Evaluation of the Antifungal Activity of Bacillus amyloliquefaciens and B. velezensis and Characterization of the Bioactive Secondary Metabolites Produced against Plant Pathogenic Fungi

Abstract

Endophytic bacteria are plant-beneficial bacteria with a broad host range. They provide numerous benefits to their hosts, helping them tolerate several biotic and abiotic stresses. An interest has recently been developed in endophytic bacteria which are producing bioactive compounds that contribute to the biological control of various phytopathogens. This research paper aimed to investigate the potentiality of new local strains of endophytic bacteria such as <i>Bacillus amyloliquefaciens</i> and <i>B. velezensis</i> and the production of several antimicrobial metabolites associated with the biocontrol of <i>Alternaria</i> sp., which cause serious diseases and affect important vegetable crops in Egypt. Twenty-five endophytic bacteria isolates were obtained from different plants cultivated in El-Sharkia Governorate, Egypt. Dual culture technique was used to evaluate the bacterial isolates' antagonistic potentiality against <i>Alternaria</i> sp. and <i>Helminthosporium</i> sp. The most active bacterial isolates obtained were selected for further screening. The antifungal activity of the most active endophytic bacterial isolate was assessed in vivo on pepper seedlings as a biocontrol agent against <i>Alternaria</i> sp. A significant antifungal activity was recorded with isolates C₁ and T₅ against <i>Alternaria</i> sp. and <i>Helminthosporium</i> sp. The bacterial endophyte discs of C₁ and T₅ showed the highest inhibitory effect against <i>Alternaria</i> sp. at 4.7 and 3.1 cm, respectively, and <i>Helminthosporium</i> sp. at 3.9 and 4.0 cm, respectively. The most active endophytic isolates C₁ and T₅ were identified and the 16S rRNA sequence was submitted to the NCBI GenBank database with accession numbers: MZ945930 and MZ945929 for <i>Bacillus amyloliquefaciens</i> and <i>Bacillus velezensis</i>, respectively. The deformity of pathogenic fungal mycelia of <i>Alternaria</i> sp. and <i>Helminthosporium</i> sp. was studied under the biotic stress of bacteria. The culture filtrates of <i>B. amyloliquefaciens</i> and <i>B. velezensis</i> were extracted with different solvents, and the results indicated that hexane was the most efficient. Gas Chromatography-Mass Spectrometry revealed that Bis (2-ethylhexyl) phthalate, Bis (2-ethylhexyl) ester, and <i>N</i>,<i>N</i>-Dimethyldodecylamine were major constituents of the endophytic crude extracts obtained from <i>B. amyloliquefaciens</i> and <i>B. velezensis</i>. The in vivo results showed that <i>Alternaria</i> sp. infection caused the highest disease incidence, leading to a high reduction in plant height and in the fresh and dry weights of pepper plants. With <i>B. amyloliquefaciens</i> application, DI significantly diminished compared to <i>Alternaria</i> sp. infected pepper plants, resulting in an increase in their morphological parameters. Our findings allow for a reduction of chemical pesticide use and the control of some important plant diseases.