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Research Article

volume 43 issue 1 (february 2020) : 126-133,   Doi: 10.18805/LR-3951

Isolation and characterization of native cowpea rhizobia from Wayanad India

D
D. Girija
P
P. S. Panchami
P
Praveena E. Jose
T
T. Saeed
S
Sneha S. Nair
1Department of Agricultural Microbiolgy, College of Horticulture, Kerala Agricultural University Vellanikkara-680 656, Kerala, India.

  • Submitted07-10-2017|

  • Accepted18-06-2018|

  • First Online 15-11-2018|

  • doi 10.18805/LR-3951

Cite article:- Girija D., Panchami S. P., Jose E. Praveena, Saeed T., Nair S. Sneha (2018). Isolation and characterization of native cowpea rhizobia from Wayanad India. Legume Research. 43(1): 126-133. doi: 10.18805/LR-3951.

ABSTRACT

In the present study, eight rhizobial strains (RH-1 to RH- 8) were  isolated from root nodules of cowpea which grows in acidic soils of Wayanad . They were characterized based on morphological, physiological, biochemical and molecular characteristics. The results on plant growth promoting traits showed all isolates positive to IAA production, four positive to ammonia production and five positive for in vitro siderophore production. Three isolates exhibited antagonistic activity   and none of them showed volatile cyanogen production.The 16S rRNA gene sequence analysis revealed all the isolates to show similarity to Rhizobium sp. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, the isolates RH-1. RH-2, RH-4 and RH-8 grew in a liquid yeast extract - mannitol agar medium at pH-4, suggesting acid tolerance in the strain of Rhizobium .The isolates were authenticated for their nodulation and growth promotion of cowpea in a paper cup experiment and four isolates (RH-2, RH-3, RH-4 and RH-5) were evaluated for their growth promotion in cowpea under field condition. It was observed that inoculation of Rhizobium increased nodulation, growth parameters and yield of cowpea compared to uninoculated control.This study showed that these rhizobial isolates with plant growth promoting traits can be used to promote the growth and yield of cow peain  acidic soils of  Kerala.

REFERENCES

  1. Arfaoui, A., Sifi, B., Boudabous, A., El Hadrami, I., Chérif, M. (2006). Identification of Rhizobium isolates possessing antagonistic activity against Fusariumoxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea. Journal of Plant Pathology, 88: 67-75.
  2. Arora, D. R. (2003). Textbook of Microbiology, New Delhi: CBS Publisher. pp. 41-48. 
  3. Arora, N.K., Kang, S.C., Maheshwari, D.K. (2001). Isolation of siderophore producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Current Sciences, 81: 673–677.
  4. Ashraf, M. and Iram, AT. (2005). Drought stress induced changes in some organic substances in nodules and other plant parts of two potential legumes differing in salt tolerance. Flora. 200: 535-546
  5. Bakker, A.W. and Schipperes, B. (1987). Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomoas spp. - mediated plant growth stimulation. Soil Biology and Biochemistry 19: 451 - 457. 
  6. Bremner, J.M. (1965). Total nitrogen. In: Methods of Soil Analysis. Part 2, Chemical and microbiological properties [(Eds.: C.A. Black, D.D. Evans, J.L. White, L.E. Ensminger and F.E. Clark)]. Agronomy 9. ASA, Madison,Wisconsin. pp. 1149-1176.
  7. Brick, J.M., Bostock, R.M., Silverstone, S.E. (1991). Rapid insitu assay for indole acetic acid production bybacteria immobilized on nitrocellulose membrane. Applied Environmental Microbiology, 57: 535–538.
  8. Cappuccino, J.C. and Sherman, N. (1992). In: Microbiology: A Laboratory Manual, New York, pp.125–179.
  9. Dekhane, S.S., Khafi, H.R., Raj, A.D. Parmar, R.M. (2011). Effect of bio fertilizer and fertility levels on yield, protein content and nutrient uptake of cowpea [Vignaunguiculata (L.)Walp.]. Legume Res., 34 (1): 51–54.
  10. Doignon-Bourcier, F., Willems, A., Coopman, R., Laguerre, G., Gillis, M., de Lajudie, P. (2000). Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses. Applied and Environmental Microbiology, 66(9), 3987-3997.
  11. Duan, J., Müller, K.M., Charles, S.,Vesely, T.C., Glick, B.R. (2009).1-Aminocyclopropane-1-carboxylate (ACC) deaminase genes in Rhizobia from southern Saskatchewan. Microbial Ecology, 57: 423-436.
  12. Evans, G.M., Furlong, J. C. (2010). Environmental Biotechnology, John Wiley and sons England, 15p.
  13. Figueiredo, A. C., Barroso, J. G., Pedro, L. G., Scheffer, J. J. (2008). Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour and Fragrance journal, 23(4), 213-226.
  14. Fischer, S. E., Fischer, S. I., Margis, S., Mori, G. B. (2007). Isolation and characterization of bacteria from rhizosphere of wheat. World Journal of Microbiology and Biotechnology, 23: 895–903.
  15. Gupta, A., Meyer, J.M., Goel, R. (2002). Development of heavy metal-resistant mutants of phosphate solubilizing Pseudomonas sp. NBRI 4014 and their characterization. Current Microbiology, 45: 323–327.
  16. Gupta, A., Saxena, A.K., Murali, G., Tilak, K.V.B.R. (1998). Effect of plant growth promoting rhizobacteria on competitive ability of introduced Bradyrhizobium sp. (Vigna) for nodulation. Journal of Scientific and Industrial Research, 57: 720-725.
  17. Hajnaa, (1945). Triple- Sugaar Ion Medium for the identification of the intestinal group of bacteria. Journal of Bacteriology, 49: 516-517. 
  18. Hirsch, A.M., Fang, Y. Asad, S., Kapuilnik, Y. (2000). The role of phytohormones in plant microbe symbioses, Plant and Soil, 194: 171-184.
  19. Hofer, A.W. (1935). Method for distinguishing between legume bacteria and their most common contaminant. Journal of American Society of Agronomy, 27: 228-30
  20. Jehangir, I.A., Mir, M.A., Bhat, M.A., Ahangar, M.A. (2017) Biofertilizers an approach to sustainability in Agriculture: A review. International Journal of Pure and Applied Bioscience, . 5 (5): 327-334.
  21. Kleczkowska, J. and Nutman, P.S. (1968). The identification and classification of rhizobia In: Identification Methods for Microbiologists Part B [ed. Gibbs BM and Shapton DA,] Academic Press, London and New York. 51.
  22. Kovaks, N. (1956). Identification of Pseudomonas pyocyaneaby the oxidase reaction. Nature, 178: 703.
  23. Lowe, G. H. (1962). The rapid detection of lactose fermentatation in paracolon organism by demonstration of 6- D-galactosidase. The Journal Medical Laboratory Technology, 19: 21-25.
  24. Mfilinge, A.K., Mtei, K., Ndakidemi, P.A. (2014). Effects of Rhizobium inoculation and supplementation with P and K, on growth, leaf chlorophyll content and nitrogen fixation of bush bean varieties. American Journal of Research Communication, 2(10): 49-87.
  25. Pandey, P. and Maheshwari, D.K. (2007). Two-species microbial consortium for growth promotion of Cajanus cajan. Current Science, 92: 1137–1142.
  26. Pindi, P.K. and Satyanarayana, S.D.V. (2012). Liquid Microbial Consortium- A Potential Tool for Sustainable Soil Health. Journal of Biofertilizers and Biopesticides, 3 (4): 1-9. 
  27. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution, 4(4), 406-425
  28. Schwyn, B. and Neilands, J.B. (1987). Universal chemical assay for detection and determination of siderophores. Annals in Biochemistry, 160: 47-56.
  29. Shahzad, F., Shafee, M., Abbas, F., Babar, S, Tariqq, M.M., Ahmad, Z. (2012). Isolation and biochemical characterization of Rhizobium meliloti from root nodules of alfalfa (Medicosativa), Journal of Plant and Animal Sciences 22(2):522-524. 
  30. Skidmore, A.M. and Dickinson, C.H. (1976). Colony interaction and hyphal interference between Septoria nodorum and phylloplane fungi. Transactions of the British Mycological Society, 66: 57–74.
  31. Sharma, V.K. and Dayal, B. (2005). Effect of organic and inorganic sources of nitrogen on growth, yield and nutrients uptake under cowpea-linseed cropping system. Legume Res. 28:79-86.
  32. Tan, Z. Y., Kan, F. L., Peng, G. X., Wang, E. T., Reinhold-Hurek, B., Chen, W. X. (2001). Rhizobium yanglingensc sp. nov.isolated from arid and semiarid regions in China. International Journal of Systematic and Evolutionary Microbiology, 51: 909–914.
  33. Tamura, K., J. Dudley, M. Nei, S. Kumar. (2007). MEGAA4: Molecular Evolutionary Genetics Anaysis (MEGA) software version 4.0. Mol. Biol., 24: 1596-1599.
  34. Vaishnavi, J. (2016). Growth and yield response of cowpea to multiaction bioinoculants. Legume Research-An International Journal, 39(6), 962-969.
  35. Vinuesa, P., Leon-Barrios, M., Silva, C., Willems, A., Jarabo-Lorenzo, A., Perez-Galdona, R., Werner D & Martinez-Romero, E. (2005). Bradyrhizobiumcanariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicumbv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int. J. Syst. Evol. Microbiol, 55: 569–575.
  36. Wolde-Meskel, E., Berg, T., Peters, N.K., Frostegard, A. (2004). Nodulation status of native woody legumes and phenotypic characteristics of associated Rhizobia in soils of southern Ethiopia. Biological Fertility of Soils, 40: 55-66.
  37. Zahran, H.H. (1999). Rhizobium–legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiology and Molecular Biology Reviews, 63: 968–989. 
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    Research Article

    volume 43 issue 1 (february 2020) : 126-133,   Doi: 10.18805/LR-3951

    Isolation and characterization of native cowpea rhizobia from Wayanad India

    D
    D. Girija
    P
    P. S. Panchami
    P
    Praveena E. Jose
    T
    T. Saeed
    S
    Sneha S. Nair
    1Department of Agricultural Microbiolgy, College of Horticulture, Kerala Agricultural University Vellanikkara-680 656, Kerala, India.

    • Submitted07-10-2017|

    • Accepted18-06-2018|

    • First Online 15-11-2018|

    • doi 10.18805/LR-3951

    Cite article:- Girija D., Panchami S. P., Jose E. Praveena, Saeed T., Nair S. Sneha (2018). Isolation and characterization of native cowpea rhizobia from Wayanad India. Legume Research. 43(1): 126-133. doi: 10.18805/LR-3951.

    ABSTRACT

    In the present study, eight rhizobial strains (RH-1 to RH- 8) were  isolated from root nodules of cowpea which grows in acidic soils of Wayanad . They were characterized based on morphological, physiological, biochemical and molecular characteristics. The results on plant growth promoting traits showed all isolates positive to IAA production, four positive to ammonia production and five positive for in vitro siderophore production. Three isolates exhibited antagonistic activity   and none of them showed volatile cyanogen production.The 16S rRNA gene sequence analysis revealed all the isolates to show similarity to Rhizobium sp. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, the isolates RH-1. RH-2, RH-4 and RH-8 grew in a liquid yeast extract - mannitol agar medium at pH-4, suggesting acid tolerance in the strain of Rhizobium .The isolates were authenticated for their nodulation and growth promotion of cowpea in a paper cup experiment and four isolates (RH-2, RH-3, RH-4 and RH-5) were evaluated for their growth promotion in cowpea under field condition. It was observed that inoculation of Rhizobium increased nodulation, growth parameters and yield of cowpea compared to uninoculated control.This study showed that these rhizobial isolates with plant growth promoting traits can be used to promote the growth and yield of cow peain  acidic soils of  Kerala.

    REFERENCES

    1. Arfaoui, A., Sifi, B., Boudabous, A., El Hadrami, I., Chérif, M. (2006). Identification of Rhizobium isolates possessing antagonistic activity against Fusariumoxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea. Journal of Plant Pathology, 88: 67-75.
    2. Arora, D. R. (2003). Textbook of Microbiology, New Delhi: CBS Publisher. pp. 41-48. 
    3. Arora, N.K., Kang, S.C., Maheshwari, D.K. (2001). Isolation of siderophore producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Current Sciences, 81: 673–677.
    4. Ashraf, M. and Iram, AT. (2005). Drought stress induced changes in some organic substances in nodules and other plant parts of two potential legumes differing in salt tolerance. Flora. 200: 535-546
    5. Bakker, A.W. and Schipperes, B. (1987). Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomoas spp. - mediated plant growth stimulation. Soil Biology and Biochemistry 19: 451 - 457. 
    6. Bremner, J.M. (1965). Total nitrogen. In: Methods of Soil Analysis. Part 2, Chemical and microbiological properties [(Eds.: C.A. Black, D.D. Evans, J.L. White, L.E. Ensminger and F.E. Clark)]. Agronomy 9. ASA, Madison,Wisconsin. pp. 1149-1176.
    7. Brick, J.M., Bostock, R.M., Silverstone, S.E. (1991). Rapid insitu assay for indole acetic acid production bybacteria immobilized on nitrocellulose membrane. Applied Environmental Microbiology, 57: 535–538.
    8. Cappuccino, J.C. and Sherman, N. (1992). In: Microbiology: A Laboratory Manual, New York, pp.125–179.
    9. Dekhane, S.S., Khafi, H.R., Raj, A.D. Parmar, R.M. (2011). Effect of bio fertilizer and fertility levels on yield, protein content and nutrient uptake of cowpea [Vignaunguiculata (L.)Walp.]. Legume Res., 34 (1): 51–54.
    10. Doignon-Bourcier, F., Willems, A., Coopman, R., Laguerre, G., Gillis, M., de Lajudie, P. (2000). Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses. Applied and Environmental Microbiology, 66(9), 3987-3997.
    11. Duan, J., Müller, K.M., Charles, S.,Vesely, T.C., Glick, B.R. (2009).1-Aminocyclopropane-1-carboxylate (ACC) deaminase genes in Rhizobia from southern Saskatchewan. Microbial Ecology, 57: 423-436.
    12. Evans, G.M., Furlong, J. C. (2010). Environmental Biotechnology, John Wiley and sons England, 15p.
    13. Figueiredo, A. C., Barroso, J. G., Pedro, L. G., Scheffer, J. J. (2008). Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour and Fragrance journal, 23(4), 213-226.
    14. Fischer, S. E., Fischer, S. I., Margis, S., Mori, G. B. (2007). Isolation and characterization of bacteria from rhizosphere of wheat. World Journal of Microbiology and Biotechnology, 23: 895–903.
    15. Gupta, A., Meyer, J.M., Goel, R. (2002). Development of heavy metal-resistant mutants of phosphate solubilizing Pseudomonas sp. NBRI 4014 and their characterization. Current Microbiology, 45: 323–327.
    16. Gupta, A., Saxena, A.K., Murali, G., Tilak, K.V.B.R. (1998). Effect of plant growth promoting rhizobacteria on competitive ability of introduced Bradyrhizobium sp. (Vigna) for nodulation. Journal of Scientific and Industrial Research, 57: 720-725.
    17. Hajnaa, (1945). Triple- Sugaar Ion Medium for the identification of the intestinal group of bacteria. Journal of Bacteriology, 49: 516-517. 
    18. Hirsch, A.M., Fang, Y. Asad, S., Kapuilnik, Y. (2000). The role of phytohormones in plant microbe symbioses, Plant and Soil, 194: 171-184.
    19. Hofer, A.W. (1935). Method for distinguishing between legume bacteria and their most common contaminant. Journal of American Society of Agronomy, 27: 228-30
    20. Jehangir, I.A., Mir, M.A., Bhat, M.A., Ahangar, M.A. (2017) Biofertilizers an approach to sustainability in Agriculture: A review. International Journal of Pure and Applied Bioscience, . 5 (5): 327-334.
    21. Kleczkowska, J. and Nutman, P.S. (1968). The identification and classification of rhizobia In: Identification Methods for Microbiologists Part B [ed. Gibbs BM and Shapton DA,] Academic Press, London and New York. 51.
    22. Kovaks, N. (1956). Identification of Pseudomonas pyocyaneaby the oxidase reaction. Nature, 178: 703.
    23. Lowe, G. H. (1962). The rapid detection of lactose fermentatation in paracolon organism by demonstration of 6- D-galactosidase. The Journal Medical Laboratory Technology, 19: 21-25.
    24. Mfilinge, A.K., Mtei, K., Ndakidemi, P.A. (2014). Effects of Rhizobium inoculation and supplementation with P and K, on growth, leaf chlorophyll content and nitrogen fixation of bush bean varieties. American Journal of Research Communication, 2(10): 49-87.
    25. Pandey, P. and Maheshwari, D.K. (2007). Two-species microbial consortium for growth promotion of Cajanus cajan. Current Science, 92: 1137–1142.
    26. Pindi, P.K. and Satyanarayana, S.D.V. (2012). Liquid Microbial Consortium- A Potential Tool for Sustainable Soil Health. Journal of Biofertilizers and Biopesticides, 3 (4): 1-9. 
    27. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution, 4(4), 406-425
    28. Schwyn, B. and Neilands, J.B. (1987). Universal chemical assay for detection and determination of siderophores. Annals in Biochemistry, 160: 47-56.
    29. Shahzad, F., Shafee, M., Abbas, F., Babar, S, Tariqq, M.M., Ahmad, Z. (2012). Isolation and biochemical characterization of Rhizobium meliloti from root nodules of alfalfa (Medicosativa), Journal of Plant and Animal Sciences 22(2):522-524. 
    30. Skidmore, A.M. and Dickinson, C.H. (1976). Colony interaction and hyphal interference between Septoria nodorum and phylloplane fungi. Transactions of the British Mycological Society, 66: 57–74.
    31. Sharma, V.K. and Dayal, B. (2005). Effect of organic and inorganic sources of nitrogen on growth, yield and nutrients uptake under cowpea-linseed cropping system. Legume Res. 28:79-86.
    32. Tan, Z. Y., Kan, F. L., Peng, G. X., Wang, E. T., Reinhold-Hurek, B., Chen, W. X. (2001). Rhizobium yanglingensc sp. nov.isolated from arid and semiarid regions in China. International Journal of Systematic and Evolutionary Microbiology, 51: 909–914.
    33. Tamura, K., J. Dudley, M. Nei, S. Kumar. (2007). MEGAA4: Molecular Evolutionary Genetics Anaysis (MEGA) software version 4.0. Mol. Biol., 24: 1596-1599.
    34. Vaishnavi, J. (2016). Growth and yield response of cowpea to multiaction bioinoculants. Legume Research-An International Journal, 39(6), 962-969.
    35. Vinuesa, P., Leon-Barrios, M., Silva, C., Willems, A., Jarabo-Lorenzo, A., Perez-Galdona, R., Werner D & Martinez-Romero, E. (2005). Bradyrhizobiumcanariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicumbv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int. J. Syst. Evol. Microbiol, 55: 569–575.
    36. Wolde-Meskel, E., Berg, T., Peters, N.K., Frostegard, A. (2004). Nodulation status of native woody legumes and phenotypic characteristics of associated Rhizobia in soils of southern Ethiopia. Biological Fertility of Soils, 40: 55-66.
    37. Zahran, H.H. (1999). Rhizobium–legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiology and Molecular Biology Reviews, 63: 968–989. 
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