Phenylacetic Acid Is ISR Determinant Produced by Bacillus fortis IAGS162, Which Involves Extensive Re-modulation in Metabolomics of Tomato to Protect against Fusarium Wilt

Abstract

Bacillus fortis IAGS162 has been previously shown to induce systemic resistance in tomato plants against Fusarium wilt disease. In the first phase of current study, the ISR determinant was isolated from extracellular metabolites of this bacterium. ISR bioassays combined with solvent extraction, column chromatography and GC/MS analysis proved that phenylacetic acid (PAA) was the potential ISR determinant that significantly ameliorated Fusarium wilt disease of tomato at concentrations of 0.1 and 1 mM. In the second phase, the biochemical basis of the induced systemic resistance (ISR) under influence of PAA was elucidated by performing non-targeted whole metabolomics through GC/MS analysis. Tomato plants were treated with PAA and fungal pathogen in various combinations. Exposure to PAA and subsequent pathogen challenge extensively re-modulated tomato metabolic networks along with defense related pathways. In addition, various phenylpropanoid precursors were significantly up-regulated in treatments receiving PAA. This work suggests that ISR elicitor released from B. fortis IAGS162 contributes to resistance against fungal pathogens through dynamic reprogramming of plant pathways that are functionally correlated with defense responses.

Keywords: Bacillus; fusarium wilt; induced systemic resistance (ISR); phenylacetic acid; tomato.

FIGURE 1

Extraction procedure and ISR activity of crude and purified metabolites from B. fortis IAGS162.

FIGURE 2

Elucidation of biochemicals presents in ISR active sub-fraction in cell free culture filtrates of B. fortis IAGS162. (A) Chromatogram of Gas Chromatography and Mass Spectrometry analysis (GC/MS) for identification of the ISR determinant/s of B. fortis IAGS162 present in the ISR-active sub-fraction. (B) The mass spectrum analysis obtained by electrospray ionization of Phenylacetic acid.

FIGURE 3

Influence of root treatment with chemicals present in ISR active sub-fraction on the disease development on tomato plants after inoculation with fusarium wilt pathogen. Sterile distilled water was used as positive control. ISR eliciting sub-fraction was subjected to GC/MS analysis and chemicals present were purchased and subjected to ISR bioassay. Vertical bar represents standard errors. Asterisks indicate statistically significant reduction in disease index as compared to pathogen control as governed by ANOVA at P ≤ 0.05.

FIGURE 4

Schematic representations of tomato metabolites in fold change as compared to untreated control plants. Different colors represent levels of metabolite fold change where blue is increasing and red is decreasing. Mean values of two independent experiments are presented here.

FIGURE 5

Principal component analysis (PCA) score plot of metabolite finger printing of tomato shoots 7 days after treatment applications. FOL, F. oxysporum f.sp. lycopersici. PAA, phenylacetic acid. UC, non-treated control.

FIGURE 6

An overview of changes in phenylpropenoid pathway of tomato plants under influence of PAA and fusarium wilt pathogen. Comparison was made between two treatments viz: 1 = Plants receiving PAA and F. oxysporum, 2 = plants receiving F. oxysporum alone (considered as control). Metabolites in red font color show significant increase over control as governed by ANOVA at P ≤ 0.05. Black font represent no significant change as compared to control. Gray color represent metabolites not detected.

FIGURE 7

Heat map illustrating the different metabolite levels in tomato plants under different treatments. Each row represents differentially expressed metabolites while each column represents a treatment. Mean values of two independent experiments are presented here. Metabolites are clustered using average linkage hierarchical clustering. The colors in the heat map represent the intensity of the log²-fold change in metabolite levels. FOL, F. oxysporum f.sp. lycopersici. PAA, phenylacetic acid. UC, non-treated control.