Isolation of antagonistic bacterial strains against fungi

H.  Karimov; N.  Azimova; N.  Khaytbayeva; F.  Kobilov; K.  Khamidova; O.  Shukurov; J.  Razzokov

doi:10.15421/0224113

H. Karimov Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan
N. Azimova Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan
N. Khaytbayeva Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan
F. Kobilov Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan
K. Khamidova Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan
O. Shukurov Institute of Fundamental and Applied Research at the National Research University TIIAME
J. Razzokov Institute of Fundamental and Applied Research at the National Research University TIIAME

Keywords: Bacillius; bacteria; microflora; pathogens; antagonistic activity

Abstract

This study focused on isolating and identifying antagonistic bacterial strains from diseased vegetable crops and evaluating their potential as biocontrol agents against phytopathogenic fungi. Samples were collected from tomato, sweet pepper, and potato plants across various regions of Uzbekistan. Several bacterial strains were identified using morphological, molecular, and biochemical analyses, with Bacillus licheniformis 6.25 emerging as the most potent antagonist. Molecular characterization through 16S rRNA sequencing confirmed a 99.86% similarity to known B. licheniformis species. The antagonistic activity of B. licheniformis 6.25 was tested against Fusarium solani, Alternaria alternata, and Verticillium dahliae. The strain suppressed fungal growth by over 50%, demonstrating strong antifungal properties. Metabolite analysis via GC-MS revealed bioactive compounds with antifungal, anti-inflammatory, and antimicrobial effects. Antifungal assays showed inhibition zones of 26 mm against A. alternata and 21 mm against F. solani. These findings highlight the potential of B. licheniformis 6.25 as a biocontrol agent for sustainable agriculture and pharmaceutical applications.

References

Azimova, N., Xamidova, X., Karimov, H., Xaytbaeva, N., Ravshanov, B., & Yesenova, D. (2023). Bacterial flora of infected vegetable crops. E3S Web of Conferences, 421, 04011.

Bilay, V. I. (1977). Fuzarii [Fusaria]. Naukova Dumka, Kiev (in Russian).

Bosco de Oliveira, A. (Ed.). (2019). Abiotic and biotic stress in plants. Intech Open, London.

Connor, N., Sikorski, J., Rooney, A. P., Kopac, S., Koeppel, A. F., Burger, A., Cole, S. G., Perry, E. B., Krizanc, D., Field, N. C., Slaton, M., & Cohan, F. M. (2010). Ecology of speciation in the genus Bacillus. Applied and Environmental Microbiology, 76(5), 1349–1358.

Eshboev, F., Karakozova, M., Abdurakhmanov, J., Bobakulov, K., Dolimov, K., Abdurashidov, A., Baymirzaev, A., Makhnyov, A., Terenteva, E., Sasmakov, S., Piyakina, G., Egamberdieva, D., Nazarov, P. A., & Azimova, S. (2023). Antimicrobial and cytotoxic activities of the secondary metabolites of endophytic fungi isolated from the medicinal plant Hyssopus officinalis. Antibiotics, 12(7), 1201.

Falardeau, J., Wise, C., Novitsky, L., & Avis, T. J. (2013). Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens. Journal of Chemical Ecology, 39(7), 869–878.

Felske, A. D. M., Tzeneva, V., Heyrman, J., Langeveld, M. A., Akkermans, A. D. L., & De Vos, P. (2004). Isolation and biodiversity of hitherto undescribed soil bacteria related to Bacillus niacini. Microbial Ecology, 48(1), 111–119.

Fira, D., Dimkić, I., Berić, T., Lozo, J., & Stanković, S. (2018). Biological control of plant pathogens by Bacillus species. Journal of Biotechnology, 285, 44–55.

Guarrasi, V., Sannino, C., Moschetti, M., Bonanno, A., Di Grigoli, A., & Settanni, L. (2017). The individual contribution of starter and non-starter lactic acid bacteria to the volatile organic compound composition of Caciocavallo Palermitano cheese. International Journal of Food Microbiology, 259, 35–42.

He, L., Chen, W., & Liu, Y. (2006). Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12. Microbiological Research, 161(4), 321–326.

Holt, J. G., Krieg, N. R., Sneath, P. H. A., Stanley, J. T., & William, S. T. (1994). Bergey’s manual of determinative bacteriology. Williams and Wilkins, Baltimore.

Hucker, G. J., & Conn, H. J. (1927). Further studies on the methods of Gram staining. New York State Agricultural Experiment Station. Technical Bulletin, 128.

Imad, H. H., Hussein, J. H., Muhanned, A. K., & Nidaa, S. H. (2015). Identification of five newly described bioactive chemical compounds in methanolic extract of Mentha viridis by using gas chromatography – mass spectrometry (GC-MS). Journal of Pharmacognosy and Phytotherapy, 7(7), 107–125.

Jasmine, J. M., Latha, K., & Vanaja, R. (2013). Determination of bioactive components of decholestrate, a polyherbal formulation by GC-MS analysis. New York Science Journal, 1(e8), 12–39.

Karandashov, V., Nagy, R., Wegmüller, S., Amrhein, N., & Bucher, M. (2004). Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences, 101(16), 6285–6290.

Khaitbaeva, N., Azimova, N., Sherkulova, J., Khamidova, X., Karimov, K., Kodirova, R., & Zufarova, Z. (2023). Study of phytotoxic properties of pathogenic fungi in eggplants. International Journal of Multidisciplinary and Current Research, 11, 200–206.

Lane, D. J. (1991). 16S/23S rRNA sequencing. In: Stackebrandt, E., & Goodfellow, M. (Eds.). Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Inc., New York. Pp. 115–176.

Leslie, J. F., & Summerell, B. A. (Eds.). (2006). The Fusarium laboratory manual. Blackwell Publishing, Hoboken.

Logan, N. A. (1988). Bacillus species of medical and veterinary importance. Journal of Medical Microbiology, 25(3), 157–165.

Lorenzini, G., Guidi, L., Nali, C., Ciompi, S., & Soldatini, G. F. (1997). Photosynthetic response of tomato plants to vascular wilt diseases. Plant Science, 124(2), 143–152.

Mao, T., Chen, X., Ding, H., Chen, X., & Jiang, X. (2020). Pepper growth promotion and Fusarium wilt biocontrol by Trichoderma hamatum MHT1134. Biocontrol Science and Technology, 30(11), 1228–1243.

Nogués, S., Cotxarrera, L., Alegre, L., & Trillas, M. I. (2002). Limitations to photosynthesis in tomato leaves induced by Fusarium wilt. New Phytologist, 154(2), 461–470.

Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: Versatile weapons for plant disease biocontrol. Trends in Microbiology, 16(3), 115–125.

Payne, M., Champagne, S., Lowe, C., Leung, V., Hinch, M., & Romney, M. G. (2018). Evaluation of the Film Array Blood Culture Identification Panel compared to direct Maldi-Tof MS identification for rapid identification of pathogens. Journal of Medical Microbiology, 67(9), 1253–1256.

Pignatelli, M., Moya, A., & Tamames, J. (2009). EnvDB, a database for describing the environmental distribution of prokaryotic taxa. Environmental Microbiology Reports, 1(3), 191–197.

Romero, D., de Vicente, A., Rakotoaly, R. H., Dufour, S. E., Veening, J.-W., Arrebola, E., Cazorla, F. M., Kuipers, O. P., Paquot, M., & Pérez-García, A. (2007). The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Molecular Plant-Microbe Interactions, 20(4), 430–440.

Sagdullaeva, M. S., Kirgizbaeva, K. M., Ramazanova, S. S., Gulyamova, M. G., & Fayzieva, F. K. (1990). Flora gribov Uzbekistana. Tom 1. Muchnistorosyanyye griby [Mushroom flora of Uzbekistan. Vol. 1. Powdery mildew fungi]. Fan, Tashkent (in Russian).

Salvà-Serra, F., Svensson-Stadler, L., Busquets, A., Jaén-Luchoro, D., Karlsson, R., R. B. Moore, E., & Gomila, M. (2018). A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: A modified version of the Marmur procedure. Protocol Exchange.

Saravanakumar, K., Raj, A., & Umaiyambigai, D. (2016). GC-MS and FT-IR profiling of leaves methanol extract from the Pleiospermium alatum (Wall. ex Wt. & Arn.) Swingle Rutaceae family. The Journal of Phytopharmacology, 5(5), 201–204.

Shavkiev, J., Kholmamatovich, K. H., Ismoilovna, T. B., Kizi, A. N. S., Khodjakbarovich, N. M., & Muminovna, K. K. (2022). Some volatile metabolites produced by the antifungal-Trichoderma asperellum UZ-A4 micromycete. International Journal of Phytopathology, 11(3), 239–251.

Silva, M. L., David, J. P., Silva, L. C. R. C., Santos, R. A. F., David, J. M., Lima, L. S., Reis, P. S., & Fontana, R. (2012). Bioactive oleanane, lupane and ursane triterpene acid derivatives. Molecules, 17(10), 12197–12205.

Sokirko, V. P., Gorkovenko, V. S., & Zazimko, M. I. (2014). Phytopathogenic fungi (morphology and systematics). KubSAU, Krasnodar.

Song, B., Rong, Y.-J., Zhao, M.-X., & Chi, Z.-M. (2013). Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic. Applied Microbiology and Biotechnology, 97(16), 7141–7150.

Stein, T. (2005). Bacillus subtilis antibiotics: Structures, syntheses and specific functions. Molecular Microbiology, 56(4), 845–857.

Stepanov, A. S., & Vasilyeva, N. V. (2018). New opportunities to identify and type Staphylococcus spp. by using Maldi-Tof mass spectrometry. Russian Journal of Infection and Immunity, 8(4), 489–496.

Stracquadanio, C., Quiles, J. M., Meca, G., & Cacciola, S. O. (2020). Antifungal activity of bioactive metabolites produced by Trichoderma asperellum and Trichoderma atroviride in liquid medium. Journal of Fungi, 6(4), 263.

Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24(8), 1596–1599.

Tsuchiya, K., (1990). Problems on allelopathy in vegetable cropping. Agriculture and Horticulture, 65, 9–16 (in Japanese).

Turaeva, B. I., Soliev, A. B., Karimov, H. K., Azimova, N. S. Q., Kutlieva, G. J., Khamidova, K. M., & Zuxritdinova, N. Y. (2021). Disease causing phytopathogenic micromycetes in citrus in Uzbekistan. Pakistan Journal of Phytopathology, 33(2), 383–393.

Vardhan, S., Kaushik, R., Saxena, A. K., & Arora, D. K. (2010). Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species. Antonie van Leeuwenhoek, 99(2), 283–296.

Zeriouh, H., Romero, D., García-Gutiérrez, L., Cazorla, F. M., de Vicente, A., & Pérez-García, A. (2011). The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular Plant-Microbe Interactions, 24(12), 1540–1552.

Zhang, X., Li, B., Wang, Y., Guo, Q., Lu, X., Li, S., & Ma, P. (2013). Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1. Applied Microbiology and Biotechnology, 97(21), 9525–9534.

Zhao, Y., Selvaraj, J. N., Xing, F., Zhou, L., Wang, Y., Song, H., Tan, X., Sun, L., Sangare, L., Folly, Y. M. E., & Liu, Y. (2014). Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS One, 9(3), e92486.