Molecular identification of some plant growth promoting microorganisms and their evaluation on growth characteristic of wheat

Document Type : Complete scientific research article

Authors

1 Associate professor, Soil and Water Research Institute

2 Assistant professor, Soil and Water Research Institute, Agricultural Research, Education and Extension Organization (AREEO)

3 Associate professor, Soil and Water Research Institute, Agricultural Research, Education and Extension Organization (AREEO)

4 Former M.Sc. student, Department of Soil Science, College of Agriculture, Isfahan University of Technology, Isfahan, Iran

Abstract

Background and objectives: Wheat is one of the most important food crops. In modern agriculture, Due to the increasing human population and the detrimental effects of chemical fertilizer such as environmental pollution and concerns about human health, adapting suitable alternatives like production of bio-fertilizers which have none of these dangerous effects would be necessary. Plant growth-promoting fungi and bacteria used in bio-fertilizers are the beneficial microorganisms that can enhance plant growth, yield and resistance to biotic and abiotic stresses directly or indirectly through a wide variety of mechanisms. Although bio-fertilizers have become more noticeable in recent years, the lack of accurate identification of microorganisms used in the production of these fertilizers is one of the problems of bio-fertilizers in Iran. Failure to accurate identification of species is due to deficiencies in morphological or biochemical methods in fungi and bacteria. Therefore, the purpose of this study was to identify of fungal and bacterial isolates molecularly and also to investigate the effect of these isolates alone and in combination with arbuscular mycorrhizal fungus on some growth characteristics of the wheat plant.

Materials and methods: After the preparation of fungal and bacterial isolates from the Soil and Water Research Institute, which are known as plant growth promoting microorganisms, accurate identification of each isolate was performed based on morphological and molecular methods. Extraction of genomic DNA in arbuscular mycorrhizal fungus was done by single-spore method and in other bacterial and fungal isolates by CTAB method. Amplification of 16S rDNA region in bacterial isolates, TEF-1α and ITS1-5.8S-ITS2 regions in Trichoderma species, ITS1-5.8S-ITS2 region in Serendipita indica and SSU-ITS1-5.8S-ITS2- LSU region by nested PCR method in the arbuscular mycorrhizal fungus was performed. To investigate the effect of fungal and bacterial isolates alone or in combination with arbuscular mycorrhizal fungus in a greenhouse, their effect on wheat growth indices such as plant height, shoot and root dry weight, head height and weight and dry weights of seeds were measured.

Results: Based on morphological and molecular investigation, three species of Bacillus including Bacillus velezensis, B. pumilus, and B. subtilis and three species of Trichoderma including Trichoderma atroviride, T. longibrachiatum, and T. harzianum and S. indica and Rhizophagus irregularis were identified. T. harzianum + R. irregularis and T. longibrachiatum showed the maximum dry weight and head weight. T. longibrachiatum + R. irregularis and T. atroviride showed the highest effect on plant height. Pots treated with T. harzianum + R. irregularis, T. harzianum, T. atroviride, and S. indica+ R. irregularis showed the highest weight of seeds compared to control plants.

Conclusion: The result showed that the effect of fungal treatments such as T. harzianum + R. irregularis and Trichoderma spp. on growth characteristics of wheat was significant. According to the results of this study, the use of an appropriate combination of inoculants and the preparation of primary formulations for use in farm experiments is proposed to increase the availability of nutrients and improve the growth of wheat plants.

Keywords: Arbuscular mycorrhizal fungus, Bacillus, Plant growth promoting fungi, Trichoderma

Keywords

References

1.Al-Karaki, G.N., and Clark, R.B. 1998. Growth, mineral acquisition, and water use by mycorrhizal wheat grown under water stress. J. Plant Nutr. 21: 2. 263-276.
2.Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Research. 25: 17. 3389-3402.
3.An, Z.Q., Hendrix, J.W., Hershman, D.E., and Henson, G.T. 1990. Evaluation of the "most probable number"(MPN) and wet-sieving methods for determining soil-borne populations of endogonaceous mycorrhizal fungi. Mycologia. 82: 5. 576-581.
4.Barazani, O., Benderoth, M., Groten, K., Kuhlemeier, C., and Baldwin, I.T. 2005. Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata. Oecologia. 146: 234-243.
5.Bhuvaneswari, G., Reetha, S., Sivaranjani, R., and Ramakrishnan, K. 2014. Effect of AM fungi and Trichoderma species as stimulations of growth and morphological character of chilli (Capsicum annuum L.). Inter. J. Curr. Microbiol. Appl. Sci. 3: 3. 447-455.
6.Błaszkowski, J., Chwat, G., and Góralska, A. 2015. Acaulospora ignota and Claroideoglomus hanlinii, two new species of arbuscular mycorrhizal fungi (Glomeromycota) from Brazil and Cuba. Mycological Progress. 14: 4. 18.
7.Bolton, H., Fredrickson, J.K., and Elliot, L.F. 1993. Microbial ecology of the rhizosphere. Microbial production of plant growth regulators. In Soil microbial ecology. Applications in agricultural and enviromental management.F.B. Metting, Jr. (edit) Marcel Dekker, Inc., New York, Pp: 27-63.    
8.Buszewski, B., Rogowska, A., Pomastowski, P., Złoch, M., and Railean-Plugaru, V. 2017. Identification of microorganisms by modern analytical techniques. J. AOAC Inter.100: 6. 1607-1623.
9.Contreras-Cornejo, H.A., Macías-Rodríguez, L., Alfaro-Cuevas, R., and López-Bucio, J. 2014. Trichoderma spp. improve growth of Arabidopsis seedlings under salt stress through enhanced root development, osmolite production and Na+ elimination through root exudates. Molecular Plant-Microbe Interactions.27: 6. 503-514.
10.Contreras-Cornejo, H.A., Macías-Rodríguez, L., Cortés-Penagos, C., and López-Bucio, J. 2009. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiology. 149: 3. 1579-1592.
11.De Jaeger, N., de la Providencia, I.E., Dupré de Boulois, H., and Declerck, S. 2011. Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi. FEMS Microbiology Ecology. 77: 3. 558-567.
12.Dolatabadi, H.K., Goltapeh, E.M., Jaimand, K., Rohani, N., and Varma, A. 2011a. Effects of Piriformospora indica and Sebacina vermifera on growth and yield of essential oil in fennel (Foeniculum vulgare) under greenhouse conditions. J. Basic Microbiol. 51: 33-39.
13.Dolatabadi, H.K., Goltapeh, E.M., Moieni, A., Jaimand, K., Sardrood, B.P., and Varma, A. 2011b. Effect of Piriformospora indica and Sebacina vermifera on plant growth and essential oil yield in Thymus vulgaris in vitro and in vivo experiments. Symbiosis. 53: 29-35.
14.Doyle, J.J., and Doyle, J.L. 1990. Isolation of plant DNA from fresh tissue. Focus. 12: 13-15. 15.Druzhinina, I.S., Seidl-Seiboth, V., Herrera-Estrella, A., Horwitz, B.A., Kenerley, C.M., Monte, E., Mukherjee, P.K., Zeilinger, S., Grigoriev, I.V., and Kubicek, C.P. 2011. Trichoderma: the genomics of opportunistic success. Nature Reviews  Microbiology. 9: 10. 749-759.
16.Dubsk'y, M., Sr'amek, M., and Vos'atka, M. 2002. Inoculation of Cyclamen (Cyclamen persicum) and Euphorbia pulcherrina with arbuscular mycorrhizal fungi and Trichoderma harzianum. Rostlinna Vyroba. 48: 63-68.
17.Ehgartner, D., Herwig, C., and Fricke, J. 2017. Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry - the fast alternative to microscopic image analysis. Applied Microbiology and Biotechnology. 101: 20. 7675-7688.
 18.Fulchieri, M., and Frioni, L. 1994. Azospirillum inoculation on maize (Zea mays): effect on yield in a field experiment in central Argentina. Soil Biology and Biochemistry. 26: 7. 921-923.
19.Ghimire, S.R., Charlton, N.D., and Craven, K.D. 2009. The mycorrhizal fungus, Sebacina vermifera, enhances seed germination and biomass production in Switchgrass (Panicum virgatum L.). Bioenergy Research.2: 51-58.
20.Glick, B.R., Jacobson, C.B., Schwarze, M.M.K., and Pasternak, J.J. 1994.1-aminocyclopropane-1-carboxylic acid deaminase mutants of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 do not stimulate canola root elongation. Can. J. Microbiol.40: 911-915.
21.Harman, G.E. 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology. 962: 190-194.
22.He, X., and Nara, K. 2007. Element biofortification: can mycorrhizas potentially offer a more effective and sustainable pathway to curb human malnutrition?. Evolution. 57: 2742-2752.
23.Howell, C.R. 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Disease. 87: 1. 4-10.
24.Jayaraj, J., Radhakrishnan, N.V., and Velazhahan, R. 2006. Development of formulations of Trichoderma harzianum strain M1 for control of damping-off
of tomato caused by Pythium aphanidermatum. Archives of Phytopathology and Plant Protection. 39: 1. 1-8.
25.Jiang, H., Dong, H., Zhang, G., Yu, B., Chapman, L.R., and Fields, M.W. 2006. Microbial diversity in water and sediment of LakeChaka, an athalassohaline lake in northwestern China. Applied and Environmental Microbiology. 72: 6. 3832-3845.
26.Kafer, E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Advances in Genetic. 19: 33-131.
27.Kloepper, J.W., Lifshitz, R., and Zablotowicz, R.M. 1989. Free-living bacterial inocula for enhancing crop productivity. Trends in Biotechnology. 7: 2. 39-44.
28.Krüger, M., Stockinger, H., Krüger, C., and Schüßler, A. 2009. DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytologist. 183: 1. 212-223.
29.Kumar, R., Tapwal, A., da Silva, J.A.T., Pandey, S., and Borah, D. 2013. Diversity of arbuscular mycorrhizal fungi associated in a mixed natural forest of Jeypore, Assam. Bioremediation, Biodiversity and Bioavailability. 7: 1. 91-93.
30.Kumari, R., Yadav, H.K., Bhoon, Y.K., and Varma, A. 2003. Colonization of cruciferous plants by Piriformospora indica. Current Science. 85: 1672-1674.
31.Martínez-Medina, A., Pascual, J.A., Lloret, E., and Roldán, A. 2009. Interactions between arbuscular mycorrhizal fungi and Trichoderma harzianum and their effects on Fusarium wilt in melon plants grown in seedling nurseries. J. Sci. Food Agric.89: 11. 1843-1850.
32.Martens, M., Dawyndt, P., Coopman, R., Gillis, M., De Vos, P., and Willems, A. 2008. Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Inter. J. Syst. Evol. Microbiol. 58: 1. 200-214.
33.Medina, A., Probanza, A., Mañero, F.G., and Azcón, R. 2003. Interactions of arbuscular-mycorrhizal fungi and Bacillus strains and their effects on plant growth, microbial rhizosphere activity (thymidine and leucine incorporation) and fungal biomass (ergosterol and chitin). Applied Soil Ecology. 22: 1. 15-28.
34.Mohamed, H., and Haggag, W. 2006. Boicontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum. Brazil. J. Microbiol. 37: 181-191.
 35.Monte, E. 2001. Understanding Trichoderma: between biotechnology and microbial ecology. International Microbiology. 4: 1. 1-4.
36.Moreira, B.C., Mendes, F.C., Mendes, I.R., Paula, T.A., Junior, P.P., Salomao, L.C.C., Stürmer, S.L., Otoni, W.C., and Kasuya, M.C.M. 2015. The interaction between arbuscular mycorrhizal fungi and Piriformospora indica improves the growth and nutrient uptake in micropropagation-derived pineapple plantlets. Scientia Horticulturae. 197: 183-192.
37.Mwangi, M.W., Monda, E.O., Okoth, S.A., and Jefwa, J.M. 2011. Inoculation of tomato seedlings with Trichoderma harzianum and arbuscular mycorrhizal fungi and their effect on growth and control of wilt in tomato seedlings. Brazil. J. Microbiol. 42: 2.508-513.
38.Oelmuller, R., Sherameti, I., Tripathi, S., and Varma, A. 2009. Piriformospora indica, a cultivable root endophyte with multiple biotechnological applications. Symbiosis. 49: 1-17.
39.O’Donnell, K., Kistler, H.C., Cigelnik, E., and Ploetz, R.C. 1998. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proceedings of the NationalAcademy of Sciences. 95: 5. 2044-2049.
40.Peskan-Berghoefer, T., Shahollaria, B., Giong, P.H., Hehl, S., Markerta, C., Blanke, V., Kost, G., Varma, A., and Oelmeuller, R. 2004. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmatic reticulum and at the plasma membrane. Physiologia Plantarum. 122: 465-477.
41.Pham, G.H., Singh, A., Kumari, R., Malla, R., Prasad, R., Sachdev, M., Rexer, K.H., Kost, G., Luis, P., Kaldorf, M., Buscot, F., Herrrmann, S., Peskan, T., Oelmüller, R., Saxena, A.K., Declerck, S., Mittag, M., Stabentheinerv, E., Hehl, S., and Varma, A. 2004. Interactive of Piriformospora indica with diverse microorganisms in plants. In: Varma, A., Abbott, L., Werner, D. and Hampp, R. (eds). Plant Surface Microbiology. Springer-Verlag, Berlin, Pp: 237-265.
42.Rai, M., Acharya, D., Singh, A., and Varma, A. 2001. Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza. 11: 123-128.
43.Samuels, G.J. 1996. Trichoderma: a review of biology and systematics of the genus. Mycological Research. 100: 923-935.
44.Shahollari, B., Vadassery, J., Varma, A., and Oelmuller, R. 2007. A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana. Plant J. 50: 1-13.
45.Shoresh, M., Harman, G.E., and Mastouri, F. 2010. Induced systemic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology. 48: 21-43.
46.Sirrenberg, A., Göbel, C., Grond, S., Czempinski, N., Ratzinger, A., Karlovsky, P., Santos, P., Feussner,I., and Pawlowski, K. 2007. Piriformospora indica affects plant growth by auxin production. Physiologia Plantarum. 131: 581-589.
47.Srinivasan, M., Kumar, K., Kumutha, K., and Marimuthu, P. 2014. Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus intraradices through root organ culture. J. Appl. Natur. Sci. 6: 1. 290-293.
48.Srivastava, N.H., Bhandari, V., and Bhatt, A.B. 2014. PGPR Isolated from rhizospheric soil of Zanthoxylum armatum DC. in Garhwal Himalaya. Inter. J. Herbal Med. 2: 1. 100-108.
49.Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution. 30: 12. 2725-2729.
50.Tannock, G.W. 1999. Identification of lactobacilli and bifidobacteria. Current Issues in Molecular Biology. 1: 1. 53-64.
51.Tsvetkov, I., Dzhambazova, T., Kondakova, V., and Batchvarova, R. 2014. Mycorrhizal fungi Glomus spp. and Trichoderma spp. in viticulture. Bulgari. J. Agric. Sci. 20: 4. 849-855.
52.Ueda, K., Seki, T., Kudo, T., Yoshida, T., and Kataoka, M. 1999. Two distinct mechanisms cause heterogeneity of 16S rRNA. J. Bacteriol. 181: 1. 78-82.
53.Vadassery, J., Ritter, C., Venus, Y., Camehl, I., Varma, A., Shahollari, B., Novák, O., Strnad, M., Ludwig-Müller, J., and Oelmüller, R. 2008. The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and  Piriformospora indica. Molecular Plant-Microbe Interactions. 21: 10. 1371-1383.
54.Verma, S., Varma, A., Rexer, K.H., Kost, G., Sarbhoy, A., Bisen, P., Butehorn, B., and Franken, P. 1998. Piriformospora indica, gen. et sp. nov., a new root-colonizing fungus. Mycologia. 95: 896-903.
55.White, T.J., Bruns, T., Lee, S.J.W.T., and Taylor, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a Guide to Methods and Applications. 18: 1. 315-322.
56.Yang, X., and Hong, C. 2018. Differential usefulness of nine commonly used genetic markers for identifying Phytophthora species. Frontiers in Microbiology. 9. 2334.
Journal of Soil Management and Sustainable Production
Volume 9, Issue 3
December 2020
Pages 47-68

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