Iranian Journal of Plant Protection Science

Iranian Journal of Plant Protection Science

Biocontrol potential of Bacillus strains in interaction with Rhizoctonia solani pathogen of potato

Document Type : Research Paper

Authors
Hossein Norozi 1
Sareh Baghaee-Ravari 2
Shideh Mojerlou 3
1 1. Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
2 Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
3 3. Department of Horticulture and Plant Protection, Faculty of Agriculture, Shahrood University of Technology, Shahrood, Iran
10.22059/ijpps.2023.357079.1007026
Abstract
During the growth period, the potato crop is exposed to a combination of biotic and abiotic stresses. Rhizoctonia solani Kuhn is the causal agent of potato stem canker disease and one of the most important soil-borne plant pathogens that reduce the yield and quality of potatoes worldwide. Antagonistic bacteria seem to be a good alternative to chemicals in controlling plant pathogens. This study evaluated the biocontrol properties (hydrolyzing enzymes, siderophore, surfactant, biofilm) of 20 Bacillus isolates against R. solani AG3. In total, 23 to 64.9 % of isolates in dual culture assay, 38.2 to 85.4% in volatile compound production, and 76.9 to 90% in antibiotic production showed inhibition against the tested fungus. The isolates were variable in terms of biocontrol properties. Based on the in vitro results, five isolates including Ba1, Ba5, Ba8, Ba11, and Ba12 were selected for identification and additional investigations under greenhouse experiments. The selected isolates inhibited the germination of fungal sclerotium and caused mycelial fragmentation and cytoplasmic coagulation. Strains Ba1, Ba5, Ba8, Ba11, and Ba12 belonged to B. mojavensis, Bacillus sp., B. pumilus, Bacillus sp., and B. velezensis, respectively. In the greenhouse trials, the disease severity index was zero in treatments Ba5 (Bacillus sp.) and Ba8 (B. pumilus), similar to Rovral -TS fungicide and completely controlled stem canker disease. Therefore, they are suggested for additional investigations and preparation of optimal formulations.
Keywords
Bacillus sp
Biocontrol
Potato
Rhizoctonia Solani AG3
Stem Canker

Extended Abstract

Introduction

  Stem canker disease caused by Rhizoctonia solani is one of the destructive diseases of potatoes that leads to the loss of the annual yield of the product all over the world. Disease control methods include planting disease-free seed tubers and using chemical compounds that inhibit the fungus. The continuous use of fungicides causes the emergence of resistant strains and environmental pollution. Bacillus species have a unique ability to multiply rapidly, compete for space and nutrients with the Rhizoctonia pathogen, and survive under adverse environmental conditions. They secrete different types of antimicrobial compounds such as lipopeptides, antibiotics, enzymes, and volatile organic substances. They also strengthen plant growth and induce systemic resistance. The current study was conducted to investigate the biocontrol and growth of Bacillus strains in reducing potato stem canker disease and improving the growth characteristics of the plant.  

Materials and Methods   

  To evaluate the inhibitory mechanisms of Bacillus strains on Rhizoctonia pathogen, three methods including dual culture assay, production of antifungal volatile substances, and antibiotic production were used. All experiments were performed in two separate experiments based on a completely randomized design with 20 treatments and three repetitions. The biocontrol characteristics of the antagonists consisting of the production of hydrolyzing enzymes, siderophore, biosurfactant, and biofilm development were evaluated. Five selected strains were identified using 16S rRNA sequencing. Also, their effect on the germination of sclerotia of the pathogenic fungus and the morphology of its mycelium was checked. In pot experiments, in addition to candidate strains (Ba1, Ba5, Ba8, Ba11, Ba12, Ba5 + Ba11), other defined treatments including Carbendazim, Iprodione-Carbendazim, potassium phosphite, negative control (healthy plant), and positive control (fungus infected plant) were determined and disease scale calculated. The results of biological tests were analyzed by SPSS software version 24.

Results and Discussion

   In dual culture screening, strains Ba12 and in the production tests of volatile and antibiotic compounds, strains Ba11 and Ba8 respectively showed the highest inhibition against the studied fungal pathogen. Apart from the 3 mentioned strains, Ba1 and Ba5 candidates are in the next degrees of inhibiting the growth of the fungus. In evaluating the production of extracellular enzymes, Ba8 had the highest amount of chitinase and Ba12 had the highest production of cellulase, protease, and pectinase. Also, strains Ba1 and Ba5 secreted all the examined enzymes except pectinase. Five candidate strains also belong to moderate and strong biofilm-producing groups. All of them led to mycelium swelling and fragmentation and cytoplasmic coagulation in R. solani AG3 mycelia. According to the sequencing data, selected isolates including Ba1, Ba5, Ba8, Ba11, and Ba12 belonged to B. mojavensis, Bacillus sp., B. pumilus, Bacillus sp., and B. velezensis, respectively. Among the selected Bacillus strains, Ba5 and Ba8 showed zero disease index and controlled the disease, just like the Iprodione-Carbendazim fungicide. Another strain (Ba8) which controlled the pathogen 100% in greenhouse experiments, also produced high chitinase, which is effective in destroying the fungal wall and also completely prevented the germination of Rhizoctonia sclerotia. The combined treatment did not show an additive effect compared to the single treatment. 

Conclusion

   Due to their genetic and metabolic diversity, Bacillus species are well-adapted to a wide range of environmental conditions. Based on the results of the present research, it seems that the usage of Ba8 (B. pumilus) and Ba5 (Bacillus sp.) strains in combination with other available control methods should be appropriate for stem canker reduction. However, achieving the optimal formulation and validating the results requires field experiments on a larger scale.

انتصاری، محمد.، کامکار، بهنام.، قادری فر، فرشید و احمدزاده، مسعود (1396). تأثیر جدایه‌های Pseudomonas fluorescens بر شدت بیماری, صفات فیزیولوژیکی و بیوشیمیایی در غده‌های بذری تولیدی ارقام سیب‌زمینی آلوده به Rhizoctonia solani در شرایط گلخانه. مهار زیستی در گیاه پزشکی، 5 (2)،43-54.
Andric, S., Meyer, T., & Ongena, M. (2020). Bacillus responses to plant-associated fungal and bacterial communities. Frontiers in Microbiology, 11, 1350.
Asaka, O., & Shoda, M. (1996). Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilus RB14. Applied and Environmental Microbiology, 62, 397–404.
Atkinson, D., Thornton, M.K., & Miller, J.S. (2010). Development of Rhizoctonia solani on stems, stolons and tubers of potatoes I. Effect of inoculum source. American Journal of Potato Research, 87(4), 374-381.
Bacon, C.W., Palencia, E.R., & Hinton, D.M. (2015). Abiotic and biotic plant stress-tolerant and beneficial secondary metabolites produced by endophytic Bacillus species. In Plant Microbes Symbiosis: Applied Facets.Springer, New Delhi.
Bradshaw, J.E., & Ramsay, G. (2009). Potato origin and production. In Advances in Potato Chemistry and Technology. Academic Press. ‏
Bredow, C., Azevedo, J.L., Pamphile, J.A., Mangolin, C.A., & Rhoden, S.A. (2015). In silico analysis of the 16S rrna gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops. Genetics and Molecular Research, 14, 9703– 9721.
Brewer, M.T., & Larkin, R.P. (2005). Efficacy of several potential biocontrol organisms against Rhizoctonia solani on potato. Crop Protection, 24(11), 939-950.
Chowdhury, S.P., Hartmann, A., Gao, X., & Borriss, R. (2015). Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42–a review. Frontiers in Microbiology, 6, 780.
Entesari, M., Kamkar, B., Ghaderifar, F., & Ahmadzadeh, M. (2018). Effects of Pseudomonas fluorescens on the disease severity, physiological, and biochemical traits in the tubers of potato cultivars infected with Rhizoctonia solani under greenhouse condition. Biocontrol in Plant Protection. 5(2),43-54. (In Persian)
Fernando, W.D., Ramarathnam, R., Krishnamoorthy, A.S., & Savchuk, S.C. (2005). Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biology and Biochemistry, 37(5), 955-964.
Hankin, L., & Anagnostakis, S.L. (1977). Solid media containing carboxymethylcellulose to detect Cx cellulase activity of micro-organisms. Microbiology, 98(1), 109-115.
Hussain, T., & Khan, A.A. (2020). Bacillus subtilis hussaint-AMU and its Antifungal activity against Potato Black scurf caused by Rhizoctonia solani on seed tubers. Biocatalysis and Agricultural Biotechnology, 23, 101443.
Idris, E.E., Iglesias, D.J., Talon, M., & Borriss, R. (2007). Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Molecular Plant-Microbe Interactions, 20(6), 619-626.
Jayasankar, N.P., & Graham, P.H. (1970). An agar plate method for screening and enumerating pectinolytic microorganisms. Canadian journal of Microbiology, 16(10), 1023-1023.
Kazempour, M.N. (2004). Biological control of Rhizoctonia solani, the causal agent of rice sheath blight by antagonistic bacteria in greenhouse and field conditions. Plant Pathology Journal, 3, 88-96.
Keswani C., Singh H.B., Garcıa-Estrada, C., Caradus, J., He, Y.W., & Mezaache-Aichour, S. (2020) Antimicrobial secondary metabolites from agriculturally important bacteria as next-generation pesticides. Applied and Microbiology Biotechnology, 104,1013–1034.
Khedher, S.B., Kilani-Feki, O., Dammak, M., Jabnoun-Khiareddine, H., Daami-Remadi, M., & Tounsi, S. (2015). Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato. Comptes Rendus Biologies, 338(12), 784-792.
Kraus, J., & Loper, J.E. (1991). Biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens PF-5: mechanistic studies pyoluteorin, pyoverdine, siderophore. Bulletin OILB SROP (France).‏
Lamichhane, J.R., You, M.P., Laudinot, V., Barbetti, M.J., & Aubertot, J.N. (2020). Revisiting Sustainability of Fungicide Seed Treatments for Field Crops. Plant Disease, 104(3), 610–623.
Lee, Y.S., Cho, J.Y., Moon, J.H., & Kim, K.Y. (2017). Isolation and identification of N-butyl-tetrahydro-5-oxofuran-2-carboxamide produced by Bacillus sp. L60 and its antifungal activity. Journal of Bbasic Microbiology, 57(3), 283-288.
Mehmood, S., Muneer, M.A., Tahir, M., Javed, M.T., Mahmood, T., Afridi, M. S., Pakar, N.P., Abbasi, H.A., Munis, M., & Chaudhary, H.J. (2021). Deciphering distinct biological control and growth promoting potential of multi-stress tolerant Bacillus subtilis PM32 for potato stem canker. Physiology and Molecular Biology of Plants: An International Journal of Functional Plant Biology, 27(9), 2101–2114.
Morikawa, M., Hirata, Y., & Imanaka, T. (2000). A study on the structure–function relationship of lipopeptide biosurfactants. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1488(3), 211-218.‏
Murthy, N., & Bleakley, B. (2012). Simplified method of preparing colloidal chitin used for screening of chitinase-producing microorganisms. International Journal of Microbiology, 10(2), e2bc3.
Nyenje, M.E., Green, E., & Ndip, R.N. (2013). Evaluation of the effect of different growth media and temperature on the suitability of biofilm formation by Enterobacter cloacae strains isolated from food samples in South Africa. Molecules, 18(8), 9582-9593.
Olanrewaju, O.S., Ayangbenro, A.S., Glick, B.R., & Babalola, O.O. (2019). Plant health: feedback effect of root exudates-rhizobiome interactions. Applied Microbiology and Biotechnology, 103(3), 1155-1166‏.
O'Toole, G.A., & Kolter, R. (1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Molecular Microbiology, 28(3), 449-461.
Pandin, C., Le Coq, D., Canette, A., Aymerich, S., & Briandet, R. (2017). Should the biofilm mode of life be taken into consideration for microbial biocontrol agents. Microbial Biotechnology, 10(4), 719-734.
Papavizas, G.C., & Lewis, J.A. (1986). Isolating, identifying, and producing inoculum of Rhizoctonia solani. In: Hickey, K.D. (Ed.), Methods for Evaluating Pesticides for Control of Plant Pathogens. The American Phytopathological Society Press, St. Paul, MN, pp. 50–53.
Pinhero, G.P.& Yada, R.Y. (2016). Postharvest storage of potatoes. In: Singh, J., Kaur, L. (Eds.), Advances in Potato Chemistry and Technology. Academic Press, London, pp. 283–314.
Rahman, S.F.S., Singh, E., Pieterse, C.M., & Schenk, P.M. (2018) Emerging microbial biocontrol strategies for plant pathogens. Plant Science, 267, 102–111.
Saber, W.I., Ghoneem, K.M., Al-Askar, A.A., Rashad, Y.M., Ali, A.A., & Rashad, E.M. (2015). Chitinase production by Bacillus subtilis ATCC 11774 and its effect on biocontrol of Rhizoctonia diseases of potato. Acta Biologica Hungarica, 66(4), 436-448.
Sacherer, P., Defago, G., & Haas, D. (1994). Extracellular protease and phospholipase C are controlled by the global regulatory gene gaca in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiology Letters, 116(2), 155-160.
Saxena, A.K., Kumar, M., Chakdar, H., Anuroopa, N., & Bagyaraj, D.J. (2020). Bacillus species in soil as a natural resource for plant health and nutrition. Journal of Applied Microbiology, 128(6), 1583-1594.
Schaad, N. W., Jones, J. B., & Chun, W. (2001). Laboratory guide for the identification of plant pathogenic bacteria (No. Ed. 3). American Phytopathological Society (APS Press).
Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry, 160(1), 47-56.
Shafi, J., Tian, H., & Ji,M. (2017). Bacillus species as versatile weapons for plant pathogens: a review. Biotechnology and Biotechnological Equipment, 31(3), 446-459.
Shahrokhi, S., Bonjar, G.S., & Saadoun, I. (2005). Biological control of potato isolates of Rhizoctonia solani by Streptomyces olivaceus strain 115. Biotechnology, 4(2), 132-138.‏
Singh, M., Singh, D., Gupta, A., Pandey, K.D., Singh, P.K., & Kumar, A. (2019). Plant growth promoting rhizobacteria: application in biofertilizers and biocontrol of phytopathogens. In: PGPR Amelioration In Sustainable Agriculture (pp. 41-66). Woodhead Publishing.
Srilakshmi, P., Thakur, R. P., & Satyaprasad, K. (2011). Mechanism of biocontrol of Aspergillus flavus in groundnut by species of Trichoderma. Journal of Mycology and Plant Pathology, 41(2), 249–254.
Solanki, M.K., Robert, A.S., Singh, R.K., Kumar, S., Pandey, A.K., Srivastava, A.K., & Arora, D.K. (2012). Characterization of mycolytic enzymes of Bacillus strains and their bio-protection role against Rhizoctonia solani in tomato. Current Microbiology, 65(3), 330-336.
Soltani, H, zafari, D., & rouhani, H. (2006). A study on biological control of the crown, root and tuber fungal diseases of potato by Trichoderma harzianum under in-vivo and field condition in Hamadan.‏ Agricultural Research, 5 (3), 13-25
Srivastava, S., Bist, V., Srivastava, S., Singh, P.C., Trivedi, P.K., Asif, M.H., & Nautiyal, C.S. (2016). Unraveling aspects of Bacillus amyloliquefaciens mediated enhanced production of rice under biotic stress of Rhizoctonia solani. Frontiers in Plant Science, 7, 587.
Taheri, P., and Tarighi, S. 2010. Riboflavin induces resistance in rice against Rhizoctonia solani via jasmonate-mediated priming of phenylpropanoid pathway. Journal of Plant Physiology, 167(3), 201-208.
Tran, H., Ficke, A., Asiimwe, T., Höfte, M., & Raaijmakers, J.M. (2007). Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens. The New Phytologist, 175(4), 731–742.
Wale, S.J., Platt, H.W., & Cattlin, N.D. (2008). Diseases, pests and disorders of potatoes: a color handbook. London Pub, Elsevier. 176p.
Wharton, P.S. And Wood, E. (2013). Rhizoctonia stem canker and black scurf of potato. Idao, USA: University of Idaho, Extension CIS 1198.
Woodhall, A. L., Peters, J., Kiezebrink, D., Sparrow, L., & Ophelkeller, K. (2013). Informing management of potato diseases through epidemiology and diagnostics Rhizoctonia solani Reviews. Potato council agriculture & horticulture development board. 46 p.
Zhang, D., Yu, S., Yang, Y., Zhang, J., Zhao, D., Pan, Y., & Zhu, J. (2020). Antifungal effects of volatiles produced by Bacillus subtilis against Alternaria solani in potato. Frontiers in Microbiology, 11, 1196.
Iranian Journal of Plant Protection Science
Volume 54, Issue 1
September 2023
Pages 187-205

  • Receive Date 04 April 2023
  • Revise Date 18 September 2023
  • Accept Date 26 September 2023