Isolation, screening and identification of growth-promoting rhizobacteria resistant to abiotic stresses from the microbiome of Alfalfa (Medicago sativa) in saline and arid soils in Kerman province

Document Type : Research Paper

Authors

1 Department of Soil Science, Agriculture Faculty, Shahid Bahonar University of Kerman, Kerman, Iran.

2 Department of Soil Science, Faculty of Agriculture, Vali-e-asr University of Rafsanjan, Kerman, Iran.

Abstract

The purpose of this study is to investigate the characteristics of plant growth promoting rhizobacteria that stimulate plant growth, such as biological nitrogen fixation, phosphate solubilization, and produce hydrogen cyanide and exopolysaccharides, to prepare suitable and efficient biofertilizers. In this research, 135 Rhizobacteria strains were isolated, screened and identified from saline and arid soils in Kerman Province, and some of their growth-promoting characteristics were studied. The Rhizobacteria strains were studied with respect to their ability to fix nitrogen, solubilize insoluble organic and inorganic phosphates in solid and liquid culture media, and produce hydrogen cyanide and exopolysaccharides. Their growth at various sodium chloride concentrations (0, 1, 3, and 5%) and at different polyethylene glycol 6000 concentrations (water potential 0, -1, -2, and -3.5 MPa) was also investigated. According to the results, 72% and 76% of the Rhizobacteria strains exhibited almost no growth at 5% sodium chloride concentration and at water potential -3.5 MPa, respectively. In addition, 50% of them were able to produce hydrogen cyanide under saline conditions. Three of the strains (SM16, SM65, and SM89) were the most efficient producers of exopolysaccharides and had high phosphate solubilization ability (102, 98.5 and 121.7 mL/L, respectively) and were also among Rhizobacteria strains resistant to abiotic stresses. Three strain safe and efficient strains can have great potential to be used as a high quality biofertilizer for improving growth of crop plants in the region.

Keywords

Main Subjects


EXTENDED ABSTRACT

Introduction:

Microorganisms are often exposed to environmental stresses such as limited nutrient availability, salinity and sudden changes in osmolarity, drought stress, and temperature rise or fall. Proper responses of bacteria under these conditions are necessary for them to adapt effectively to environmental stresses and changes. Sinorhizobium bacteria inside alfalfa roots can be used for various purposes in agriculture. Some of these bacteria are also among plant growth-promoting Rhizobacteria that stimulate and promote plant growth often via one or more mechanisms, such as biological nitrogen fixation, phosphate solubilization, siderophore and the exopolysaccharide production, and increased availability of nutrients required by plants.  

 

Materials and methods:

In this research, 135 Rhizobacteria strains were isolated, screened and identified from saline and arid soils in Kerman Province, and some of their growth-promoting characteristics were studied. The Rhizobacteria strains were studied with respect to their ability to fix nitrogen, solubilize insoluble organic and inorganic phosphates in solid and liquid culture media, and produce hydrogen cyanide and exopolysaccharides. Their growth at various sodium chloride concentrations (0, 1, 3, and 5%) and at different polyethylene glycol 6000 concentrations (water potential 0, -1, -2, and -3.5 MPa) was also investigated.  

 

Results and discussion:

 According to the results, 72% and 76% of the Rhizobacteria strains exhibited almost no growth at 5% sodium chloride concentration and at water potential -3.5 MPa, respectively. In addition, 50% of them were able to produce hydrogen cyanide under saline conditions. Three of the strains (SM16, SM65, and SM89) were the most efficient producers of exopolysaccharides (364, 318.7 and 198.3 µg/mL, respectively) at 3% sodium chloride stress level, had high phosphate solubilization ability (102, 98,5 and 121.7 mL/L, respectively) and were also among Rhizobacteria strains resistant to abiotic stresses.  

 

Conclusions:

The viability and effectiveness of native Sinorhizobium bacteria are reduced due to salt and osmotic stress, water scarcity, high soil temperature, and soil acidity and alkalinity. Sinorhizobium strains differ in their tolerance to environmental factors. Inoculation of alfalfa with native strains of Sinorhizobium adapted to specific environments and resistant to abiotic stresses, on the one hand, can increase nodulation and nitrogen fixation under stress conditions, on the other hand, to improve plant growth and yield through their potential to stimulate plant growth. Inoculating alfalfa with effective strains of Sinorhizobium has significant economic and environmental benefits. The main purpose of this study was to select native Sinorhizobium strains isolated from saline and dry regions in Kerman Province that were tolerant to unfavorable environmental conditions and were also able to stimulate plant growth. These safe and efficient strains can have great potential to be used as a high quality biofertilizer for improving growth of crop plants in the region.

Agbodjato, N. A., Noumavo, P. A., Baba-Moussa, F., Salami, H. A., Sina, H., Sezan, A., Bankole, H., Adjanohoun, A., & Baba-Moussa, L. (2015). Characterization of potential plant prowth promoting rhizobacteria isolated from maize (Zea mays L.) in central and northern benin (west africa). Applied and Environtal Soil Science, Article ID 901656, 9 pages. https://doi.org/10.1155/2015/901656.
Ahmad, M., Nadeem, S. M., Naveed, M., & Zahir, Z. A. (2016). Potassium-solubilizing bacteria and their application in agriculture. In: Meena, V., Maurya, B., Verma, J., Meena, R. (eds). Potassium solubilizing microorganisms for sustainable agriculture. Springer, New Dehli, pp, 293-313. https://doi.org/10.1007/978-81-322-2776-2_21.
Ali, S. Z.; Sandhya, V., & Rao, L. V. (2013). Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide producing fluorescent Pseudomonas sp. Annals of Microbiology, 64, 493–502. https://doi.org/10.1007/s13213-013-0680-3.
Alori, E. T., Emmanuel, O. C., Glick, B. R., & Babalola, O. O. (2020). Plant-archaea relationships: A potential means to improve crop production in arid and semiarid regions. World Journal Microbiolology and Biotechnology, 36(9), 133. https://doi.org/10.1007/s11274-020-02910-6. PMID: 32772189.
Altieri, M. A. (2004). Linking ecologists and traditional farmers in the search for sustainable agriculture. Frontiers in Ecology and the Environment, 2 (1), 35-42. https://doi.org/10.1890/1540-9295.
Asghari, B., Khademian, R., & Sedaghati, B. (2019). Plant growth promoting rhizobacteria (PGPR) confer drought resistance and stimulate biosynthesis of secondary metabolites in pennyroyal (Mentha pulegium L.) under water shortage condition. Scientia Horticculturae, 263, 109132. https://doi.org/10.1016/j.scienta.2019.109132.
Azib, S., Cheloufi, H., Attb, S., Bouras, N., & Holtz, M. D. (2022). Phenotypic and genotypic diversity of microsymbionts nodulating Medicago sativa (L.) in the Algerian Sahara. Jordan Journal of biological Sciences, 15(2), 227-238. https://doi.org/10.54319/jjbs/150210.
Badri, D. V., & Vivanco, J. M. (2009). Regulation and function of root exudates. Plant, Cell & Environment, 32, 666–681. https://doi.org/10.1111/j.1365-3040.
Beck, D. P., Materon, L. A., & Afandi, F. (1993). Practical rhizobium legume technology manual. ICARDA. pp, 1-104. https://hdl.handle.net/20.500.11766/67561.
Bogati, K., & Walczak, M. (2022). The impact of drought stress on soil microbial community, enzyme activities and plants. Agronomy, 12 (1), 189. https://doi.org/10.3390/agronomy12010189.
Calvo, P., Nelson, L., & Kloepper, J. W. (2014). Agricultural uses of plant biostimulants. Plant and Soil, 383(12), 3-41. https://doi.org/10.1007/s11104-014-2131-8.
Castric, P. A. (1974). Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa. Canadian Journal of Microbiology, 21(5), 613-618. https://doi.org/10.1139/m75-088.
Chakraborty, B. N., Chakraborty, U., Saha, A., Sunar, K., & Dey, P. L. (2010). Evaluation of phosphate solubilizes from soils of north Bengal and their diversity analysis. World Journal of Agricultural Sciences, 6(2), 195-200. https://www.researchgate.net/publication/242630621.
Compant, S., Samad, A., Faist, H., & Sessitsch, A. (2019). A review on the plant microbiome: Ecology, functions, and emerging trends inmicrobial application. Journal of Advanced Research, 19, 29–37. https://doi.org/10.1016/j.jare.2019.03.004.
Cotteni, I. (1980). Methods of plant analysis. In: Soil and Plant Testing as a base for fertilizer recommendation. FAO Soils Bulletin, 38(2), 67-100.
Crowley, D. E. (2006). Microbial siderophores in the plant rhizospheric. In: Barton, L. L., Abadia, J. (eds) Iron Nutrition in Plants and Rhizospheric Microorganisms; Springer, Dordrecht, The Netherlands, pp. 169–198. https://doi.org/10.1007/1-4020-4743-6_8.
Dalal, R. C. (1977). Soil organic phosphorus. Advances in Agronomy, 29, 83-117. https://doi.org/10.1016/S0065-2113(08)60216-3.
Egamberdiyeva, D. (2009). Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat. Acta Physiologiae Plantarum, 31(4), 861-864. https://doi.org/10.1007/s11738-009-0297-0
Farissi, M., Ghoulam, C., & Bouizgaren, A. (2014). The effect of salinity on yield and forage quality of alfalfa populations in the Marrakech region (Morocco). Fourrages, 219, 271-275.
Gamalero, E., & Glick, B. R. (2019). Plant growth promoting bacteria in agriculture and stressed environments. In: Van Elsas, J. D., TreVors, J. T., Soares Rosado, A. & Nannipieri, P., (eds). Modern Soil Microbiology, (3rd ed.); CRC Press: Boca Raton, FL, USA; pp. 361–380. https://doi.org/10.1201/9780429059186.
Gauri, S. S., Mandal, S. M., Mondal, K. C., Dey, S., & Pati, B. R. (2009). Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81. Bioresource Technology, 100, 4240-4243. https://doi.org/10.1016/j.biortech.2009.03.064.
Glick, B. R. (2012). Plant growth-promoting bacteria: Mechanisms and applications. Scientifica, Article ID 963401. https://doi.org/10.6064/2012/963401.
Gupta, R., Anand, G., Gaur, R., & Yadav, D. (2021). Plant-microbiome interactions for sustainable agriculture: A review. Physiology and Molecular Biology of Plants, 27(1), 165–179. https://doi.org/10.1007/s12298-021-00927-1.
Ilyas, N., Mumtaz, K., Akhtar, N., Yasmin, H., Sayyed, R., Khan, W., Enshasy, H., Dailin, D., Elsayed, E., & Ali, Z. (2020). Exopolysaccharides producing bacteria for the amelioration of drought stress in wheat. Sustainability, 12, 8876. https://doi.org/10.3390/su12218876.
 Intorne, A. C., Oliveira, M. V. V., Lima, M. L., Silva, J. F., Olivares, F. L., & Filho, G. A. D. (2009). Identification and characterization of gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc. Archives of Microbiology, 191(5), 477–483. https://doi.org/10.1007/s00203-009-0472-0.
Kapadia, C., Sayyed, R. Z., El Enshasy, H. A., Vaidya, H., Sharma, D., Patel, N., Malek, R. A., Syed, A., Elgorban, A. M., & Ahmad, K. (2021). Halotolerant microbial consortia for sustainable mitigation of salinity stress, growth promotion, and mineral uptake in tomato plants and soil nutrient enrichment. Sustainability, 13(25), 8369. https://doi.org/10.3390/su13158369.
Kavamura, V. N., Santos, S. N., Silva, J. L., Parma, M. M., Avila, L. A., Visconti, A., Zucchi, T. D., Taketani, R. G., Andreote, F. D., & Melo, I. S. (2013). Screening of Brazilian cacti rhizobacteria for plant growth promotion under drought. Microbiological Research, 168(4), 183–191. https://doi.org/10.1016/j.micres.2012.12.002.
Korenblum, E., Massalha, H., & Aharoni, A. (2022). Plant–microbe interactions in the rhizosphere via a circular metabolic economy. The Plant Cell, 34(9), 3168-3182. https://doi.org/10.1093/plcell/koac163.
Kumar, A., Verma, H., Singh, V. K., Singh, P. P., Singh, S. K., Ansari, W. A. (2017). Role of pseudomonas sp. in sustainable agriculture and disease management. In: Meena, V., Mishra, P., Bisht, J., Pattanayak, A. (eds) Agriculturally important microbes for sustainable agriculture. Springer, Singapore, pp. 195-215. https://doi.org/10.1007/978-981-10-5343-6_7.
Kumar, J. C., & Saraf, M. (2015). Plant growth promoting rhizobacteria (PGPR): A review. Journal of Agricultural Research and Development, 5(2), 0108-0119. https://doi.org/10.13140/RG.2.1.5171.2164.
Latrach, L., Mouradi, M., Farissi, M., Bouizgaren, A., & Ghoulam, C. (2017). Physiological characterization of rhizobial strains nodulating alfalfa (Medicago sativa) isolated from soils of Southeastern Morocco. Applied Journal of Envionmentalr Engineeering Science, 3(4), 353-364. https://doi.org/10.48422/IMIST.PRSM/ajees-v3i4.10032.
Lau, J. A., & Lennon, J. T. (2012). Rapid response of soil microorganisms improve plant fitness in novel environments. Proceeding of the National Academy of Sciences, 109(35), 14058–14062. https://doi.org/10.1073/pnas.1202319109. ·
Leontidou, K., Genitsaris, S., Papadopoulou, A., Kamou, N., Bosmali, I., Matsi, T., Madesis, P., Vokou, D., Karamanoli, K., & Mellidou, I. (2020). Plant growth promoting rhizobacteria isolated from halophytes and drought-tolerant plants: Genomic characterisation and exploration of phyto-beneficial traits. Scientific Reports, 10, 14857. https://doi.org/10.1038/s41598-020-71652-0.
Liu, W., Wang, Q., Hou, J., Tu, C., Luo, Y., & Christie, P. (2016). Whole genome analysis of halotolerant and alkalotolerant plant growthpromoting rhizobacterium Klebsiella sp. D5A. Scientific Reports, 6, 26710. https://doi.org/10.1038/srep26710.
Mahantesh, P., & Patil, C. S. (2011). Isolation and biochemical characterization of phosphate solubilizing microbes. International Journal of Microbiology Research, 3(1), 67-70.
Mahgoub, H. A., Fouda, A., Eid, A. M., Ewais, E. E., & Hassan, S. E. (2021). Biotechnological application of plant growth-promoting endophytic bacteria isolated from halophytic plants to ameliorate salinity tolerance of Vicia faba L. Plant Biotechnology Reports, 15, 819–843. https://doi.org/10.1007/s11816-021-00716-y.
Mansour, E., Mahgoub, H. A. M., Mahgoub, S. A., El-Sobky, E., Abdul-Hamid, M. I., Kamara, M. M., Abu Qamar, S. F., El-Rarabily, K. A., & Desoki, E. S. M. (2021). Enhancement of drought tolerance in diverse Vicia faba cultivars by inoculation with plant growth-promoting rhizobacteria under newly reclaimed soil conditions. Scientific Reports, 11, 24142. https://doi.org/10.1038/s41598-021-02847-2.
Mavrodi, D. V., Mavrodi, O. V., Parejko, J. A., Bonsall, R. F., Kwak, Y. S., Paulitz, T. C., Thomashow, L. S., & Weller, D. M. (2012). Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland cereals. Applied and Environmental Microbiology, 78(3), 804–812. https://doi.org/10.1128/AEM.06784-11.
Merabet, C., Bekki, A., Benrabah, N., Bey, M., Bouchentouf, L., Ameziane, H., Rezki, M. A., Domergue, O., Cleyet Marel, J. C., Avarre, J. C., Bena, G., Bailly, X., & De Lajudie, P. (2006). Distribution of Medicago species and their microsymbionts in a saline region of Algeria. Arid Land Research and Managment, 20(3), 219-231. https://doi.org/10.1080/15324980600705685.
Morcillo, R., & Manzanera, M. (2021). The Effects of plant-associated bacterial exopolysaccharides on plant abiotic stress Tolerance. Metabolites, 11(6), 337. https://doi.org/10.3390/metabo11060337.
Mumtaz, Z. M., Ahmad, M., Jamil, M., Asad, S. A., & Hafeez, F. (2018). Bacillus strains as potential alternate for zinc biofortification of maize grains. International Journal of Agriculture and Biology, 20(8), 1779–1786. https://doi.org/10.17957/IJAB/15.0690.
Nadeem, S. M., Ahmad, M., Tufail, M. A., Asghar, H. N., Nazli, F., & Zahir, Z. A. (2020). Appraising the potential of EPS-producing rhizobacteria with ACC-deaminase activity to improve growth and physiology of maize under drought stress. Physiologia Plantarum, 172(2), 463–476. https://doi.org/10.1111/ppl.13212.
Nazli, F., Wang, X., Ahmad, M., Hussain, A., Bushra Dar, A., Nasim, M., Jamil, M., Panpluem, N., & Mustafa, A. (2021). Efficacy of indole acetic acid and exopolysaccharides-producing Bacillus safensis strain FN13 for inducing Cd-stress tolerance and plant growth promotion in Brassica juncea (L.). Applied Sciences, 11(9), 4160. https://doi.org/10.3390/app11094160.
Nguyen, P. T., Nguyen, T. T., Bui, D. C., Hong, P. T., Hoang, Q. K., & Nguyen, H. T. (2020). Exopolysaccharide production by lactic acid bacteria: The manipulation of environmental stresses for industrial applications. AIMS Microbiology, 6(4), 451–469. https://doi.org/10.3934/microbiol.2020027.
Oteino, N., Lally, R. D., Kiwanuka, S., Lloyd, A., Ryan, D., & Germaine, K. J. (2015). Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Frontiers in Microbiology, 6, 745. https://doi.org/10.3389/fmicb.2015.00745.
Paustian, K., Lehmann, J., Ogle, S., Reay, D., Robertson, G.P., Smith, P. (2016). Climate smart soils. Nature, 532, 49-57. https://doi.org/10.1038/nature17174.
Primo, E., Bogino, P., Cossovich, S., Foresto, E., Nievas, F., & Giordano, W. (2020). Exopolysaccharide II is relevant for the survival of Sinorhizobium meliloti under water deficiency and salinity stress. Molecules, 25(21), 4876. https://doi.org/10.3390/molecules25214876.
Rubiano-Labrador, C., Bland, C., Miotello, G., Armengaud, J., & Baena, S. (2015). Salt stress induced changes in the exoproteome of the halotolerant bacterium Tistlia consotensis deciphered by proteogenomics. PLOS ONE, 10, 135065. https://doi.org/10.1371/journal.pone.0135065.
Saleem, M., Nawaz, F., Hussain, M. B., & Ikram, R. M. (2021). Comparative effects of individual and consortia Plant Growth Promoting Bacteria on physiological and enzymatic mechanisms to confer drought tolerance in maize (Zea mays L.). Journal Soil Science and Plant Nutrition, 21, 3461–3476. https://doi.org/10.1007/s42729-021-00620-y.
Sharath, S., Triveni, S., Nagaraju, Y., Latha, P. C., & Vidyasagar, B. (2021). The role of phyllosphere bacteria in improving cotton growth and yield under drought conditions. Frontiers in Agronomy, 3, 466. https://doi.org/10.3389/fagro.2021.680466.
Sharma, A., Shahzad, B., Kumar, V., Kohli, S. K., Sidhu, G. P. S., Bali, A. S., Handa, N., Kapoor, D., Bhardwaj, R., & Zheng, B. (2019). Phytohormones regulate accumulation of osmolytes under abiotic stress. Biomolecules, 9(7), 285. https://doi.org/10.3390/biom9070285.
Sharon, J. A., Hathwaik, L. T., Glenn, G. M., Imam, S. H., & Lee, C. C. (2016). Isolation of efficient phosphate solubilizing bacteria capable of enhancing tomato plant growth. Journal of Soil Science and Plant Nutrition, 16(2), 525-536. http://dx.doi.org/10.4067/S0718-95162016005000043.
Singh, V. K., Singh, A. K., & Kumar, A. (2017). Disease management of tomato through PGPB: current trends and future perspective. 3 Biotech, 7(4), 255. https://doi.org/10.1007/s13205-017-0896-1.
Sperber, J. I. (1958). The incidence of apatite solubilizing organisms in the rhizosphere and soil. Australian Journal of Agricultural Research. 9(6), 778-781. https://doi.org/10.1071/AR9580778.
Surange, S., Wollum, A.G., Kumar, N., & Natutiyal, C.S. (1997). Characterisation of Rhizobium from root noudules of leguminous trees growing in alkaline soils. Canadian Journal of Microbiology, 43, 891-894 https://doi.org/10.1139/m97-130.
Timmusk, S., Paalme, V., Pavlicek, T., Bergquist, J., Vangala, A., Danilas, T., & Nevo, E. (2011). Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates. PLOS ONE, 6, 17968. https://doi.org/10.1371/journal.pone.0017968.
Whalley, W. A., Bengough, A. G., & Dexter, A.R. (1998). Water stress induced by PEG decreases the maximum growth pressure of the roots of pea seedling. Journal of Experimental Botany, 49(327), 1689-1694. https://doi.org/10.1093/jxb/49.327.1689.
Wood, N. T. (2001). Nodulation by numbers: the role of ethylene in symbiotic nitrogen fixation. Trends in Plant Science, 6(11), 501-502. https://doi.org/10.1016/S1360-1385(01)02128-8.
Yanez-Yazlle, M. F., Romano-Armada, N., Acreche, M. M., Rajal, V. B., Irazusta, V. P. (2021). Halotolerant bacteria isolated from extreme environments induce seed germination and growth of chia (Salvia hispanica L.) and quinoa (Chenopodium quinoa Willd.) under saline stress. Ecotoxicology and Environmental Safety, 218, 112273. https://doi.org/10.1016/j.ecoenv.2021.112273.
Yasmin, H., Naeem, S., Bakhtawar, M., Jabeen, Z., Nosheen, A., Naz, R., Keyani, R., Mumtaz, S., & Hassan, M.N. (2020). Halotolerant rhizobacteria Pseudomonas pseudoalcaligenes and Bacillus subtilis mediate systemic tolerance in hydroponically grown soybean (Glycine max L.) against salinity stress. PLOS ONE, 15, 0231348. https://doi.org/10.1371/journal.pone.0231348.
Zahran, H. H. (2001). Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology. Journal of Biotechnology, 91(2-3), 143-153. https://doi.org/10.1016/s0168-1656(01)00342-x.