Cyclic Lipopeptides of Bacillus amyloliquefaciens subsp plantarum Colonizing the Lettuce Rhizosphere Enhance Plant Defense Responses Toward the Bottom Rot Pathogen Rhizoctonia solani

The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2 mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.