ABB  Vol.2 No.4 , August 2011
Mutational search for high temperature (60oC) tolerant variant of Rhizobium species CWP G34A—Mutation generates high temperature variant of Rhizobium species Cwp G34A
ABSTRACT
This study focused on the development of thermophilic strain/s of a cowpea (Vigna unguiculata) compatible nitrogen fixing bacterium. A preliminary plant screening was carried out using some strains of tropical rhizobia and cowpea. Rhizobium species CWP G34A that formed Fix+ nodules repeatedly was selected for further studies. First, it was tested for growth at high temperatures of 40 to 55oC at 5oC interval with 28oC as the control temperature. Mutagenesis was conducted on the bacterium with ethylmethane sulphonate (EMS). The wildtype and mutants generated were tested for high temperature tolerance by growing them individually in nutrient broth at 60oC for 24 hours. Optical density (670 nm) was read before and after incubation. The mutants were grouped into classes based on percentage difference in OD values obtained before and after exposure to 60oC. Rhizobium species CWP G34A produced functional pink nodules on the cowpea consistently in three different plant tests. There was no growth at all the temperatures tested except at 28oC and 40oC after 24 hours of incubation. It grew better at former (51 × 1010 Cfu/ml) than latter (11 Cfu/ml) temperature. Like the parental strain, all the mutants but one, did not grow after exposure to 60oC. Sixty degree centigrade caused various reductions in optical density (OD) values of the variants. Eleven classes of the mutants were formed with membership percentage ranging from 1 to 22%. Class 1 contains only one member while class 11 has the highest mutant population of 22% with OD difference of 0 to 10% and –90 to –100% respectively. The high percentage reduction in the OD of variants in class 11 is similar to that of the unmutated cells (–94.56%). The only mutant that survived the 60oC and grew was MU70. An increase of 1.67% in OD was obtained for MU70. Mutant MU70 therefore appeared a promising strain that can be further tested to inoculate cowpea in the dry and warm season for increased nitrogen fixation. This will provide encouraging information for farmers to grow the cowpea throughout the year particularly under high temperatures in summer in order to boost the yield of the legume.

Cite this paper
nullBoboye, B. , Ogundeji, B. and Evbohoin, H. (2011) Mutational search for high temperature (60oC) tolerant variant of Rhizobium species CWP G34A—Mutation generates high temperature variant of Rhizobium species Cwp G34A. Advances in Bioscience and Biotechnology, 2, 255-262. doi: 10.4236/abb.2011.24037.
References
[1]   Marsh, L.E., Baptiste, R., Marsh, D.B., Trinklein, D., Kremer, R.J. (2006) Temperature effects on Bradyrhizobium spp. growth and symbiotic effectiveness with pigeon pea and cowpea. Journal of Plant Nutrition, 29(3), 331-346.

[2]   Zahran, H.H. (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in arid climate. Microbiology Molecular Biology Review, 63(4), 968-989.

[3]   Jensen, J.B., Peters, N.K., and Bhuvaneswari, T.V. (2002) Redundancy in periplasmic binding protein-dependent transport systems for trehalose, sucrose, and maltose in Sinorhizobium meliloti. Journal of Bacteriology, 184, 2978-2986.

[4]   Graham, P.H. (1992) Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Canadian Journal of Microbiology, 38, 475-484.

[5]   Allison, F.E. and Minor, F.W. (2010) The effects of temperature on growth rates of Rhizobia. HUwww.ncbi.nlm.nih.gov/pmc/articles/pmc3745/78UH.

[6]   Eaglesham, A.R.J. and Ayanaba, A. (1984) Tropical stress ecology of rhizobia, root-nodulation and legume fixaton. In: Current Developments in Biological Nitrogen Fixation. Edited by: Subba Rao N.S. Edward Arnold Publishers, London, UK; pp.:1-35.

[7]   Hartel, P.G. and Alexander, M. (1984) Temperature and desiccation tolerance of cowpea rhizobia. Canadian Journal of Microbiology, 30, 820-823.

[8]   Karanja, N.K. and Wood, M. (1988) Selecting Rhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya. Tolerance of high temperature and antibiotic resistance. Plant Soil, 112, 15-22.

[9]   Elboutahiri, N., Thami-Alami, I. and Udupa, S.M. (2010) Phenotypic and genetic diversity in Sinorhizobium meliloti and S. medicae from drought and salt affected regions of Morocco. BMC Microbiology, 10, 15.

[10]   Segovia, L., Pinero, D., Palacios, R. and Martinez Romero, E. (1991) Genetic structure of a soil population of non-symbiotic Rhizobium leguminosarum. Applied and Environmental Microbiology, 57, 426-433.

[11]   Munevar, F. and Wollum, A.G. (1982) Response of soybean plants to high root temperature as affected by plant cultivar and Rhizobium strain. Agronomy Journal, 74, 138-142.

[12]   Arayankoon, T., Schomberg, H.H. and Weaver, R.W. (1990) Nodulation and N2 fixation of guar at high root temperature. Plant Soil, 126, 209-213.

[13]   Kishinevsky, B.D., Sen, D. and Weaver, R.W. (1992) Effect of high root temperature on Bradyrhizobium- peanut symbiosis. Plant Soil, 143, 275-282.

[14]   Rainbird, R.M., Akins, C.A. and Pate, J.J.S. (1983). Effect of temperature on nitrogenase functioning in cowpea nodules. Plant Physiology, 73, 392-394.

[15]   Hungria, M. and Franco, A.A. (1993) Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L. Plant Soil, 149, 95-102.

[16]   Piha, M.I. and Munnus, D.N. (1987) Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature. Plant Soil, 98, 183-194.

[17]   Roughley, R.J. (1970) The influence of root temperature, Rhizobium strain and host selection on the structure and nitrogen-fixing efficiency of the root nodules of Trifolium subterraneum. Annals of Botany, 34, 631-646.

[18]   Roughley, R.J. and Dart, P.J. (1970) Root temperature and root-hair infection of Trifolium subterraneum L. cv. Cranmore. Plant Soil, 32, 518-520.

[19]   Munns, D.N., Keyser, H.H., Fogle, V.W., Hohenberg, J.S., Righetti, T.L., Lauter, D.L., Zaruog, M.G., Clarkin, K.L. and Whitacre, K.W. (1979) Tolerance of soil acidity in symbiosis of mung bean with rhizobia. Agronomy Journal, 71, 256-260.

[20]   Hernandez-Armenta, R., Wien, H.C. and Eaglesham, A.R.J. (1989) Maximum temperature for nitrogen fixation in common bean. Crop Science, 29, 1260-1265.

[21]   Broughton, W.J. and Dilworth, M.J. (1971) Control of leghemoglobin in Snake beans. Biochemical Journal, 125, 1075-1080.

[22]   Parkinson, J.S. (1976) Che A, Che B and Che C genes of Escherichia coli and their role in chemotaxis. Journal of Bacteriology, 126, 758-770.

[23]   Boboye, B. and Alao, A. (2008) Effect of mutation on Trehalose-catabolic-enzyme synthesized by a tropical Rhizobium species F1. Research Journal of Microbiology, 3(4), 269-275.

[24]   Powar, C.B. and Diganawala, H.F. (1992) General Microbiology. Eight edition. Himalay Publishing House, 598-600.

[25]   Hirsch. A.M., Lum, M.R. and Downie, J.A. (2001) What makes the rhizobia-legume symbiosis so special?. Plant Physiology, 127, 1484-1492.

[26]   Pelczar, M.J., Reid, R.D. and Chan, E.C.S. (1983) Microbiology. Fourth Edition. Tata Mcgraw-Hill Publishing Company, 111.

[27]   Brock, T. D., Smith, D. W., and Madigan, M. T. (1984). Biology of Microorganisms. Fourth Edition. Prentice-Hall International, Inc. 244, 310.

[28]   Prescott, L.M., Harley, J.P. and Klein, D.A. (2005) Microbiology. Sixth International Edition. Mcgraw-Hill Publishing Company, UK, 652-668.

[29]   Suzuki, D.T., Griffin, J.E., Miller, H.J. and Lewontin, C.R. (2001) An Introduction to Genetic Analysis. 7th Edn. Freeman Publishers, USA, 101-105.

 
 
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