Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation

*Shweta Nandanwar, Yogesh Yele, Anil Dixit, Dennis Goss-Souza, Ritesh Singh, Arti Shanware and Lalit Kharbikar*

#### **Abstract**

Nitrogen is a vital component of atmosphere and plays important roles in the biochemistry of all life forms on the earth. Various mechanisms of biological nitrogen fixation and recycling in the environment have been evolved in all known ecosystems. For example, symbiotic nitrogen fixation is the major N2-fixing mechanism in the agroecosystems. Symbiotic nitrogen fixation is dependent on the biotic factors, such as host plant genotypes and the microbial strains. However, the interaction of these biotic factors is influenced by abiotic factors, such as climate and environmental conditions. The effects of various environmental variables, such as pesticides, temperature, and light as well as acidity, alkalinity, salinity, phosphorus, and water content status of the soils on the nitrogen fixation have been discussed briefly in this chapter.

**Keywords:** nitrogen fixation, salinity, ammonification, nitrite, denitrification

#### **1. Introduction**

The nutrient nitrogen is an essential element required by all life forms including the plants due to the presence in essential molecules such as proteins, amino acids, and enzymes. Although, this nutrient is in a large amount in the atmosphere, a small group of microorganisms is able to fix it becoming them available to plants.

The biological and physical processes in an ecosystem convert the nitrogen into multiple chemical forms and the phenomenon is called as nitrogen cycle. Biological nitrogen fixation accounts for about 70% of the atmospheric nitrogen. The four steps involved in a nitrogen cycle, namely nitrogen fixation, ammonification, nitrifications, and denitrification are described in **Figure 1**.

This process of nitrogen fixation is influenced by several aspects of biotic and abiotic variables. In this chapter, the effects of various factors especially the pesticide applications, temperature, and light as well as acidity, alkalinity, salinity, phosphorus, and water content status of the soils on the nitrogen fixation have been discussed briefly.

This chapter has addressed each environmental variable and discussed the effect of each one on the nitrogen fixation process and brings information related to it, for

**Figure 1.** *The nitrogen cycle.*

example, regarding the use of pesticides in an indiscriminate way. Usually, when the pesticides are used in the recommended dose, it does not harm the soil microbiota, but when the pesticides are overused, it may affect the physiological and growth characteristics of both rhizobia and legume plants. They also inhibit more or less the symbiotic nitrogen fixation by blocking biochemical communication and dialogues between the legumes and rhizobial symbionts.

#### **2. Effects of pesticides on nitrogen fixation**

In the current scenario of modern agriculture, agrochemicals play a key role in improving plant protection and production of food crops. Chemical pesticides are extensively used against a range of pests infesting agricultural crops worldwide. Due to the indiscriminate use of pesticides, the issue of the impact of these chemicals on the environment and soil has gained attention. Only about 0.1% of the total applied pesticides reach the target site/organism, while the remaining bulk contaminates the soil environment [1]. Consequently, sizable amounts of pesticides reach soil directly and indirectly, which affects the composition of soil microflora [2].

One of the most important and potentially limiting factors to biological nitrogen fixation is the use of chemical pesticides. Recommended doses of pesticides, in general, do not have any harmful effect on nitrogen fixation.

At higher doses, various chemical pesticides affect the physiological and growth characteristics of both rhizobia and legume plants. They also inhibit more or less the symbiotic nitrogen fixation by blocking biochemical communication and dialogues between the legumes and rhizobial symbionts [3].

Herbicides applied to leguminous crops constitute a potential hazard to the establishment and performance of the N2-fixing root nodules. Soil and foliar application of herbicide at the recommended rates altered the morphology of root hairs and reduced nodule numbers and nitrogenase activity [4]. Some rhizobium strains also shown resistance towards herbicides when isolated and grown in laboratory media.

**149**

plants.

*Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation*

**3. Effects of temperature and light on nitrogen fixation**

formation and nitrogen fixation in temperate legumes.

successfully adapted to arctic conditions.

Legume seeds are inoculated with specific rhizobium to increase yield and at the same time they are also treated with fungicides to disinfect and guard it against seed and soil-borne pathogens. It is imperative to know the effect of seed disinfectants on the efficacy of rhizobia. Earlier studies show fungicidal seed treatment is safe as far as nitrogen fixation by rhizobium in symbiosis with legumes is concerned. Contradictory, Osman et al. reported a very strong negative effect of the dose of the

Temperature has a significant influence on survival and persistence of N-fixing microbes in soils [6]. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate legumes. Whereas in tropical legumes, competitive ability and N fixation efficiency of legume symbionts are mainly affected. Similarly, low temperatures reduce nodule

In the extreme environment (−40 to −68°C) of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates [7]. This indicates that both the plants and their rhizobia have

At constant temperature (20°C) under long photoperiod (14 hours), plants may not show a significant decrease in N fixation in terms of fixation per plant or per unit nodule mass [8]. However, under a short photoperiod (6 hours) at the same temperature, plants show a significant decrease in N fixation. Under short photoperiod, plants compensate for reduced photosynthesis by maintaining only half the root nodule mass and N fixation activity as that of long photoperiod. However, similar rates of N2 fixation per unit mass of nodule may be observed in some plants under both the long and short photoperiods. This is because the shoot reserves for sustaining nitrogenase activity may compensate for reduced N fixation ability of

**4. Effects of acidity, alkalinity, and salinity on nitrogen fixation**

at low pH upon the addition of combined nitrogen.

A major problem associated with many acidic soils is metal toxicity. Highly acidic soils (pH < 4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high levels of aluminium and manganese which are often toxic for both plants and symbiotic N-fixing bacteria [7]. Nodulation by this N-fixing bacteria is more affected than the normal growth of the host-plants in such conditions. It is a well-known fact that most leguminous plants grow less favourably in acidic conditions than in neutral or slightly alkaline conditions [9]. This is mainly due to a reduced nitrogen fixation as may be concluded from the improved growth

Highly alkaline soils (pH > 8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduces symbiotic nitrogen fixation [4]. However, the symbiotic N-fixing rhizobia, showing significantly higher salt tolerance, have been isolated from alkaline soils [10]. This suggests that the possession of high salt tolerance trait might be of some evolutionary significance for the survival of rhizobia in alkaline soils. Phosphate solubilising, alkaline tolerant rhizobia have also been isolated from wild legumes grown in dune systems of the southwest coast of India [11]. The stress tolerance traits of these rhizobia are of potential value in strain improvement of symbiotic bacteria for efficient N-fixing ability.

*DOI: http://dx.doi.org/10.5772/intechopen.86128*

thiram on *Rhizobium meliloti* [5]*.*

*Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation DOI: http://dx.doi.org/10.5772/intechopen.86128*

Legume seeds are inoculated with specific rhizobium to increase yield and at the same time they are also treated with fungicides to disinfect and guard it against seed and soil-borne pathogens. It is imperative to know the effect of seed disinfectants on the efficacy of rhizobia. Earlier studies show fungicidal seed treatment is safe as far as nitrogen fixation by rhizobium in symbiosis with legumes is concerned. Contradictory, Osman et al. reported a very strong negative effect of the dose of the thiram on *Rhizobium meliloti* [5]*.*

#### **3. Effects of temperature and light on nitrogen fixation**

Temperature has a significant influence on survival and persistence of N-fixing microbes in soils [6]. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate legumes. Whereas in tropical legumes, competitive ability and N fixation efficiency of legume symbionts are mainly affected. Similarly, low temperatures reduce nodule formation and nitrogen fixation in temperate legumes.

In the extreme environment (−40 to −68°C) of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates [7]. This indicates that both the plants and their rhizobia have successfully adapted to arctic conditions.

At constant temperature (20°C) under long photoperiod (14 hours), plants may not show a significant decrease in N fixation in terms of fixation per plant or per unit nodule mass [8]. However, under a short photoperiod (6 hours) at the same temperature, plants show a significant decrease in N fixation. Under short photoperiod, plants compensate for reduced photosynthesis by maintaining only half the root nodule mass and N fixation activity as that of long photoperiod. However, similar rates of N2 fixation per unit mass of nodule may be observed in some plants under both the long and short photoperiods. This is because the shoot reserves for sustaining nitrogenase activity may compensate for reduced N fixation ability of plants.

#### **4. Effects of acidity, alkalinity, and salinity on nitrogen fixation**

A major problem associated with many acidic soils is metal toxicity. Highly acidic soils (pH < 4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high levels of aluminium and manganese which are often toxic for both plants and symbiotic N-fixing bacteria [7]. Nodulation by this N-fixing bacteria is more affected than the normal growth of the host-plants in such conditions. It is a well-known fact that most leguminous plants grow less favourably in acidic conditions than in neutral or slightly alkaline conditions [9]. This is mainly due to a reduced nitrogen fixation as may be concluded from the improved growth at low pH upon the addition of combined nitrogen.

Highly alkaline soils (pH > 8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduces symbiotic nitrogen fixation [4]. However, the symbiotic N-fixing rhizobia, showing significantly higher salt tolerance, have been isolated from alkaline soils [10]. This suggests that the possession of high salt tolerance trait might be of some evolutionary significance for the survival of rhizobia in alkaline soils. Phosphate solubilising, alkaline tolerant rhizobia have also been isolated from wild legumes grown in dune systems of the southwest coast of India [11]. The stress tolerance traits of these rhizobia are of potential value in strain improvement of symbiotic bacteria for efficient N-fixing ability.

*Nitrogen Fixation*

**Figure 1.** *The nitrogen cycle.*

example, regarding the use of pesticides in an indiscriminate way. Usually, when the pesticides are used in the recommended dose, it does not harm the soil microbiota, but when the pesticides are overused, it may affect the physiological and growth characteristics of both rhizobia and legume plants. They also inhibit more or less the symbiotic nitrogen fixation by blocking biochemical communication and dialogues

In the current scenario of modern agriculture, agrochemicals play a key role in improving plant protection and production of food crops. Chemical pesticides are extensively used against a range of pests infesting agricultural crops worldwide. Due to the indiscriminate use of pesticides, the issue of the impact of these chemicals on the environment and soil has gained attention. Only about 0.1% of the total applied pesticides reach the target site/organism, while the remaining bulk contaminates the soil environment [1]. Consequently, sizable amounts of pesticides reach soil directly and indirectly, which affects the composition of soil microflora [2].

One of the most important and potentially limiting factors to biological nitrogen

At higher doses, various chemical pesticides affect the physiological and growth characteristics of both rhizobia and legume plants. They also inhibit more or less the symbiotic nitrogen fixation by blocking biochemical communication and dialogues

Herbicides applied to leguminous crops constitute a potential hazard to the establishment and performance of the N2-fixing root nodules. Soil and foliar application of herbicide at the recommended rates altered the morphology of root hairs and reduced nodule numbers and nitrogenase activity [4]. Some rhizobium strains also shown resistance towards herbicides when isolated and grown in laboratory media.

fixation is the use of chemical pesticides. Recommended doses of pesticides, in

general, do not have any harmful effect on nitrogen fixation.

between the legumes and rhizobial symbionts [3].

between the legumes and rhizobial symbionts.

**2. Effects of pesticides on nitrogen fixation**

**148**

Soil salinity, which extremely increases in the protected cultivation, may occur when there are irregular irrigation schedules, inadequate drainage systems, wrong fertiliser applications, etc. [12]. The specific sensitivity of leguminous crops, where the N fixation is predominated by symbiotic bacteria, to soil salinity is well documented for initiation, development, and function of nodules [13]. An increase in salinity decreases the nodule permeability in these crops. This decrease is associated with a contraction of nodule inner-cortex cells and an increase in abscisic acid content of the nodule [14]. This phenomenon may lead to less survival of the symbiotic N-fixing bacteria associated with these crops. Even if the bacteria are survived, they may be less viable with decreased N-fixing ability. However, survival of the viable *Rhizobium* sp. under extreme conditions of salinity has been reported recently in root nodules of *Sesbania aculeata* [10].

#### **5. Effects of phosphorus (P) status of soil on nitrogen fixation**

Phosphorus deficiency in soils affects nodule functioning; however, it does not inhibit the other aspects of plant growth and metabolism. There exists an interaction between the plant's growth and N fixation in response to increasing P supply [15]. This interaction may be positive, zero, or negative. A negative interaction suggests a greater need for P by N2-fixing plants; a zero interaction indicates that the plants have the same P requirement for growth and N fixation, while a positive interaction indicates that higher P supply may be inhibitory to N2 fixation (**Figure 2**).

Legume tissues do not appear to have higher P content than those of other plants. Therefore a positive interaction exists between legume's growth and N fixation in response to P supply (**Figure 2**). For example, in both soybean and lucerne (alfalfa), nodules from plants grown under P deficiency may have a higher concentration of P than those grown with sufficient P and may fix more N2 per unit of P [16].

**Figure 2.**

*Effects of phosphorus (P) status of soil on nitrogen fixation.*

#### **6. Effects of soil water availability on nitrogen fixation**

Water influences the growth of soil micro-organisms through processes of diffusion, mass flow, and nutrient concentration [17]. Soil water retention is related to soil pore space, and soils containing larger pores and pore spaces retain less water. Thus, soil aggregates having smaller internal pore spaces offers more favourable environments for the growth of N-fixing microbes.

There is a high degree of correlation between soil water content and N-fixing activity [16]. Maximum N fixation occurs at about field capacity of soil and above this, the N-fixing activity may be reduced due to water logging. Slow natural drying of soil over a 6 week period may result in a progressive reduction of N-fixing activity, which can be restored by irrigation. This indicates that soil water availability is

**151**

*Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation*

the major environmental factor affecting nitrogen fixation. It is plausible that water stress directly affects nodule activity. This effect may further be aggravated by

Environmental variables such as pesticides, temperature, and light as well as acidity, alkalinity, salinity, phosphorus, and water content status of the soils have a significant impact on nitrogen fixation. Despite many decades of progress and the acquisition of ample information, the physiological and molecular bases of the effects of these environmental variables on the symbiotic and non-symbiotic N2 fixation systems remains largely unknown and empirical in nature. Although understanding these processes was originally thought to be straightforward and tractable, we now have learned that the flavonoid nod-gene inducers are specific for a particular N2 fixation system [19]. Needless to say that, the production of these inducers is influenced by environmental variables. Therefore, more work needs to be done to understand the underlying molecular bases for tolerance to environmental variables in both N2 fixation systems. Further, the genomics and proteomics tools need to be combined with traditional plant breeding and microbial selection studies in order to rapidly define and utilise their genetic loci involved in tolerance

We would like to thank all colleagues who have done work on nitrogen fixation and related fields. We apologise to colleagues whose work in this rapidly changing field was not directly cited in this review due to space limitations and timing. We

would also like to thank anonymous reviewers for helpful comments.

reduced supplies of photosynthate due to wilted plant leaves [18].

*DOI: http://dx.doi.org/10.5772/intechopen.86128*

**7. Conclusion**

to environmental stresses.

**Acknowledgements**

the major environmental factor affecting nitrogen fixation. It is plausible that water stress directly affects nodule activity. This effect may further be aggravated by reduced supplies of photosynthate due to wilted plant leaves [18].

#### **7. Conclusion**

*Nitrogen Fixation*

(**Figure 2**).

unit of P [16].

**Figure 2.**

root nodules of *Sesbania aculeata* [10].

Soil salinity, which extremely increases in the protected cultivation, may occur when there are irregular irrigation schedules, inadequate drainage systems, wrong fertiliser applications, etc. [12]. The specific sensitivity of leguminous crops, where the N fixation is predominated by symbiotic bacteria, to soil salinity is well documented for initiation, development, and function of nodules [13]. An increase in salinity decreases the nodule permeability in these crops. This decrease is associated with a contraction of nodule inner-cortex cells and an increase in abscisic acid content of the nodule [14]. This phenomenon may lead to less survival of the symbiotic N-fixing bacteria associated with these crops. Even if the bacteria are survived, they may be less viable with decreased N-fixing ability. However, survival of the viable *Rhizobium* sp. under extreme conditions of salinity has been reported recently in

**5. Effects of phosphorus (P) status of soil on nitrogen fixation**

Phosphorus deficiency in soils affects nodule functioning; however, it does not inhibit the other aspects of plant growth and metabolism. There exists an interaction between the plant's growth and N fixation in response to increasing P supply [15]. This interaction may be positive, zero, or negative. A negative interaction suggests a greater need for P by N2-fixing plants; a zero interaction indicates that the plants have the same P requirement for growth and N fixation, while a positive interaction indicates that higher P supply may be inhibitory to N2 fixation

Legume tissues do not appear to have higher P content than those of other plants. Therefore a positive interaction exists between legume's growth and N fixation in response to P supply (**Figure 2**). For example, in both soybean and lucerne (alfalfa), nodules from plants grown under P deficiency may have a higher concentration of P than those grown with sufficient P and may fix more N2 per

**6. Effects of soil water availability on nitrogen fixation**

environments for the growth of N-fixing microbes.

*Effects of phosphorus (P) status of soil on nitrogen fixation.*

Water influences the growth of soil micro-organisms through processes of diffusion, mass flow, and nutrient concentration [17]. Soil water retention is related to soil pore space, and soils containing larger pores and pore spaces retain less water. Thus, soil aggregates having smaller internal pore spaces offers more favourable

There is a high degree of correlation between soil water content and N-fixing activity [16]. Maximum N fixation occurs at about field capacity of soil and above this, the N-fixing activity may be reduced due to water logging. Slow natural drying of soil over a 6 week period may result in a progressive reduction of N-fixing activity, which can be restored by irrigation. This indicates that soil water availability is

**150**

Environmental variables such as pesticides, temperature, and light as well as acidity, alkalinity, salinity, phosphorus, and water content status of the soils have a significant impact on nitrogen fixation. Despite many decades of progress and the acquisition of ample information, the physiological and molecular bases of the effects of these environmental variables on the symbiotic and non-symbiotic N2 fixation systems remains largely unknown and empirical in nature. Although understanding these processes was originally thought to be straightforward and tractable, we now have learned that the flavonoid nod-gene inducers are specific for a particular N2 fixation system [19]. Needless to say that, the production of these inducers is influenced by environmental variables. Therefore, more work needs to be done to understand the underlying molecular bases for tolerance to environmental variables in both N2 fixation systems. Further, the genomics and proteomics tools need to be combined with traditional plant breeding and microbial selection studies in order to rapidly define and utilise their genetic loci involved in tolerance to environmental stresses.

#### **Acknowledgements**

We would like to thank all colleagues who have done work on nitrogen fixation and related fields. We apologise to colleagues whose work in this rapidly changing field was not directly cited in this review due to space limitations and timing. We would also like to thank anonymous reviewers for helpful comments.

## **Author details**

Shweta Nandanwar1 , Yogesh Yele2 , Anil Dixit2 , Dennis Goss-Souza<sup>3</sup> , Ritesh Singh4 , Arti Shanware5 and Lalit Kharbikar2 \*

1 Princess Margaret Science Laboratories, Harper Adams University, Newport, Shropshire, United Kingdom

2 ICAR—National Institute of Biotic Stress Management, Raipur, India

3 Santa Catarina State University—UDESC, Florianópolis, Brazil

4 Thakur Chhedilal Barrister College of Agriculture and Research Station, Bilaspur, India

5 Rajiv Gandhi Biotechnology Centre, RTM Nagpur University, Nagpur, India

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

© 2020 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.

**153**

*Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation*

[9] Hartwig UA. The regulation of symbiotic N2 fixation: A conceptual model of N feedback from the

and Systematics. 1998;**1**:92-120

2000;**41**(6):402-409

2005;**5**(2):134-138

2012;**7**(7):1073-1086

2001;**21**:27-34

les colloques

1983. pp. 36-55

1997;**201**:463-469

[10] Kulkarni S. Crossing the limits of *Rhizobium* existence in extreme conditions. Current Microbiology.

[11] Arun B, Sridhar KR. Growth tolerance of rhizobia isolated from sand dune legumes of the southwest coast of India. Engineering in Life Sciences.

[12] Adil AA, Kant C, Turan M. Humic acid application alleviate salinity stress of bean (*Phaseolus vulgaris* L.) plants decreasing membrane leakage. African Journal of Agricultural Research.

[13] Saadallah K, Drevon JJ, Abdelly C. Nodulation et croissance nodulaire chez le Haricot (*Phaseolus vulgaris* L.) sous contrainte saline. Agronomie.

[14] Irekti H, Drevon JJ. Acide abcissique

et conductance á la diffusion de l'oxygène dans les nodositeś de haricot soumises à un choc salin. In: Drevon JJ, Sifi B, editors. Fixation Symbiotique de l'Azote et Développement Durable dans le Bassin Méditerranéen. Vol. 100. INRA, Versailles Cedex, France; 2003.

[15] Robson AD. Mineral nutrition. In: Broughton WJ, editor. Nitrogen Fixation. Oxford: Clarendon Press;

[16] Drevon JJ, Hartwig UA. Phosphorus

induced decline in nodule nitrogenase activity in soybean and alfalfa. Planta.

deficiency increases the argon-

ecosystem to the gene expression level. Perspectives in Plant Ecology, Evolution

*DOI: http://dx.doi.org/10.5772/intechopen.86128*

[1] Carriger JF, Rand GM, Gardinali PR, Perry WB, Tompkins MS, Fernandez AM. Pesticides of potential ecological concern in sediment from South Florida Canals: An ecological risk prioritization for aquatic arthropods. Soil and Sediment Contamination.

[2] Andrea MM, Peres TB, Luchini LC, Pettinelli A. Impact of long-term pesticide application on some soil biological parameters. Journal of

Environmental Science and Health, Part

[3] Ahemad M, Khan MS. Pesticides as antagonists of rhizobia and the Legume- *Rhizobium* Symbiosis: A paradigmatic and mechanistic outlook. Biochemistry and Molecular Biology.

[4] Ljunggren H, Martensson A. Herbicide effect on leguminous symbiosis. In: Proceedings of the *21st* Swedish Weed Conference. Uppsala: Sve-riges Lantbruksuniversitet; 1980.

[5] Osman AG, Sherif AM, Elhussein AA, Mohamed AT. Sensitivity of some nitrogen fixers and the target pest *Fusarium oxysporum* to fungicide thiram. Interdisciplinary Toxicology.

[6] Lie TA. Symbiotic nitrogen fixation under stress conditions. Plant and Soil.

**References**

2006;**15**:21-45

B. 2000;**35**:297-307

2013;**1**(4):63-75

pp. 99-106

2012;**5**:25-29

1971;**35**(1):117-127

1994;**161**:115-125

1986;**95**(3):399-409

[7] Bordeleau LM, Prevost D. Nodulation and nitrogen fixation in extreme environments. Plant and Soil.

[8] Murphy PM. Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes. Plant and Soil. *Effects of Pesticides, Temperature, Light, and Chemical Constituents of Soil on Nitrogen Fixation DOI: http://dx.doi.org/10.5772/intechopen.86128*

#### **References**

*Nitrogen Fixation*

**Author details**

Arti Shanware5

Bilaspur, India

Shweta Nandanwar1

Shropshire, United Kingdom

, Yogesh Yele2

and Lalit Kharbikar2

**152**

provided the original work is properly cited.

© 2020 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,

, Anil Dixit2

1 Princess Margaret Science Laboratories, Harper Adams University, Newport,

\*

2 ICAR—National Institute of Biotic Stress Management, Raipur, India

4 Thakur Chhedilal Barrister College of Agriculture and Research Station,

5 Rajiv Gandhi Biotechnology Centre, RTM Nagpur University, Nagpur, India

3 Santa Catarina State University—UDESC, Florianópolis, Brazil

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

, Dennis Goss-Souza<sup>3</sup>

, Ritesh Singh4

,

[1] Carriger JF, Rand GM, Gardinali PR, Perry WB, Tompkins MS, Fernandez AM. Pesticides of potential ecological concern in sediment from South Florida Canals: An ecological risk prioritization for aquatic arthropods. Soil and Sediment Contamination. 2006;**15**:21-45

[2] Andrea MM, Peres TB, Luchini LC, Pettinelli A. Impact of long-term pesticide application on some soil biological parameters. Journal of Environmental Science and Health, Part B. 2000;**35**:297-307

[3] Ahemad M, Khan MS. Pesticides as antagonists of rhizobia and the Legume- *Rhizobium* Symbiosis: A paradigmatic and mechanistic outlook. Biochemistry and Molecular Biology. 2013;**1**(4):63-75

[4] Ljunggren H, Martensson A. Herbicide effect on leguminous symbiosis. In: Proceedings of the *21st* Swedish Weed Conference. Uppsala: Sve-riges Lantbruksuniversitet; 1980. pp. 99-106

[5] Osman AG, Sherif AM, Elhussein AA, Mohamed AT. Sensitivity of some nitrogen fixers and the target pest *Fusarium oxysporum* to fungicide thiram. Interdisciplinary Toxicology. 2012;**5**:25-29

[6] Lie TA. Symbiotic nitrogen fixation under stress conditions. Plant and Soil. 1971;**35**(1):117-127

[7] Bordeleau LM, Prevost D. Nodulation and nitrogen fixation in extreme environments. Plant and Soil. 1994;**161**:115-125

[8] Murphy PM. Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes. Plant and Soil. 1986;**95**(3):399-409

[9] Hartwig UA. The regulation of symbiotic N2 fixation: A conceptual model of N feedback from the ecosystem to the gene expression level. Perspectives in Plant Ecology, Evolution and Systematics. 1998;**1**:92-120

[10] Kulkarni S. Crossing the limits of *Rhizobium* existence in extreme conditions. Current Microbiology. 2000;**41**(6):402-409

[11] Arun B, Sridhar KR. Growth tolerance of rhizobia isolated from sand dune legumes of the southwest coast of India. Engineering in Life Sciences. 2005;**5**(2):134-138

[12] Adil AA, Kant C, Turan M. Humic acid application alleviate salinity stress of bean (*Phaseolus vulgaris* L.) plants decreasing membrane leakage. African Journal of Agricultural Research. 2012;**7**(7):1073-1086

[13] Saadallah K, Drevon JJ, Abdelly C. Nodulation et croissance nodulaire chez le Haricot (*Phaseolus vulgaris* L.) sous contrainte saline. Agronomie. 2001;**21**:27-34

[14] Irekti H, Drevon JJ. Acide abcissique et conductance á la diffusion de l'oxygène dans les nodositeś de haricot soumises à un choc salin. In: Drevon JJ, Sifi B, editors. Fixation Symbiotique de l'Azote et Développement Durable dans le Bassin Méditerranéen. Vol. 100. INRA, Versailles Cedex, France; 2003. les colloques

[15] Robson AD. Mineral nutrition. In: Broughton WJ, editor. Nitrogen Fixation. Oxford: Clarendon Press; 1983. pp. 36-55

[16] Drevon JJ, Hartwig UA. Phosphorus deficiency increases the argoninduced decline in nodule nitrogenase activity in soybean and alfalfa. Planta. 1997;**201**:463-469

[17] Turco RF, Sadowsky MJ. Understanding the microflora of bioremediation. In: Skipper HD, Turco RF, editors. Bioremediation: Science and Applications. Soil Science (Special Publication). Vol. 43. Madison, WI: Soil Science Society of America Journal; 1995. pp. 87-103

[18] Sprent IJ. The effects of water stress on nitrogen-fixing root nodules. New Phytologist. 1972;**71**(4):603-611

[19] Schmidt PE, Broughton WJ, Werner D. Nod factors of Bradyrhizobium japonicum and Rhizobium sp. NGR 234 induce flavonoid accumulation in soybean root exudates. Molecular Plant Microbe Interaction. 1994;**7**:384-390

**155**

**Chapter 10**

**Abstract**

symbiotic performance

**1. Introduction**

Philippines

Distribution and Characterization

Nodulating Bradyrhizobia in the

The research about the indigenous soybean bradyrhizobia in the Philippines is scarce, and this greatly influences the improvement of soybean production in the country. Thus, soil samples were collected from 11 locations in the country and were used to isolate the indigenous bradyrhizobia in the soil. Through the use of polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP) and sequence analysis of the 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS) region and *rpoB* housekeeping gene, the most abundant and dominant indigenous bradyrhizobia in the country were identified. Then, the representative isolates of the most dominant species per location were used to test their symbiotic efficiency and N-fixation ability with the local soybean cultivars. The results showed that among all the tested indigenous strains, the *B. elkanii* IS-2 is the most effective and efficient microsymbiont of the Philippines' local soybean cultivars. This report was able to provide necessary information on the distribution of soybean bradyrhizobia in the Philippines and characterized the symbiotic performance of the indigenous strains.

Soybean (*Glycine max* [L.]) is a leguminous plant that can form a symbiotic relationship with the nitrogen-fixing group of bacteria living in the rhizosphere, which are generally termed as rhizobia. In the Philippines, soybean production has been limited by the poor grain yield which leads to the importation of more than 90% of the country's demand. Thus, it is essential to look for an alternative way to

The research about tropical bradyrhizobia indicated a high diversity of species and their distribution has been reported to be due to several abiotic and biotic factors such as soil acidity [1–3], alkalinity [3, 4], temperature [1, 5–11], climate [12, 13], soil water status [14, 15], soil type [2, 14, 16–18], and soil management or cultural practices [2, 14, 19–22]. In case of the Philippines, the pioneer research that was able to identify the most dominant species of bradyrhizobia in the country reported that *B. elkanii* species was the most abundant, followed by the

of the Indigenous Soybean-

*Maria Luisa Tabing Mason and Yuichi Saeki*

**Keywords:** tropical rhizobia, genomic diversity, N-fixation,

increase the volume of production per unit area.

#### **Chapter 10**

*Nitrogen Fixation*

1995. pp. 87-103

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[18] Sprent IJ. The effects of water stress on nitrogen-fixing root nodules. New Phytologist. 1972;**71**(4):603-611

[19] Schmidt PE, Broughton WJ, Werner D. Nod factors of Bradyrhizobium japonicum and Rhizobium sp. NGR 234 induce flavonoid accumulation in soybean root exudates. Molecular Plant Microbe

Interaction. 1994;**7**:384-390

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