Biological control of leaf pathogens of tomato plants by <em>Bacillus subtilis</em> (strain FZB24)

Abstract

<i>Bacillus subtilis</i> reisolated from the biological control agents FZB24^® and Phytovit^® has shown promising results against several pathogens causing important foliar tomato diseases (late blight, early blight, powdery mildew, and leaf mold) with higher activity when applied prior pathogen infection. Since most previous studies focused primarily on the degree of disease reduction, further investigations on the mechanisms contributed to disease suppression and enhancement of plant resistance are attractive properties explored further and in more detail in the current study at microbial, histological, and molecular levels. This will help to optimize the application strategies of <i>B. subtilis</i> as a biological control agent or their metabolites as biopesticides.<br /> Application of <i>B. subtilis</i> cells and their excreted metabolites resulted in a significant reduction in disease severity of tested pathogens. In spite of <i>B. subtilis</i> cells significantly reduced late blight severity on the entire plant by 44%, but when they applied merely on the lower leaves they showed no systemic protection on the upper leaves. Using qRT-PCR, cells showed as well no induction in the expression of PR1a gene, which is an indicator of SAR. In addition, no changes in other responses of plant defense were observed demonstrating the antagonistic effect of bacterial cells and non-involvement in plant resistance.<br /> Metabolites formed by <i>B. subtilis</i> strains FZB24 and Phytovit inhibited the development of diseases and the pathogen better than the bacteria itself revealing their important role as effective substances in disease suppression. This was in favor of metabolites produced by FZB24 strain harvested 72 hours of culturing. The highest destructive effect of metabolites proved to be against <i>Phytophthora infestans</i> restricting its developmental structures and decreasing its biomass in leaf tissue by 83% and resulted in more than 70% reduction in late blight severity. They strongly inhibited the inter- and intracellular growth of <i>P. infestans</i> and resulted in superficial horizontal colonization of <i>P. infestans</i> with no progress in deeper tissue layers, besides to reduce the formation of haustoria, which are responsible for pathogen establishment. Moreover, metabolite application on the lower leaves resulted on the upper leaves in systemic protection associated with PR1a gene activation at 12 hpi.<br /> The susceptible tomato plants (cv. Money Maker) could not limit the colonization by <i>P. infestans</i> that effects on the essential activities of the plant cells changing host metabolism and activating the basal immunity after 12 hours of inoculation. All those responses were proved to be insufficient to limit <i>P. infestans</i> growth because infection resulted in more than 80% disease severity 6 days after inoculation. However, the number of differentially expressed genes after pathogen inoculation investigated using microarray analysis were reduced by 50% in metabolite-treated plants after 12 hours of inoculation. Therefore, such reduction in plant responses reflect less susceptibility, which depends on modified patterns of gene responses during the attempts of the pathogen to establish the infection structure. In addition, other changes in plant responses were exclusively unregulated after metabolite application involved in hormone signaling and photosynthesis function, besides to suppression in stress responses.<br /> Systemic protection achieved by <i>B. subtilis</i> metabolites was correlated to certain changes in gene expression under the influence of this type of resistance inducer affecting on the ability of the pathogen to form the haustoria, which is necessary for development of the pathogen and disease establishment. That indicates haustoria provide ideal targets for late blight control.