Integrated Genomic and Metabolomic Analysis Illuminates Key Secreted Metabolites Produced by the Novel Endophyte Bacillus halotolerans Cal.l.30 Involved in Diverse Biological Control Activities.

The endophytic strain Cal.l.30, isolated from the medicinal plant Calendula officinalis, was selected among seven Bacillus strains with plant growth promoting activity and strong biological potential against the postharvest fungal pathogen Botrytis cinerea. Treatment by inoculating Cal.l.30 bacterial cell culture or cell free supernatant on harvested grapes and cherry tomato fruits, significantly reduced gray mold disease severity index and disease incidence. Based on 16S rRNA sequence analysis and whole genome phylogeny, Cal.l.30 was identified as Bacillus halotolerans. Genome mining revealed that B. halotolerans Cal.l.30 is endowed with a diverse arsenal of secondary metabolite biosynthetic gene clusters (SM-BGCs) responsible for metabolite production with antimicrobial properties. A sub-set of the identified SM-BGCs (mojavensin A, 'bacillunoic acid') appears to be the result of recent horizontal gene transfer events. Its genome was also mined for CAZymes associated with antifungal activity. Further UHPLC-HRMS analysis indicated that Cal.l.30 synthesizes and secretes secondary metabolites with antimicrobial activity, including the lipopeptides, fengycin, surfactin and mojavensin A, bacillaene isoforms, L-dihydroanticapsin and bacillibactin. Other compounds with known antimicrobial activity were also detected, such as azelaic acid, 15- hydroxypentadecanoid acid and 2-hydroxyphenylacetic acid. The genomic and metabolomic features of the B. halotolerans Cal.l.30 provided new perspectives on the exploitation of novel Bacillus sp. as a biocontrol agent.

Genomic and Metabolomic Insights into Secondary Metabolites of the Novel Bacillus halotolerans Hil4, an Endophyte with Promising Antagonistic Activity against Gray Mold and Plant Growth Promoting Potential.

The endophytic bacterial strain Hil4 was isolated from leaves of the medicinal plant Hypericum hircinum. It exhibited antifungal activity against Botrytis cinerea and a plethora of plant growth promoting traits in vitro. Whole genome sequencing revealed that it belongs to Bacillus halotolerans and possesses numerous secondary metabolite biosynthetic gene clusters and genes involved in plant growth promotion, colonization, and plant defense elicitation. The Mojavensin cluster was present in the genome, making this strain novel among plant-associated B. halotolerans strains. Extracts of secreted agar-diffusible compounds from single culture secretome extracts and dual cultures with B. cinerea were bioactive and had the same antifungal pattern on TLC plates after bioautography. UHPLC-HRMS analysis of the single culture secretome extract putatively annotated the consecutively produced antimicrobial substances and ISR elicitors. The isolate also proved efficient in minimizing the severity of gray mold post-harvest disease on table grape berries, as well as cherry tomatoes. Finally, it positively influenced the growth of Arabidopsis thaliana Col-0 and Solanum lycopersicum var. Chondrokatsari Messinias after seed biopriming in vitro. Overall, these results indicate that the B. halotolerans strain Hil4 is a promising novel plant growth promoting and biocontrol agent, and can be used in future research for the development of biostimulants and/or biological control agents.