Induced resistance in tomato plant against Alternaria solani by Bacillus subtilis strain 30B-B6

Early blight (EB) caused by the necrotrophic fungus Alternaria solani is a devastating plant disease of tomato crops, currently mainly controlled by synthetic chemical fungicides. In the framework of sustainable agriculture, a promising new control strategy is the induction of plant defense responses by elicitors. These last decades, numerous researches on new elicitors have emerged, and recent findings have reported a promising strain of Bacillus subtilis, 30B-B6, as a potential elicitor of tomato plants. The present study investigates the systemic induction of defense response in tomato (Solanum lycopersicum var. Moneymaker) against A. solani strain MBC9282, using B. subtilis 30B-B6 as an elicitor. Trials were conducted under controlled greenhouse conditions. Results revealed that root application of the eliciting bacterium 6 hours before the fungal leaf inoculation significantly increased tomato plant protection by 83% against EB compared to control plants. Disease protection was assumed to be due to the induction of resistance in the host plant since experimental protocols were designed in order to avoid any direct contact between the biocontrol agent and the pathogen. A second bioassay with the bacterial supernatant (filtered 30B-B6 suspension) highlighted an inability of the metabolite suspension to trigger tomato resistance against A. solani, when the bacterium was not present. A third bioassay was performed to evaluate the expression of tomato defense-related genes by RT-qPCR on four treatment groups: (1) bacterial elicitation, (2) fungal inoculation, (3) both bacterial elicitation and fungal inoculation and (4) control plants. Gene expression levels were assessed separately on tomato’s lower-older leaves and upper-younger leaves. The fungal inoculations were performed on the lower leaves, while the upper ones served for a systemicity assessment. The target genes analyzed were assigned to specific plant metabolic pathways related to the induced systemic resistance (ISR) and/or the systemic acquired resistance (SAR): the SAR marker PR1 gene, the ISR markers LOX and PI-1 genes, the NPR1 gene linked to the crosstalk between both signaling pathways and PR2-b gene, responsive to salicylic acid and/or jasmonic acid. The fungal inoculation of non-elicited plants led to a systemic significant increase in the PR2-b gene expression compared to non-inoculated plants, at 6 hours post inoculation (hpi). No activation of PR1 gene was detected in plants challenged by the pathogen. The 30B-B6 application triggered significant increases in LOX and PI-1 gene expression in tomato upper leaves, respectively at 6 hpi and 9hpi, compared to the other treatments. However, when both the bacterium and the fungus were present, only the PR2-b gene expression was up regulated in the systemic leaves compared to uninoculated plants, at 24 hpi. Our results indicate that the 30B-B6 root application enhances tomato resistance to EB, deriving from an induction of defense genes, but the signaling pathways involved remain equivocal. The promising features of 30B-B6 highlighted in the present work make it a potential biological agent in the control of tomato crops against EB and encourage its practical use in agriculture.