Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae

Abstract

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant <i>Glycyrrhiza uralensis</i> (licorice) were screened for their <i>in vitro</i> antimicrobial activities against common fungal pathogens of tomato (<i>Fusarium oxysporum</i> f. sp., <i>Fulvia fulva</i>, <i>Alternaria solani</i>), cotton (<i>Fusarium oxysporum</i> f. sp. Vesinfectum, <i>Verticillium dahliae</i>), pomegranite (<i>Ceratocystis fimbriata</i>), <i>Cymbidinium</i> (<i>Colletotrichum gloeosporioides</i>), and Tsao-ko (<i>Pestalotiopsis microspora</i> and <i>Fusarium graminearum</i>) and the common bacteria <i>Staphylococcus aureus</i>, <i>Bacillus cereus</i>, <i>Salmonella enteritidis</i>, and <i>Escherichia coli</i>. Several <i>Bacillus</i> strains, particularly <i>Bacillus atrophaeus</i> and <i>Bacillus mojavensis</i>, had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing <i>Bacillus atrophaeus</i> and <i>Bacillus mojavensis</i>, were selected for plant growth chamber experiments based on strong <i>in vitro</i> antifungal activities. All five strains significantly reduced disease severity in <i>Arabidopsis thaliana</i> plants challenged with <i>V. dahlia</i> infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of <i>Bacillus atrophaeus</i> strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with <i>V. dahlia</i>, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from <i>G. uralensis</i> have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly <i>B. atrophaeus</i>, and suggest this species may a promising candidate as a biocontrol agent to confer resistance to <i>Verticillium</i> wilt disease and other phytopathogens in cotton and other crops.