Insights into the molecular basis of biocontrol of Brassica pathogens by Bacillus amyloliquefaciens UCMB5113 lipopeptides

Abstract

Background and aims: Certain micro-organisms can improve plant protection against pathogens. The protective effect may be direct, e.g. due to antibiotic compounds, or indirect, by priming of plant defence as induced systemic resistance (ISR). The plant growth-promoting rhizobacterium Bacillus amyloliquefaciens UCMB5113 shows potential for disease management of oilseed rape. To investigate the mode of action of this protection, especially in relation to jasmonic acid-dependent ISR, Bacillus UCMB5113 was tested with Arabidopsis thaliana mutants and several important fungal pathogens of Brassica species.

Methods: Secreted lipopeptide fractions from Bacillus UCMB5113, together with synthetic peptide mimics, were evaluated for their effects on fungal phytopathogens and A. thaliana . The structures of secreted lipopeptides were analysed using mass spectrometry. Plant mutants and reporter lines were used to identify signalling steps involved in disease suppression by lipopeptides.

Key results: In plate tests Bacillus UCMB5113 and lipopeptide extracts suppressed growth of several fungal pathogens infecting Brassica plants. Separation of secreted lipopeptides using reversed-phase high-performance liquid chromatography revealed several fractions that inhibited fungal growth. Analysis by mass spectrometry identified the most potent compounds as novel linear forms of antifungal fengycins, with synthetic peptide mimics confirming the biological activity. Application of the lipopeptide extracts on Arabidopsis roots provided systemic protection against Alternaria brassicicola on leaves. Arabidopsis signalling mutants and PDF1.2 and VSP2 promoter-driven GUS lines indicated that the lipopeptide fraction involved jasmonic-acid-dependent host responses for suppression of fungal growth indicative of ISR.

Conclusions: The ability of Bacillus UCMB5113 to counteract pathogens using both antagonistic lipopeptides and through ISR provides a promising tool for sustainable crop production.

Keywords: Antagonism; Arabidopsis thaliana; Bacillus amyloliquefaciens; beneficial bacteria; biocontrol; lipopeptide; rhizosphere.

Fig. 1.

Screening of antagonistic activity of Bacillus amyloliquefaciens UCMB5113 against fungal phytopathogens. (A) A single B. amyloliquefaciens UCMB5113 colony was streaked on one side of the PDA plate and fungal strains were inoculated at the centre. Growth inhibition was studied 3–14 d later. (B) LP extract was tested for antifungal activity to phytopathogens grown on PDA. The pathogen was inoculated at the centre of the plate and aliquots of the LP extract (1) or methanol (solvent control) (2) were added at the periphery of the dish. The plates were incubated at room temperature and growth was recorded after 3–14 d.

Fig. 2.

Fractionation of Bacillus amyloliquefaciens UCMB5113 LP extract and antifungal tests. (A) RP-HPLC profile of LP extract obtained after acid precipitation of B. amyloliquefaciens UCMB5113 growth medium was obtained using a C₁₈ reversed phase column using a water/acetonitrile/TFA gradient and the eluent monitored at 215 nm. (B) Growth inhibitory activity of the LP extract fractions isolated by RP-HPLC was tested on the phytopathogens (a) A. brassicicola, (b) A. brassicae, (c) B. cinerea, (d) S. sclerotiorum and (e) V. longisporum. HPLC fractions 1–14 or methanol as control (C) were added to different parts of the Petri dish and growth was studied 3–14 d later.

Fig. 3.

Analysis of HPLC fraction 9 by UPLC-ESI-MS. (A) LC-MS chromatogram revealing one major peak eluting at 1·95 min. (B) MS spectrum showing that this peak mainly contained a compound with m/z 1495·8 that was further structurally characterized by MS-MS as illustrated in C. (C) Fragmentation pattern obtained and typical y- and b-type ions as well as internal fragments that were unambiguously assigned leading to the linear peptide sequence represented. Note that the analysis cannot distinguish Leu from Ile so Leu was arbitrarily chosen in the molecular structure. Further, the isomery of the fatty acid chain cannot be determined so the chain is written as a linear form for convenience.

Fig. 4.

Effects of synthetic peptides on growth of fungal pathogens. (A) Synthetic peptide with acetyl side chain (a1, b3), synthetic peptide with fatty acid side chain (a2, b2), B. amyloliquefaciens UCMB5113 LP extract (a3, b1) or methanol (a4, b4) was dropped on discs in the peripheral part of a PDA plate containing A. brassicicola or V. longisporum in the centre. Effects on fungal growth were recorded 5 d later. (B) Suppressive effect of synthetic peptides against A. brassicicola growth on Arabidopsis thaliana Col-0 leaves. One-week-old seedlings grown on MS media were treated with (a) water, (b) 5 % methanol, (c) synthetic peptide with acetyl side chain and (d) synthetic peptide with fatty acid side chain compounds. Two weeks after treatment, four leaves of each plant were inoculated with A. brassicicola. The presence of necrotic lesions was recorded 1 week after fungal inoculation. (C) Quantification of fungal level in plants treated with synthetic peptides. Five days after fungal inoculation, the level of Alternaria DNA on plants was quantified by qPCR where cutinase levels relative to ubiquitin5 expression are plotted. ANOVA was run to compare DNA levels among different treatments (n = 4). Plants were untreated (‘C’), or treated with methanol as solvent control (‘M’), synthetic peptide with acetyl side chain (‘AcePEP’) and synthetic peptide with fatty acid side chain (‘MyrPEP’).

Fig. 5.

qPCR analysis of A. brassicicola levels in B. amyloliquefaciens UCMB5113 primed Arabidopsis plants. One-week-old seedlings of Arabidopsis wildtype or the mutants coi1-16, sid2, jar1, myc2, myb72 and npr1 were tested. Roots of one-week-old seedlings grown on MS media were treated with water, 5 % methanol or an LP extract. Two weeks after treatment, four leaves of each plant were inoculated with A. brassicicola. Five days after fungal inoculation, the level of Alternaria DNA was quantified by qPCR. ANOVA was run to compare DNA levels among the treatments for each plant line where different letters indicate significant differences. Plants were untreated (white bar), treated with methanol as solvent control (grey bar) or treated with LP extract (black bar).

Fig. 6.

Effects on JA reporter plants by LP extract and A. brassicicola. One-week-old GUS reporter plants with different root treatments by water, methanol or LP extract were challenged with A. brassicicola spores on leaves 2 weeks later. Three days after fungal inoculation, plants were stained for reporter gene GUS expression. (A) VSP2:GUS without fungal treatment; (B) VSP2:GUS with fungal treatment; (C) VSP2:GUS roots after fungal treatment; (D) PDF1.2:GUS without fungal treatments; (E) PDF1.2:GUS with fungal treatment. Arrows indicate site of fungal inoculation on leaves and the enhanced expression in roots.