Molecular characterization and antagonistic activity of Bacillus strains against Sclerotium rolfsii
Abstract
The devastating soil-borne pathogen Sclerotium rolfsii has become increasingly significant worldwide, affecting several crops. However, biological disease management, particularly using antagonistic microbes, offers an environmentally friendly alternative to chemical control. Bacillus species, in particular, have demonstrated strong antagonistic potential against various fungal pathogens. In this study, the morpho-microscopic and biochemical characterization of Bacillus isolates obtained from rhizosphere soil was conducted. Furthermore, their antagonistic activities were assessed in vitro using dual culture and well diffusion techniques against S. rolfsii. During primary screening, four isolates viz. S3C9, S1C9, S2C9, and S6C8 strongly suppressed fungal mycelial growth, with inhibition zones measuring 18.3, 14.7, 14.3, and 14.7 mm, respectively. Three isolates (S2C9, S3C9, and S4C9) were further evaluated for inhibitory properties using cell-free supernatant through the well diffusion method, yielding inhibition zones of 6.3, 4.7, and 4.7 mm, respectively. Moreover, molecular analysis identified two isolates, S1C9 and S3C9, as strains of Bacillus subtilis, with GenBank accession codes OP962345 and OQ180051, respectively. This study provides evidence that the local soil of Kot Diji, Sindh Province, Pakistan, harbors beneficial microbes with significant antifungal activity, which could be utilized for future biocontrol strategies against phytopathogenic fungi.
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Ahmad, A.G.M., Attia, A.Z.G., Mohamed, M.S., Elsayed, H,E., 2019. Fermentation, formulation and evaluation of PGPR Bacillus subtilis isolate as a bioagent for reducing occurrence of peanut soil-borne diseases. Journal of Integrative Agriculture 18(9), 2080-2092.
Al-Ali, A., Deravel, J., Krier, F., Béchet, M., Ongena, M., 2018. Biofilm formation is determinant in tomato rhizosphere colonization by Bacillus velezensis FZB42. Environmental Science and Pollution Research 25, 29910-29920.
Almoneafy, A.A., Kakar, K.U., Nawaz, Z., Li, B., Saand MA., Chun-lan, Y., Xie, G.L., 2014. Tomato plant growth promotion and antibacterial related-mechanisms of four rhizobacterial Bacillus strains against Ralstonia solanacearum. Symbiosis 63, 59-70.
Athukorala, S.N., Fernando, W.D., Rashid, K.Y., De, K.T., 2010. The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum. Biocontrol Science and Technology 20(8), 875-890.
Aziz, S., Jamshed, S.A., Mukhtar, T., Irshad, G., Ijaz, S.S., Raja, M.U., 2024. Evaluation of Bacillus spp. as biocontrol agents against chili leaf spot caused by Xanthomonas vesicatoria. Journal of Plant Diseases and Protection 131, 987-997.
Bidima, M.G., Chtaina, N., Ezzahiri, B., El Guilli, M., 2021. Evaluation of the antagonistic potential of bacterial strains isolated from Moroccan soils for the biological control of Sclerotium rolfsii Sacc. International Journal of Food Science and Agriculture 5(4), 608-616.
Billah, K.M., Hossain, M.B., Prince, M.H., Sumon, M.M., 2017. Pathogenicity of Sclerotium rolfsii on different host, and its over wintering survival; A mini review. International Journal of Advances in Agriculture Sciences 2(1), 1-6.
Cheesbrough, M., 1981. Medical laboratory manual for tropical countries. Volume 1 (pp. xii+-520).
Chen, L., Wu, Y.D., Chong, X.Y., Xin, Q.H., Wang, D.X., 2020. Seed‐borne endophytic Bacillus velezensis LHSB1 mediate the biocontrol of peanut stem rot caused by Sclerotium rolfsii. Journal of Applied Microbiology 128(3), 803-813.
Dodia, S.M., Joshi., B., Gangurde, S.S., Thirumalaisamy, P.P., Mishra, G.P., Narandrakumar, D., Soni, P., Rathnakumar, A.L., Dobaria, J.R., Sangh, C., Chitikineni, A., 2019. Genotyping-by-sequencing based genetic mapping reveals large number of epistatic interactions for stem rot resistance in groundnut. Theoretical and Applied Genetics 132, 1001-1016.
Doty, S.L., Joubert, P.M., Firrincieli, A., Sher, A.W., Tournay, R., Kill, C., Parikh, S.S., Okubara, P., 2022. Potential biocontrol activities of Populus endophytes against several plant pathogens using different inhibitory mechanisms. Pathogens 12(1).
Etesami, H., Jeong, B.R., Glick, B.R., 2023. Potential use of Bacillus spp. as an effective biostimulant against abiotic stresses in crops-A review. Current Research in Biotechnology 5, 100128.
Gao, S., Wu, H., Wang, W., Yang, Y., Xie, S., Xie, Y., Gao, X., 2013. Efficient colonization and harpins mediated enhancement in growth and biocontrol of wilt disease in tomato by Bacillus subtilis. Letters in Applied Microbiology 57(6), 526-533.
Gao, Z., Zhang, B., Liu, H., Han, J., Zhang, Y., 2017. Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea. Biological Control 105, 27-39.
Gholami, M., Khakvar, R., Niknam, G., 2014. Introduction of some new endophytic bacteria from Bacillus and Streptomyces genera as successful biocontrol agents against Sclerotium rolfsii. Archives of Phytopathology and Plant Protection 47(1), 122-130.
Guenoun, K., Chattaoui, M., Bouri, M., Rhouma, A., Naghmouchi, K., Raies, A., 2019. Biological control of growth promoting rhizobacteria against verticillium wilt of pepper plant. Biologia 74, 237-250.
Hadri, S.H., Asad, M.J., Hyder, M.Z., Naqvi, S.M.S., Mukhtar, T., Zafar, M., Shah, S.H., Mehmood, R.T., Wu, J.H.D., 2019. Characterization of a novel thermophilic endopolygalacturonase produced by Bacillus licheniformis IEB-8. BioResources 14(2), 2873-2884.
Hägg, P., de, Pohl, J.W., Abdulkarim, F., Isaksson, L.A., 2004. A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli. Journal of Biotechnology 111(1), 17-30.
Hashem, A., Tabassum, B., Abd_Allah, E.F., 2019. Bacillus subtilis: a plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences 26(6), 1291-1297.
Iqbal, U., Mukhtar, T., 2020a. Inhibitory effects of some fungicides against Macrophomina phaseolina causing charcoal rot. Pakistan Journal of Zoology 52(2), 709-715.
Iqbal, U., Mukhtar, T., 2020b. Evaluation of biocontrol potential of seven indigenous Trichoderma species against charcoal rot-causing fungus, Macrophomina phaseolina. Gesunde Pflanzen 72(2), 195-202.
Iquebal, M.A., Tomar, R.S., Parakhia, M.V., Singla, D., Jaiswal, S., Rathod, V.M., Padhiyar, S.M., Kumar, N., Rai, A., Kumar, D., 2017. Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence. Scientific Reports 7(1), 5299.
Javaid, A., Afzal, R., Shoaib, A., 2020. Biological management of southern blight of chili by Penicillium oxalicum and leaves of Eucalyptus citriodora. International Journal of Agriculture and Biology 23(1), 93-102.
Jia, S., Song, C., Dong, H., Yang, X., Li, X., Ji, M., Chu, J., 2023. Evaluation of efficacy and mechanism of Bacillus velezensis CB13 for controlling peanut stem rot caused by Sclerotium rolfsii. Frontiers in Microbiology 14, 1111965.
Karimi, K., Amini, J., Harighi, B., Bahramnejad, B., 2012. Evaluation of biocontrol potential of ‘Pseudomonas’ and ‘Bacillus’ spp. against Fusarium wilt of chickpea. Australian Journal of Crop Science 6(4), 695-703.
Keinath, A.P., DuBose, V.B., 2017. Management of southern blight on tomato with SDHI fungicides. Crop Protection 101, 29-34.
Khan, A.R., Mustafa, A., Hyder, S., Valipour, M., Rizvi, Z.F., Gondal, A.S., Yousuf, Z., Iqbal, R., Daraz, U., 2022. Bacillus spp. as bioagents: uses and application for sustainable agriculture. Biology, 11(12), 1763.
Kour, D., Rana, K.L., Yadav, N., Yadav, A.N., Kumar, A., Meena, V.S., Singh, B., Chauhan, V.S., Dhaliwal, H.S., Saxena, A.K., 2019. Rhizospheric microbiomes: biodiversity, mechanisms of plant growth promotion, and biotechnological applications for sustainable agriculture. Plant growth promoting rhizobacteria for agricultural sustainability: From Theory to Practices, pp. 19-65.
Krishnan, G.V., Abraham, B., Lankalapalli, R.S., Bhaskaran Nair Saraswathy Amma, D.K., Bhaskaran, K., 2024. Rice sheath blight disease control by native endophytic Bacillus subtilis from Kuttanad, a globally important agricultural heritage system. New Zealand Journal of Botany, 1-23.
Kumari, P., Bishnoi, S.K., Chandra, S., 2021. Assessment of antibiosis potential of Bacillus sp. against the soil-borne fungal pathogen Sclerotium rolfsii Sacc. (Athelia rolfsii (Curzi) Tu & Kimbrough). Egyptian Journal of Biological Pest Control 31, 1-11.
Li, L., Wang, J., Liu, D., Li, L., Zhen, J., 2023. The antagonistic potential of peanut endophytic bacteria against Sclerotium rolfsii causing stem rot. Brazilian Journal of Microbiology 54(1), 361-370.
Meena, P.N., Meena, A.K., Tiwari, R.K., Lal, M.K., Kumar, R., 2024. Biological control of stem rot of groundnut induced by Sclerotium rolfsii Sacc. Pathogens 13(8), 632.
Mohamed, I., Eid, K.E., Abbas, M.H., Salem, A.A., Ahmed, N., Ali, M., Shah, G.M., Fang, C., 2019. Use of plant growth promoting Rhizobacteria (PGPR) and mycorrhizae to improve the growth and nutrient utilization of common bean in a soil infected with white rot fungi. Ecotoxicology and Environmental Safety 171, 539-548.
Monica, C., 1991. Medical Laboratory manual for Tropical countries. VOL, 11, pp.60-63.
Mukhtar, T., Hussain, M.A., Kayani, M.Z., 2013. Biocontrol potential of Pasteuria penetrans, Pochonia chlamydosporia, Paecilomyces lilacinus, and Trichoderma harzianum against Meloidogyne incognita in okra. Phytopathologia Mediterranea 52(1), 66-76.
Nihorimbere, V., Ongena, M., Cawoy, H., Brostaux, Y., Kakana, P., Jourdan, E., Thonart, P., 2010. Beneficial effects of Bacillus subtilis on field-grown tomato in Burundi: Reduction of local Fusarium disease and growth promotion. African Journal of Microbiology Research 4(11), 1135-1142.
Saand, M.A., Xu, Y.P., Li, W., Wang, J.P., Cai, X.Z., 2015. Cyclic nucleotide gated channel gene family in tomato: genome-wide identification and functional analyses in disease resistance. Frontiers in Plant Science 6, 303.
Sabir, J.S., Abo-Aba, S.E., Sabry, A., Hussein, R.M., Bahieldin, A., 2013. Isolation, identification and comparative analysis of 16S rRNA of Bacillus subtilis grown around Rhazya stricta roots. Life Science Journal 10(12S), 980-986.
Saeed, M., Mukhtar, T., Ahmed, R., Ahmad, T., Iqbal, M.A., 2023. Suppression of Meloidogyne javanica infection in peach (Prunus persica (L.) Batsch) using fungal biocontrol agents. Sustainability 15(18), 13833. https://doi.org/10.3390/su151813833
Sagar, A., Rai, S., Sharma, S., Perveen, K., Bukhari, N.A., Sayyed, R.Z., Mastinu, A., 2024. Molecular characterization reveals biodiversity and biopotential of rhizobacterial isolates of Bacillus spp. Microbial Ecology 87(1), 83.
Saha, D., Purkayastha, G.D., Ghosh, A., Isha, M., Saha, A., 2012. Isolation and characterization of two new Bacillus subtilis strains from the rhizosphere of eggplant as potential biocontrol agents. Journal of Plant Pathology 94(1), 109-118.
Santos, R.M.D., Rigobelo, E.C., 2021. Growth-promoting potential of rhizobacteria isolated from sugarcane. Frontiers in Sustainable Food Systems 5, 596269.
Sethi, S.K., Mukherjee, A.K., 2018. Screening of biocontrol potential of indigenous Bacillus spp. isolated from rice rhizosphere against R. solani, S. oryzae, S. rolfsii and response towards growth of rice. Journal of Pure and Applied Microbiology 12(1).
Shahzaman, S., Inam-ul-Haq, M., Mukhtar, T., Naeem, M., 2015. Isolation and identification of antagonistic rhizobacterial strains obtained from chickpea (Cicer arietinum L.) fields and their in vitro evaluation against fungal root pathogens. Pakistan Journal of Botany 47(4), 1553-1558.
Sharf, W., Javaid, A., Shoaib, A., Khan, I.H., 2021. Induction of resistance in chili against Sclerotium rolfsii by plant-growth-promoting rhizobacteria and Anagallis arvensis. Egyptian Journal of Biological Pest Control 31(16). 1-11.
Shekhar, N.C., Sangeeta, M., Bahadur, S.H., 2006. Biological control and plant-growth promotion by Bacillus strains from milk. Journal of Microbiology and Biotechnology, 16(2), 184-192.
Singh, N., Pandey, P., Dubey, R.C., Maheshwar, D.K., 2008. Biological control of root rot fungus Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg.) by rhizosphere competent Bacillus subtilis BN1. World Journal of Microbiology and Biotechnology 24, 1669-1679.
Sunil, N., Thara, S.S., 2022. Evaluation of biocontrol agents and chemical fungicides against Sclerotium rolfsii causing stem rot and foliar blight of cowpea. Journal of Krishi Vigyan 10(2), 141-145.
Talpur, M.K.A., Qazi, M.A., Phulpoto, A.H., Maitlo, M.A., Phulpoto, I.A., Syed, F.H., Wassan, S.A., Saand, M.A., Kanhar, N.A., 2020. Bioprospecting actinobacterial diversity antagonistic to multidrug-resistant bacteria from untapped soil resources of Kotdiji, Pakistan. Biologia 75(1), 129-138.
Tan, Z., Lin, B., Zhang, R., 2013. A novel antifungal protein of Bacillus subtilis B25. SpringerPlus 2(543).
Todorova, S., Kozhuharova, L., 2010. Characteristics and antimicrobial activity of Bacillus subtilis strains isolated from soil. World Journal of Microbiology and Biotechnology 26, 1207-1216.
Veena, S.S., Sreekumar, J., Jeeva, M.L., Byju, G., Suja, G., Sengupta, S., Thangamani, C., Thakur, P., Narayan, A., Gudadhe, P.S., Sunitha, S., 2025. Optimizing management interventions against Sclerotium rolfsii Sacc. On elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson) in India. Crop Protection 188, 107013.
Verma, P.P., Shelake, R.M., Das, S., Sharma, P., Kim, J.Y., 2019. Plant Growth-Promoting Rhizobacteria (PGPR) and Fungi (PGPF): Potential Biological Control Agents of Diseases and Pests. In: Singh, D., Gupta, V., Prabha, R. (eds) Microbial Interventions in Agriculture and Environment. Springer, Singapore.
Wang, T., Li, W., Wang, F., Li, J., Qin, J., Song, Z., Xu, J., Qiu, H., Cheng, Y., 2024. Biocontrol potential of Bacillus velezensis SEC-024A against southern blight of industrial hemp. Industrial Crops and Products 222, 119767.
Wang, Y., Zhang, C., Liang, J., Wang, L., Gao, W., Jiang, J., Chang, R., 2020. Surfactin and fengycin B extracted from Bacillus pumilus W-7 provide protection against potato late blight via distinct and synergistic mechanisms. Applied Microbiology and Biotechnology 104, 7467-7481.
Zain, M., Yasmin, S., Hafeez, F.Y., 2019. Isolation and characterization of plant growth promoting antagonistic bacteria from cotton and sugarcane plants for suppression of phytopathogenic Fusarium species. Iranian Journal of Biotechnology 17(2), e1974.
Zou, L., Wang, Q., Wu, R., Zhang, Y., Wu, Q., Li, M., Ye, K., Dai, W., Huang, J., 2023. Biocontrol and plant growth promotion potential of endophytic Bacillus subtilis JY-7-2L on Aconitum carmichaelii Debx. Frontiers in Microbiology, 13, 1059549.
DOI: https://doi.org/10.33804/pp.009.01.5484
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