of treated and untreated rice varieties with glyphosate and glufosinate are shown in **Tables 4** and **5**, respectively. According to the tables, it is evident that there was no discernible different in growth parameters; however, yield parameters were considerably varied between the herbicide treated plants.

**Rice variety Plant height (cm) 1000-grain weight (g) Yield/plant (g)**

glufosinate treated At362 variety indicated low reduction in yield per plant (**Table 3**).

retardation is indicated by the decrease in plant height resulting stunting of glyphosate treated plants. Similarly, the yield parameters such as 1000-grain weight yield per plant also showed apparent decrease in treated plants. However, yield per plant of the treated At362 indicated comparatively less reduction of yield. These findings led to conclude that though most of the rice varieties included in the study were resistant to the glyphosate, there was a considerable yield penalty. Similarly, the response of rice varieties included in the study to glufosinate are summarized in **Table 3** and according to the table, a general trend of decreasing plant height that is stunting growth and yield parameters specially yield per plant was observed. Comparatively,

At362 66.33 (1.20) 25.00 (0.44) 84.57 (0.59) Bg359 62.00 (2.08) 22.46 (0.32) 75.37 (0.64) Bg366 46.67 (2.73) 23.17 (0.28) 72.27 (0.50)

Bg379-2 64.00 (0.58) 25.67 (0.44) 68.70 (0.29) Bg403 67.00 (1.15) 21.39 (0.38) 82.43 (0.67)

Bw364 62.33 (1.45) 24.21 (0.42) 85.17 (0.49) Ld365 59.00 (2.08) 13.26 (0.29) 81.27 (0.50) *Kaluheenati* 73.33 (0.88) 22.89 (1.51) 70.93 (0.54)

*Pachchapermal* 70.33 (1.20) 31.64 (0.38) 80.23 (1.13

At362 50.33 (0.88) 16.86 (0.32) 82.87 (0.24) Bg359 52.00 (3.06) 16.37 (0.29) 69.60 (0.38) Bg366 27.67 (3.67) 18.49 (0.90) 64.27 (0.62)

Bg379-2 48.67 (3.48) 15.27 (1.41) 65.20 (0.59) Bg403 49.00 (2.65) 17.56 (0.38) 80.57 (0.46)

Bw364 55.00 (3.40) 14.26 (0.55) 70.33 (2.50) Ld365 48.00 (2.00) 9.85 (0.20) 74.97 (2.98 *Kaluheenati* 59.83 (3.68) 14.37 (0.59) 68.50 (0.36)

*Pachchaperumal* 61.00 (3.12) 22.94 (1.34) 70.63 (0.30

**Table 3.** Summary of the parametric variables; plant height, 1000-grain weight and yield per plant of control and treated

**Control**

202 Rice Crop - Current Developments

**Treated**

Bg369 34.33 (1.67)

Bg454 52.00 (2.08)

*Kuruluthuda* 70.00 (1.15)

Bg369 48.67 (2.33)

Bg454 45.33 (1.45)

*Kuruluthuda* 64.33 (2.33)

with glufosinate (0.05 gl−1).


**Table 4.** Summary of nonparametric variables; number of tillers per plant, number of leaves per plant, number of panicles per plant and number of seeds per panicle of control and treated with glyphosate (0.5 gl−1).


and leaf length of Bg352 at 0.05 gl−1of glufosinate application showed no significant difference (p > 0.05) (data not given). In addition, the control plants and the plants resistant to 0.05 gl−1glufosinate, number of leaves per plant and number of tillers per plant (**Table 5**) were

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Analysis of the variance of yield parameters indicated no significant difference for number of the seeds per panicle at 0.05 gl−1 glufosinate application except Bg454, Bg369 and *Kuruluthuda*. Almost all varieties indicated significant differences (p ≤ 0.05) for flag leaf length, flag leaf width at0.05 gl−1glufosinate application predicting the possibility of glufosinate (at 0.05 gl−1) to cause reduction in flag leaf quality even when applied at 3–4 leaf stage of the plant (data not given). Varieties such as Bg360, Bg357, Bg369, Bg379-2, Bg450, Bg403, Bg250 and Bg 454 reported insignificant differences for thousand seed weight character at 0.05 gl−1. Significant yield reduction was observed for Bg362, Bg359, Bg94-1, Bg358, Bg300 and At362 at 0.05 gl−1.

After application of 0.05 gl−1 concentration of glufosinate, injuries were identified (**Figure 2**) as rapid chlorosis (**Figure 2B**) of treated leaves followed by wilting (**Figure 2D**), necrosis (**Figure 2C**) and ultimate death of susceptible plants. Similar symptoms have been reported for different rice varieties [19, 34, 35] and for wheat [37] (Deeds *et al.,* 2006). In addition, brown color lesions (**Figure 2A**) were also observed on leaves, and browning of leaf tips (**Figure 2E**) commonly occurred on all varieties. The injuries were significantly higher after 1 week from herbicide application. Severe chlorosis was observed in rice leaves depending on the susceptibility of the varieties within 3–6 days after herbicide treatment. Within 2 weeks after herbicide application, the observable symptoms were disappeared even in the varieties which were exposed to the highest concentration of glufosinate. Previous studies have been shown that rupture and contortion of inter-venal mesophyll cells with concomitant disorganization of bundle sheath

Comparatively, the glyphosate treated rice plants indicated that all the yield parameters (number of panicle/plant, number of seeds/panicle and 1000 grain weight) were significant

After application of 0.5 gl−1 glyphosate concentration, a number of visual injuries were observed in individuals of varieties (**Figure 3**). The injuries were promptly observable after 1 week of herbicide application. Among these injuries, general chlorosis in the upper part of the leaves was most abundant. Comparatively, severe chlorosis was observed in rice leaves that depend on the resistance of the varieties within 3–6 days after herbicide treatment. In susceptible varieties, leaf wilting leads to plant death. Newly emerged leaves of survived varieties remained in green color; however, the young emerging leaves which were subjected to treatment were often tightly curled inwardly. Multiple shoots arising from internodes of main stem (**Figure 3A**) were observed, and the secondary shoots and flag leaves were wrinkled or curled in *Kaluheenati* and *Pachchaperumal*. At the booting stage, all the leaves of the variety *Kuruluthuda* were curled and leaf discoloration had occurred. The plants remained in the same stage and indicated no maturity until the harvesting stage (**Figure 3B**). Malformation of inflorescences was also observed in certain varieties at the reproductive stage. The inflorescence of Bg369 and Bg454 was found aborted inside the flag leaf sheath and unable to emerge as a panicle (**Figure 3C**). Meanwhile, panicles of certain varieties were yet to appear in full due

not statistically significant.

cells herbicide treated plants [38, 39].

differ from the controls (**Tables 2** and **4**).

**Table 5.** Summary of nonparametric variables; number of tillers per plant, number of leaves per plant, number of panicles per plant and number of seeds per panicle of control and treated with glufosinate (0.05 gl−1).

#### **3.1. Effect of glufosinate and glyphosate on agro-morphological characters of HR resistant rice varieties**

The results of the study suggest that several specific growth parameters of certain glufosinate-resistant varieties at 0.05 gl−1showed no significant difference (p > 0.05) compared to control plants. For instance, plant height of Bg379-2 (**Table 3**), leaf blade width of Bg366, and leaf length of Bg352 at 0.05 gl−1of glufosinate application showed no significant difference (p > 0.05) (data not given). In addition, the control plants and the plants resistant to 0.05 gl−1glufosinate, number of leaves per plant and number of tillers per plant (**Table 5**) were not statistically significant.

Analysis of the variance of yield parameters indicated no significant difference for number of the seeds per panicle at 0.05 gl−1 glufosinate application except Bg454, Bg369 and *Kuruluthuda*. Almost all varieties indicated significant differences (p ≤ 0.05) for flag leaf length, flag leaf width at0.05 gl−1glufosinate application predicting the possibility of glufosinate (at 0.05 gl−1) to cause reduction in flag leaf quality even when applied at 3–4 leaf stage of the plant (data not given). Varieties such as Bg360, Bg357, Bg369, Bg379-2, Bg450, Bg403, Bg250 and Bg 454 reported insignificant differences for thousand seed weight character at 0.05 gl−1. Significant yield reduction was observed for Bg362, Bg359, Bg94-1, Bg358, Bg300 and At362 at 0.05 gl−1.

After application of 0.05 gl−1 concentration of glufosinate, injuries were identified (**Figure 2**) as rapid chlorosis (**Figure 2B**) of treated leaves followed by wilting (**Figure 2D**), necrosis (**Figure 2C**) and ultimate death of susceptible plants. Similar symptoms have been reported for different rice varieties [19, 34, 35] and for wheat [37] (Deeds *et al.,* 2006). In addition, brown color lesions (**Figure 2A**) were also observed on leaves, and browning of leaf tips (**Figure 2E**) commonly occurred on all varieties. The injuries were significantly higher after 1 week from herbicide application. Severe chlorosis was observed in rice leaves depending on the susceptibility of the varieties within 3–6 days after herbicide treatment. Within 2 weeks after herbicide application, the observable symptoms were disappeared even in the varieties which were exposed to the highest concentration of glufosinate. Previous studies have been shown that rupture and contortion of inter-venal mesophyll cells with concomitant disorganization of bundle sheath cells herbicide treated plants [38, 39].

Comparatively, the glyphosate treated rice plants indicated that all the yield parameters (number of panicle/plant, number of seeds/panicle and 1000 grain weight) were significant differ from the controls (**Tables 2** and **4**).

After application of 0.5 gl−1 glyphosate concentration, a number of visual injuries were observed in individuals of varieties (**Figure 3**). The injuries were promptly observable after 1 week of herbicide application. Among these injuries, general chlorosis in the upper part of the leaves was most abundant. Comparatively, severe chlorosis was observed in rice leaves that depend on the resistance of the varieties within 3–6 days after herbicide treatment. In susceptible varieties, leaf wilting leads to plant death. Newly emerged leaves of survived varieties remained in green color; however, the young emerging leaves which were subjected to treatment were often tightly curled inwardly. Multiple shoots arising from internodes of main stem (**Figure 3A**) were observed, and the secondary shoots and flag leaves were wrinkled or curled in *Kaluheenati* and *Pachchaperumal*. At the booting stage, all the leaves of the variety *Kuruluthuda* were curled and leaf discoloration had occurred. The plants remained in the same stage and indicated no maturity until the harvesting stage (**Figure 3B**). Malformation of inflorescences was also observed in certain varieties at the reproductive stage. The inflorescence of Bg369 and Bg454 was found aborted inside the flag leaf sheath and unable to emerge as a panicle (**Figure 3C**). Meanwhile, panicles of certain varieties were yet to appear in full due

**3.1. Effect of glufosinate and glyphosate on agro-morphological characters of HR** 

panicles per plant and number of seeds per panicle of control and treated with glufosinate (0.05 gl−1).

The results of the study suggest that several specific growth parameters of certain glufosinate-resistant varieties at 0.05 gl−1showed no significant difference (p > 0.05) compared to control plants. For instance, plant height of Bg379-2 (**Table 3**), leaf blade width of Bg366,

**Table 5.** Summary of nonparametric variables; number of tillers per plant, number of leaves per plant, number of

**resistant rice varieties**

**Rice variety Number of tillers/**

204 Rice Crop - Current Developments

Bg369 1 2

Bg454 1 3

*Kuruluthuda* 1 3

Bg369 1 2

Bg454 1 2

*Kuruluthuda* 1 4

**Control**

**Treated**

**plant**

**Number of leaves/**

**Number of panicles**

**Number of seeds/**

**panicle**

**plant**

At362 1 1 2 15 Bg359 0 3 1 14 Bg366 1 2 1 10

Bg379 1 2 1 10 Bg403 1 5 2 20

Bw364 1 1 1 15 *Kaluheenati* 1 3 2 6 Ld365 1 5 2 20

*Pachchaperumal* 1 3 1 15

At362 0 2 3 34 Bg359 1 4 2 10 Bg366 2 1 1 5

Bg379 1 4 1 9 Bg403 1 3 3 20

Bw364 1 1 2 22 *Kaluheenati* 0 1 1 11

Ld365 0 2 1 15 *Pachchaperumal* 0 2 2 10

**Figure 2.** Visual injuries caused by glufosinate: (A) brown color lesions on leaf blade, (B) severe chlorosis on leaf blade after glufosinate treatment, (C) necrotic areas of leaf blade, (D) wilting of susceptible plants, and (E) browning of leaf tips.

to the fusion of the flag leaf at the maturity stage. Malformation of inflorescence and developing grains with only bleached lemma and palea were commonly found in Bg366 (**Figure 3D**).

**Figure 3.** Visual injuries caused by glyphosate: (A) multiple shoots and roots that sprouted from the internodes, (B) leaf

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

http://dx.doi.org/10.5772/intechopen.76991

207

The rice varieties such as At362, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454 and *Pachchaperumal* were resistant to both glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) applications. Even though the herbicide resistant varieties emerged from the screening, the responses of agro-morphological and yield characters varied across the type of herbicide and the variety. Glyphosate substantially reduced the growth parameters as well as yield compared to glufosinate treated varieties. As

In the variety Bg379-2, distorted and crescent-shape spikelet were observed.

curling and discoloration, (C) fused panicle to flag leaf, and (D) bleached lemma and Palea.

**4. Conclusions**

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides… http://dx.doi.org/10.5772/intechopen.76991 207

**Figure 3.** Visual injuries caused by glyphosate: (A) multiple shoots and roots that sprouted from the internodes, (B) leaf curling and discoloration, (C) fused panicle to flag leaf, and (D) bleached lemma and Palea.

to the fusion of the flag leaf at the maturity stage. Malformation of inflorescence and developing grains with only bleached lemma and palea were commonly found in Bg366 (**Figure 3D**). In the variety Bg379-2, distorted and crescent-shape spikelet were observed.

### **4. Conclusions**

**Figure 2.** Visual injuries caused by glufosinate: (A) brown color lesions on leaf blade, (B) severe chlorosis on leaf blade after glufosinate treatment, (C) necrotic areas of leaf blade, (D) wilting of susceptible plants, and (E) browning of leaf

tips.

206 Rice Crop - Current Developments

The rice varieties such as At362, Bg359, Bg366, Bg369, Bg379-2, Bg403, Bg454 and *Pachchaperumal* were resistant to both glyphosate (0.5 gl−1) and glufosinate (0.05 gl−1) applications. Even though the herbicide resistant varieties emerged from the screening, the responses of agro-morphological and yield characters varied across the type of herbicide and the variety. Glyphosate substantially reduced the growth parameters as well as yield compared to glufosinate treated varieties. As far as yield is concerned, there was a significant yield penalty in HR rice varieties. These broadspectrum HR rice varieties have a higher potential to be utilized in rice breeding programs to breed new HR varieties and can be used to develop HR rice in future.

[8] Peters K, Breitsameter L, Gerowitt B. Impact of climate change on weeds in agriculture:

Natural Resistance of Sri Lankan Rice (*Oryza sativa* L.) Varieties to Broad-Spectrum Herbicides…

http://dx.doi.org/10.5772/intechopen.76991

209

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[19] Senseman SA. Herbicide Handbook. 9th ed. Lawrence, K.S.: Weed Science Society of

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### **Acknowledgements**

The research grants provided by the National Research Council, Sri Lanka (Grant No. NRC 12-037) and the Faculty of Natural Sciences, The Open University of Sri Lanka are greatly acknowledged. The assistance provided by Ms. Deshani Lakshika is highly appreciated.

### **Author details**

Shyama R. Weerakoon\*, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and Sachithri Munasighe

\*Address all correspondence to: shyamaweerakoon@gmail.com

Department of Botany, The Open University of Sri Lanka, Nawala, Nugegoda, Sri Lanka

### **References**


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far as yield is concerned, there was a significant yield penalty in HR rice varieties. These broadspectrum HR rice varieties have a higher potential to be utilized in rice breeding programs to

The research grants provided by the National Research Council, Sri Lanka (Grant No. NRC 12-037) and the Faculty of Natural Sciences, The Open University of Sri Lanka are greatly acknowledged. The assistance provided by Ms. Deshani Lakshika is highly appreciated.

Shyama R. Weerakoon\*, Seneviratnage Somaratne, E. M. Sachini I. Ekanayaka and

Department of Botany, The Open University of Sri Lanka, Nawala, Nugegoda, Sri Lanka

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Int. Rice Res. No. 36. Los Baños, The Philippine: Int Rice Res. Inst; 1965

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208 Rice Crop - Current Developments

**Author details**

Sachithri Munasighe

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[24] Datta SK, Datta K, Soltanifar N, Donn G, Potrykus I. Herbicide resistant indica rice plants from IRRI breeding line ER72 after PEG-mediated transformation of protoplasts. Plant Molecular Biology. 1992;**20**:619-629

**Chapter 12**

)

is the meta-

O) and chlorofluorocarbon (CFC).

**Provisional chapter**

**Methanogens Harboring in Rice Rhizosphere Reduce**

Submerged rice paddy soils are one of the major anthropogenic sources of methane (CH4

carbon dioxide. Methanogens are strictly anaerobic microorganisms and CH4

application of EDTA at suitable rate in the soil of submerged rice field.

), methane (CH4

emission to the atmosphere. Methane is the second most important greenhouse gas after

bolic end product of those methanogens. Methane is produced by methanogens through multi-step enzyme-mediated process. Methanogens convert labile organic carbon com-

determined by quantifying biomarkers namely methyl coenzyme M reductase A (mcrA) gene and coenzyme M (2-mercaptoethane sulphonate) in soil. Nickel ions are present as cofactor in enzymes involved in methanogenesis. Methane emission can be mitigated by

**Keywords:** methane emission, methanogens, biomarkers, EDTA application, rice paddy

In the era of development and globalization, emissions of greenhouse gases (GHGs) are unavoidable consequences, and that increases atmospheric temperature causing global warming. A greenhouse gas is a substrate in the atmosphere that absorbs and emits radiation within the thermal range. This process is the fundamental cause of the greenhouse effect and global warming [1]. Without GHGs, the average temperature of earth's surface would be about −18°C (0°F) [2], rather than present average of 15°C (59°F) [3]. The primary GHGs in the earth's atmosphere are water

and application of organic matter in submerged rice field significantly

emission from soil to the atmosphere. The rate of methanogenesis may be

**Methanogens Harboring in Rice Rhizosphere Reduce** 

DOI: 10.5772/intechopen.73299

© 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,

© 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,

), nitrous oxide (N2

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

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

**Labile Organic Carbon Compounds to Produce**

**Labile Organic Carbon Compounds to Produce** 

**Methane Gas**

**Abstract**

pounds in CH4

increased CH4

soil

**1. Introduction**

vapor, carbon dioxide (CO2

**Methane Gas**

Prabhat Pramanik and Pil Joo Kim

Prabhat Pramanik and Pil Joo Kim

http://dx.doi.org/10.5772/intechopen.73299

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


**Provisional chapter**

### **Methanogens Harboring in Rice Rhizosphere Reduce Labile Organic Carbon Compounds to Produce Methane Gas Labile Organic Carbon Compounds to Produce Methane Gas**

**Methanogens Harboring in Rice Rhizosphere Reduce** 

DOI: 10.5772/intechopen.73299

Prabhat Pramanik and Pil Joo Kim Prabhat Pramanik and Pil Joo Kim 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.73299

#### **Abstract**

[24] Datta SK, Datta K, Soltanifar N, Donn G, Potrykus I. Herbicide resistant indica rice plants from IRRI breeding line ER72 after PEG-mediated transformation of protoplasts.

[28] Gealy DR, Dilday RH. Biology of Red Rice (*Oyrza sativa* L.) Accessions and their Susceptibility to Glufosinate and Other Herbicides. Lawrence, Kansas: Weed Science

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[31] Croughan TP. U.S. Patent No. 5,736,629. Washington, DC: U.S. Patent and Trademark

[32] Webster EP, Masson JA. Acetolactate synthase-inhibiting herbicides on imidazolinone-

[33] Gealy DR, Mitten DH, Rutger JN. Gene flow between red rice (*Oryza sativa*) and herbicide-resistant rice (*O. sativa*): Implications for weed management. Weed Technology.

[34] Olofsdotter M, Valverde B, Madsen-Hauge K. Herbicide resistant rice (*Oryza sativa* L.) —A threat or a solution. In: FAO Report of the Global Workshop on Red Rice Control; 30

[35] Ellis JM, Griffin JL, Linscombe SD, Webster EP. Rice (*Oryza sativa*) and corn (*Zea mays*) response to simulated drift of glyphosate and glufosinate. Weed Technology.

[36] Davis B, Scott RC, Norsworthy JK, Gbur E. Response of rice (*Oryza sativa*) to low rates of

[37] Weerakoon SR, Somaratne S, Wijeratne RGD, Ekanayaka EMSI. Natural herbicide resistance (HR) to broad-spectrum herbicide, glyphosate among traditional and inbred-cultivated rice (*Oryza sativa* L.) varieties in Sri Lanka. Pakistan Journal of Biological Sciences. 2013;**16**

[38] Deeds ZA, Al-Khatib K, Peterson DE, Stahlman PW. Wheat response to simulated drift of glyphosate and imazamox applied at two growth stages. Weed Technology.

[39] Bellinder RR, Lyons RE, Scheckler SE, Wilson HP. Cellular alterations resulting from

Plant Molecular Biology. 1992;**20**:619-629

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Society of America, Allen Press; 1997. p. 34

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Office; 1998

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2003;**17**:627-645

2003;**17**:452-460

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2006;**20**:23-31

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Economic, Regulatory, and Technical Aspects. 1996. pp. 213-250

resistant rice. Louisiana Agriculture. 1994;**37**(3):25-26

August-3 September; Varadero, Cuba. 1999. pp. 121-145

glyphosate and glufosinate. Weed Technology. 2011;**25**(2):198-203

foliar applications of HOE-39866. Weed Science. 1987;**35**:27-35

Submerged rice paddy soils are one of the major anthropogenic sources of methane (CH4 ) emission to the atmosphere. Methane is the second most important greenhouse gas after carbon dioxide. Methanogens are strictly anaerobic microorganisms and CH4 is the metabolic end product of those methanogens. Methane is produced by methanogens through multi-step enzyme-mediated process. Methanogens convert labile organic carbon compounds in CH4 and application of organic matter in submerged rice field significantly increased CH4 emission from soil to the atmosphere. The rate of methanogenesis may be determined by quantifying biomarkers namely methyl coenzyme M reductase A (mcrA) gene and coenzyme M (2-mercaptoethane sulphonate) in soil. Nickel ions are present as cofactor in enzymes involved in methanogenesis. Methane emission can be mitigated by application of EDTA at suitable rate in the soil of submerged rice field.

**Keywords:** methane emission, methanogens, biomarkers, EDTA application, rice paddy soil

### **1. Introduction**

In the era of development and globalization, emissions of greenhouse gases (GHGs) are unavoidable consequences, and that increases atmospheric temperature causing global warming. A greenhouse gas is a substrate in the atmosphere that absorbs and emits radiation within the thermal range. This process is the fundamental cause of the greenhouse effect and global warming [1]. Without GHGs, the average temperature of earth's surface would be about −18°C (0°F) [2], rather than present average of 15°C (59°F) [3]. The primary GHGs in the earth's atmosphere are water vapor, carbon dioxide (CO2 ), methane (CH4 ), nitrous oxide (N2 O) and chlorofluorocarbon (CFC).

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

Human activities since the beginning of the industrial revolution (Taken as year 1750) have resulted 40% increased in the atmospheric carbon dioxide concentration from 280 ppm in 1970 to 400 ppm in 2015 [4]. Carbon dioxide (CO2 ) is the most important GHG in atmosphere in terms of its emitted volume. The other GHGs are CH<sup>4</sup> , N2 O, CFC compounds etc. Methane is the second most important GHG emitted to the atmosphere on volume basis and it has 25 times higher global warming potential (GWP) as compared to equivalent amount of CO2 [5]. The half-life of CH4 in the atmosphere is about 25 years, which is also much higher than that of CO2 . Due to these characteristics, CH4 is considered as one of the most notorious GHGs having potential of causing global warming to the atmosphere. The CH4 concentration in the earth's atmosphere has been increased by 150% since 1750. Methane accounts for 20% of the total radiative forcing from the entire long-lived and globally mixed GHGs, excluding water vapor.

**c.** Exchanges between mud and water

terface on soil.

Mn2+, Fe2+ and CH4

tion in rice paddy soil.

The average global CH4

rice paddy soil favors CH4

increase CH4

rate of CH4

**3. Methanogens and CH4**

accounts for 11% of the total anthropogenic CH<sup>4</sup>

after the soil Eh value dropped below −200 eV [17].

**d.** Soil reduction

The presence of molecular oxygen in the soil-water interface makes it a sink of several redox reactions in soil and controls availability of phosphate and other nutrients in submerged soil. The presence of oxygen in the soil-water interface profoundly affects the N economy of submerged rice paddy soils. Ammonium-N released from broadcasted chemical fertilizer or from applied organic matter is converted to nitrate in the oxygenated in-

Methanogens Harboring in Rice Rhizosphere Reduce Labile Organic Carbon Compounds…

http://dx.doi.org/10.5772/intechopen.73299

An acute reduced state makes the major difference between chemical reactions of a submerged soil and aerated soil. Excluding the thin oxygenated layer in the soil-water interface, submerged soils have a negative oxidation-reduction potential (Eh value) due to

2−, Mn4+, Fe3+ and CO2

emission from rice fields is approximately 20–40 Tg CH<sup>4</sup>

emission is generally low; however, the flux gradually increases with plant devel-

that rice production will be increased from 473 million tons of 1990 to approximately 781 million tons by 2020 to fulfill the food demand of the world population and that proportionately

Methane is mainly produced during decomposition of organic matter by strictly anaerobic methanogens under intense reduced condition [14]. At the initial state of rice cultivation, the

opment and with enhanced anaerobic condition [15, 16]. Anaerobic conditions of submerged

Both cold- and hot-water extractable organic carbon (C) compounds are labile fraction of soil organic C. Low molecular weight organic compounds namely low molecular weight organic acids, carbohydrates are considered as labile organic C compounds in soil [18]. Labile organic C compounds rather than total organic C pool acts as the energy source for heterotrophic microorganisms like methanogens in soil [19]. Methane is the metabolic end product

production and the highest CH4

.

+ , H2 S, 213

produc-

year−1, which

emissions [12]. It had already been reported

emission is generally observed

anaerobic condition. Under such condition, dominant form of elements are NH<sup>4</sup>

Under submerged condition, aerobic soil microorganisms consume oxygen during their metabolism and that in turn gradually depletes oxygen pool making the soil anaerobic in reaction [10]. The redox potential (Eh) value in submerged soil starts decreasing after 3–4 days of flooding and sharply decreases with time. The Eh values in submerged anaerobic soils vary around −200 eV values throughout the rice cultivation duration [11]. Such anaerobic reducing

environment is one of the prime factors for determining the rate and quantity of CH4

 **production**

emission from rice paddy soils by 40–50% [13].

− , SO4

instead of NO3

**2.1. Oxidation – reduction (redox) potential**
