Bacterial volatiles induce systemic resistance in Arabidopsis

Abstract

Plant growth-promoting rhizobacteria, in association with plant roots, can trigger induced systemic resistance (ISR). Considering that low-molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in plants, we examined whether volatile organic compounds (VOCs) associated with rhizobacteria can initiate ISR. In Arabidopsis seedlings exposed to bacterial volatile blends from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, disease severity by the bacterial pathogen Erwinia carotovora subsp. carotovora was significantly reduced compared with seedlings not exposed to bacterial volatiles before pathogen inoculation. Exposure to VOCs from rhizobacteria for as little as 4 d was sufficient to activate ISR in Arabidopsis seedlings. Chemical analysis of the bacterial volatile emissions revealed the release of a series of low-molecular weight hydrocarbons including the growth promoting VOC (2R,3R)-(-)-butanediol. Exogenous application of racemic mixture of (RR) and (SS) isomers of 2,3-butanediol was found to trigger ISR and transgenic lines of B. subtilis that emitted reduced levels of 2,3-butanediol and acetoin conferred reduced Arabidopsis protection to pathogen infection compared with seedlings exposed to VOCs from wild-type bacterial lines. Using transgenic and mutant lines of Arabidopsis, we provide evidence that the signaling pathway activated by volatiles from GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways. This study provides new insight into the role of bacteria VOCs as initiators of defense responses in plants.

Figure 1.

PGPR VOCs can modulate infection severity of Arabidopsis seedlings by E. carotovora subsp. carotovora strain SCC1. Plants exposed to airborne chemicals released from eight ISR-inducing PGPR strains compared with a non-ISR Escherichia coli strain DH5α and water treatment; different letters indicate significant differences between treatments according to lsd at P = 0.05. Inset, Representative seedlings 24 h after E. carotovora subsp. carotovora infection and exposure to VOCs from GB03 (left) or DH5α (right).

Figure 2.

Induced disease resistance in Arabidopsis seedlings within 4 d of PGPR VOC exposure: PGPR strains GB03 (black boxes), IN937a (white boxes), or water treatment (gray boxes). Differences in letters indicates a significant difference between treated and control samples based on Fisher's lsd test at P = 0.05.

Figure 3.

VOC profile of ISR-inducing strains GB03 (black boxes) and IN937a (white boxes) compared with the noninducing strain DH5α (gray boxes). Compounds positively identified include 3-hydroxy-2-butanone, 2,3-butanediol, tetramethyl pyrazine, and the hydrocarbons decane, undecane, decanal, dodecane, 2-undecanone, 2-tridecanone, and 2-tridecanol. Different letters indicate significant differences between treatments according to lsd at P = 0.05. An asterisk indicates that emissions were below detection limits.

Figure 4.

Chromatographic profiles of volatiles released on d 2 from B. subtilis parental strain 168 (A), an overexpresser line BSIP1171 (B), mutant lines BSIP1173 (C) and BSIP1174 (D), and uninoculated media (E). Compounds positively identified include 3-hydroxy-2-butanone (1), 2,3-butanediol (2), decanal (3), decane (4), tetramethyl pyrazine (5), and undecane (6); nonyl acetate was added as an internal standard (IS). Asterisks in the lower chromatograms designate compounds that align with numbered peaks above.

Figure 5.

Infection of Arabidopsis by E. carotovora subsp. carotovora strain SCC1 after exposure to wild (GB03 and 168) overproducing 2,3-butanediol (BSIP1171) and mutants with attenuated 2,3-butanediol synthesis (BSIP1173 and BSIP1174). E. coli strain DH5α and water treatment were used as negative controls. Different letters indicate significant differences between treatments according to lsd at P = 0.05.

Figure 6.

A dose response curve of 2,3-butanediol on the susceptibility of Arabidopsis to infection by E. carotovora subsp. carotovora strain SCC1. Volatile headspace collections from GB03 and IN977a were provided as positive controls, whereas solvent (CH₂Cl₂) provided a negative control. Different letters indicate significant differences between treatments according to lsd at P = 0.05.

Figure 7.

GB03 produced (2R,3R)-(-)-2,3-butanediol identification based on rt matches of authentic standards. GC-flame ionization detector (FID) trace of synthetic (RS)-2,3-butanediol (rt 28.6 min; A), synthetic (2R,3R)-(-)-2,3-butanediol (rt 21.1 min; B), a mixture of (2S,3S)-(+)-2,3-butanediol (rt 20.3 min) and (2R,3R)-(-)-2,3-butanediol (C), and GB03 volatile profile containing (2R,3R)-(-)-2,3-butanediol (rt 21.03 min; D).

Figure 8.

Activation of an ethylene/JA::GUS transgenic line of Arabidopsis (Pdf1.2) by GB03 VOCs, whereas other reporter transgenic lines (Pr-1a activated by SA and Jin14 activated by JA) were not activated by bacterial VOCs; the chemical elicitors SA and 1-aminocyclopropane-1-carboxylic acid (activate the Pr-1a gene and the Pdf1.2 gene respectively) were employed as positive controls, and DH5α VOCs and water treatment served as negative controls. An asterisk indicates a significant difference between treated and control samples according to lsd at P = 0.05. Inset, Transgenic seedlings Pdf 1.2 exposed to GB03 volatiles (top) and water control (bottom).