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. 2016 Feb 23:7:196.
doi: 10.3389/fmicb.2016.00196. eCollection 2016.

Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

Rouhallah Sharifi  1 , Choong-Min Ryu  2
Affiliations

Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

Rouhallah Sharifi et al. Front Microbiol. .

Abstract

Biological control (biocontrol) agents act on plants via numerous mechanisms, and can be used to protect plants from pathogens. Biocontrol agents can act directly as pathogen antagonists or competitors or indirectly to promote plant induced systemic resistance (ISR). Whether a biocontrol agent acts directly or indirectly depends on the specific strain and the pathosystem type. We reported previously that bacterial volatile organic compounds (VOCs) are determinants for eliciting plant ISR. Emerging data suggest that bacterial VOCs also can directly inhibit fungal and plant growth. The aim of the current study was to differentiate direct and indirect mechanisms of bacterial VOC effects against Botrytis cinerea infection of Arabidopsis. Volatile emissions from Bacillus subtilis GB03 successfully protected Arabidopsis seedlings against B. cinerea. First, we investigated the direct effects of bacterial VOCs on symptom development and different phenological stages of B. cinerea including spore germination, mycelial attachment to the leaf surface, mycelial growth, and sporulation in vitro and in planta. Volatile emissions inhibited hyphal growth in a dose-dependent manner in vitro, and interfered with fungal attachment on the hydrophobic leaf surface. Second, the optimized bacterial concentration that did not directly inhibit fungal growth successfully protected Arabidopsis from fungal infection, which indicates that bacterial VOC-elicited plant ISR has a more important role in biocontrol than direct inhibition of fungal growth on Arabidopsis. We performed qRT-PCR to investigate the priming of the defense-related genes PR1, PDF1.2, and ChiB at 0, 12, 24, and 36 h post-infection and 14 days after the start of plant exposure to bacterial VOCs. The results indicate that bacterial VOCs potentiate expression of PR1 and PDF1.2 but not ChiB, which stimulates SA- and JA-dependent signaling pathways in plant ISR and protects plants against pathogen colonization. This study provides new evidence for bacterial VOC-elicited plant ISR that protects Arabidopsis plants from infection by the necrotrophic fungus B. cinerea. Our work reveals that bacterial VOCs primarily act via an indirect mechanism to elicit plant ISR, and have a major role in biocontrol against fungal pathogens.

Keywords: bacterial volatile organic compounds; biofilm formation; induced systemic resistance; leaf surface attachment; phytohormones; plant growth-promoting rhizobacteria.

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Figures

FIGURE 1
FIGURE 1
Plant protection against Botrytis cinerea by volatile compounds emitted from Bacillus subtilis GB03. Bacterial volatile compounds (BVCs) elicit induced systemic resistance (ISR) against B. cinerea infection. (A) Representative photo of ISR by BVCs using by the I-plate system. The right panel = BVC treatment; left panel = water control (B) Disease severity (0 = no symptom, 10 = severe necrosis) in the presence of bacterial VOCs and water-treated controls was recorded and plates were photographed 3 days after pathogen challenge. Different letters indicate significant differences between treatments according to Fisher’s LSD test at P = 0.05.
FIGURE 2
FIGURE 2
Dose-dependent inhibition of B. cinerea mycelial growth, spore germination, and spore development by B. subtilis GB03 volatile compounds. (A) Spore germination, (B) spore formation, and (C) mycelial growth of B. cinerea were measured after 7 days in the presence of one, two, or three spot-inoculated filter disks (5 mm diameter) of B. subtilis GB03 bacterial suspension (108 CFU/ml) that were allowed to grow for 24 h before addition of fungi. Different letters indicate significant differences between treatments according to Fisher’s LSD test at P = 0.05. Error bars indicate standard error of the mean (SEM).
FIGURE 3
FIGURE 3
Direct and indirect mechanisms of B. subtilis GB03 volatile compound effects on Botrytis cinerea and ISR of Arabidopsis. (A) Schematic of experimental design to test bacterial VOC-elicited ISR using the two-chamber I-plate system. Arabidopsis seedlings were grown in one chamber and B. subtilis GB03 were grown in the other chamber. The two chambers share the same head space, which allows bacterial volatiles to be transmitted to seedlings and inoculated fungal pathogens. In one group of plates, B. subtilis GB03 were removed before fungal inoculation at 14 days (middle image); in the other group, bacteria and volatile emission were retained (left image). Water-treated filter disks were used as control. (B) Disease severity caused by B. cinerea inoculation on Arabidopsis seedlings pretreated with B. subtilis GB03 VOCs, and in the presence and absence of continuing B. subtilis VOC emission. The inset pie graph indicates percentage plant protection conferred by VOC-elicited ISR (indirect mechanism) and by VOC-induced inhibition of fungal growth (direct mechanism). The inset photos above or inside bar graph indicate that B. subtilis GB03 volatile compounds inhibit leaf attachment of B. cinerea. Fungi growth were checked after staining with Trypan blue. Different letters indicate significant differences between treatments according to Fisher’s LSD test at P = 0.05.
FIGURE 4
FIGURE 4
Bacillus subtilis GB03 volatile compounds inhibit surface biofilm formation of Botrytis cinerea. Fungal biofilm formation in the presence of different concentrations of bacterial VOCs was measured using the CV staining method. The absorbance of CV retained in the fungal biofilm was measured spectrophotometrically at 600 nm, and represents the biofilm quantity. Fungal cultures were photographed 7 days after the start of VOC exposure. Different letters indicate significant differences between treatments according to Fisher’s LSD test at P = 0.05.
FIGURE 5
FIGURE 5
Bacillus subtilis GB03 volatile compounds elicit Arabidopsis defense priming of the jasmonic acid and salicylic acid signaling pathways. Defense priming gene expression levels were measured by time-course qRT-PCR analysis of (A) PR1 in the salicylic acid signaling pathway, (B) PDF1.2 in the jasmonic acid signaling pathway, and (C) ChiB in the ethylene signaling pathway (C). The ratio of gene expression in the B. subtilis GB03-treated plants versus that in the water-treated control relative to expression of the Actin gene is computed as the mean ± SEM. Different letters indicate significant differences between treatments (A,B) according to Fisher’s LSD test at P = 0.05.
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