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. 2019 Aug;20(8):1037-1050.
doi: 10.1111/mpp.12809. Epub 2019 May 18.

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Yan Li  1 , Marie-Claire Héloir  2 , Xun Zhang  1 , Mareen Geissler  3 , Sophie Trouvelot  2 , Lucile Jacquens  2 , Marius Henkel  3 , Xin Su  1 , Xuewen Fang  1 , Qi Wang  1 , Marielle Adrian  2
Affiliations

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Yan Li et al. Mol Plant Pathol. 2019 Aug.

Abstract

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.

Keywords: Bacillus subtilis; Plasmopara viticola; defence; downy mildew; fengycin; lipopeptide; surfactin.

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Figures

Figure 1
Figure 1
B. subtilis GLB191 supernatant‐induced protection of grapevine leaves against downy mildew. Sporulation of Plasmopara viticola was assessed with the downy mildew susceptible cultivar cv. Marselan treated with water (H2O), PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S). Values correspond to the mean percentage obtained for 36 discs of six leaves from three plants (the second and third apical leaves from each plant). The data are representative of three independent experiments. Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different at P < 0.01 according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction.
Figure 2
Figure 2
Direct effect of B. subtilis GLB191 supernatant on P. viticola zoospores. Grapevine (V. vinifera cv. Marselan) leaves were treated with water (H2O), PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S) and inoculated with P. viticola sporangia 2 h later. At 24 h post inoculation, leaf discs were punched out from leaves and the number of infection sites (i.e. stomata with encysted zoospores of P. viticola) was determined by epifluorescence observations after aniline blue staining of the pathogen. Panel A: Values are the mean number of infection sites for ten discs of six leaves harvested from three plants (the second and third apical leaves from each plant). The data correspond to a representative of three independent experiments. Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction (P < 0.01). Panel B: Representative photographs of fluorescence microscopy observations. Arrows indicate the infection sites (encysted zoospores). Scale bars represent 100 μm.
Figure 3
Figure 3
Callose production induced by B. subtilis GLB191 supernatant. Grapevine (V. vinifera cv. Marselan) leaves were treated by water (H2O), PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S). Callose production (fluorescent spots) was assessed 3 days post treatment by epifluorescence observations after aniline blue staining. Panel A: Values are the mean number of fluorescent spots for six leaves from three plants (the second and third apical leaves from each plant). Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction (P < 0.01). The data is a representative of three independent experiments. Panel B: Representative photographs of fluorescence microscopy observations. Scale bars represent 100 μm.
Figure 4
Figure 4
Defence‐related gene expression in grapevine leaves treated with B. subtilis GLB191 supernatant. The relative transcript accumulation of defence genes was determined by qRT‐PCR in grapevine (V. vinifera cv. Marselan) leaves 24 h post treatment with water (H2O), PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S). Results represent relative fold expression calculated with the 2−ΔΔCt method, compared to the reference gene EF1γ and to the water control. The data are representative of three independent experiments. PR2, PR protein 2 β‐1,3‐glucanase; PR1, PR protein 1; STS, stilbene synthase; PAL, phenylalanine ammonia lyase; PR3, PR protein 3 chitinase 4c; Lox9, lipoxygenase 9; JAZ1, jasmonate ZIM‐domain protein. *The values above each column indicate the relative fold of transcript accumulation in leaves treated with GLB191‐S compared to that of PDB‐treated ones.
Figure 5
Figure 5
Protection of grapevine leaves against downy mildew induced by B. subtilis GLB191 and its derived mutants. Sporulation of Plasmopara viticola was assessed with the downy mildew susceptible cultivar cv. Marselan treated with water (H2O), PDB medium (PDB) or the supernatant of the wild‐type B. subtilis GLB191 (GLB191‐S) and its mutants ΔppsB (ΔppsB‐S), ΔsrfAA (ΔsrfAA‐S), ΔppsBsrfAA (ΔppsBsrfAA‐S). Values correspond to the mean percentage obtained for 36 discs of six leaves from three plants (the second and third apical leaves from each plant). The data are the mean of three independent experiments. Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction (P < 0.01).
Figure 6
Figure 6
Direct effect of the supernatants of B. subtilis GLB191 and its derived mutants on P. viticola zoospores. Grapevine (V. vinifera cv. Marselan) leaves were treated with water (H2O), PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S) and its mutants ΔppsB (ΔppsB‐S), ΔsrfAA (ΔsrfAA‐S), ΔppsBsrfAA (ΔppsBsrfAA‐S), and inoculated with P. viticola sporangia 2 h later. At 24 hpi, leaf discs were punched out from leaves and the number of infection sites (i.e. stomata with encysted zoospores of P. viticola) was determined by UV epifluorescence observations after aniline blue staining of the pathogen. Panel A: Values are the mean number of infection sites for ten discs of six leaves harvested from three cuttings (the second and third apical leaves from each plant). The data correspond to a representative of three independent experiments. Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction (P < 0.05). Panel B: Representative photographs of fluorescence microscopy observations. Arrows indicate the infection sites (encysted zoospores). Scale bars represent 100 μm.
Figure 7
Figure 7
Callose production induced by the supernatant of B. subtilis GLB191 and its derived mutants. Callose deposition in grapevine (V. vinifera cv. Marselan) leaves treated by water (H2O), PDB medium (PDB) or the supernatant of the wild‐type B. subtilis GLB191 (GLB191‐S) and its mutants ΔppsB (ΔppsB‐S), ΔsrfAA (ΔsrfAA‐S), ΔppsBsrfAA (ΔppsBsrfAA‐S) was assessed 3 days post treatment by epifluorescence observations after aniline blue staining. Panel A: Values are the mean estimation for leaves from three plants (the second and third apical leaves from each plant) in the same treatment. Treatments were compared by means with the non‐parametric Kruskal–Wallis approach at the 5% significance level. Means with different letters are significantly different according to the Mann–Whitney pairwise post hoc test with application of the Bonferroni correction (P < 0.05). The data are representative of three independent experiments. Panel B: Representative photographs observed using a fluorescence microscope. Scale bars represent 100 μm.
Figure 8
Figure 8
Defence‐related genes expression in grapevine leaves treated with supernatant of B. subtilis GLB191 and its derived mutants. The relative transcript accumulations of genes PR2 (encoding the pathogenesis‐related protein 2 β‐1,3‐glucanase), STS (encoding stilbene synthase) and PR3 (encoding the pathogenesis‐related protein 3 chitinase 4c) were determined by qRT‐PCR in leaves treated with water, PDB medium (PDB) or the supernatant of B. subtilis GLB191 (GLB191‐S) and its mutants ΔppsB (ΔppsB‐S), ΔsrfAA (ΔsrfAA‐S), ΔppsBsrfAA (ΔppsBsrfAA‐S). Results represent relative fold expression calculated with the 2−ΔΔCt method, compared to the reference gene EF1γ and to the water control. The data are representative of three independent experiments.
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