Fusarium fungi are globally important pathogens of plants, livestock, and humans. Reports of a polyphagous beetle/Fusarium-mediated infection causing avocado dieback disease in Southern California led us to search for natural compounds as substitutes for synthetic compounds currently used to fight Fusarium infections. Towards this end, we isolated soil and endophytic bacteria with potential to replace not only synthetic fungicides, but also fertilizers. Bacillus subtilis 30VD-1 was isolated from the Mildred E. Mathias Botanical Garden and UCLA is the licensor. This bacterial species exhibits effective antagonism against Fusarium spp. (F. oxysporum f. sp. matthioli (FOM) and F. solani (FS)) grown in vitro and in vivo. Both volatile and non-volatile compounds are synthesized by strain 30VD1, but our goal here is to identify the non-volatile microbial products that exhibit a dose-dependent response in a fungal growth bioassay. The activity is efficiently extracted from spent culture medium with n-butanol (BuOH) and a 100-µg/ml extract, when added to the growth medium, causes ca. 40% inhibition in radial growth of Fusarium spp. Phase contrast microscopy showed distortions and abnormal swellings in FOM hyphae upon co-culturing with 30VD1. Pea seed bacterization with 30VD1 led to a reduction in wilt severity in plants with ca. a 35% increase in plant dry biomass compared to uninoculated plants growing in Fusarium-infested soil. Overall, the results suggest a multivariate mode of antagonism of 30VD1 against Fusarium spp.—by producing chitinase, volatiles, and other antifungal molecules. We are performing assays to determine the chemical nature and stability of the butanol-soluble inhibitory substance. Sequential chromatographic steps such as reversed and normal phase are being conducted to purify the activity. Compound identification will rely on high resolution mass spectrometry (GC/MS and LC/MS), and if needed, NMR, followed by in planta testing of purified fractions by spray inoculation and documenting the level of protection againstFusarium infection. Lack of virulence on Caenorhabditis elegans, as well as strain 30VD1’s compatibility with other PGPBs, particularlyRhizobium, Mesorhizobium, Bacillus, and Paraburkholderia, provide evidence of the non-harmful nature of 30VD1 and its metabolites, further indicating their potential importance for biocontrol. The combination of these traits will lead to the development of a planet-friendly biofungicide.

Mining phytomicrobiomes for microbial compounds to replace synthetic fertilizers and fungicides for sustainable agriculture

2019 Fall National Meeting

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