Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean

Abstract

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Keywords: Sclerotinia sclerotiorum; antifungal peptide; bio-fungicide; modes of action; soybean.

Figure 1

In vitro and semi-in planta antifungal activities of GMA4CG_V6 against S. sclerotiorum 555 on soybean leaves. (a) Representative pictures showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 in SFM media. (b) Inhibition of fungal growth at different concentrations of GMA4CG_V6. Each data point represents the average % growth from three replications and error bars represent the standard errors of the means between replicates. (c) Representative pictures (under white light and with CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on detached soybean leaves. (d) Relative disease lesion area following GMA4CG_V6 application on soybean leaf surface at different concentrations. Each data point represents the average lesion size from five leaves relative to control (no peptide, 0 µM), with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with p < 0.05. This experiment was repeated three times with similar results.

Figure 2

Semi-in planta antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on soybean pods and stems. (a) Soybean pods detachment assay showing the effect of GMA4CG_V6 on disease development at 3 dpi. Representative pictures (under white light and with CropReporter). (b) Relative disease lesion area at 3 dpi following the application of GMA4CG_V6 on the pods. (c) Soybean stems detachment assay showing the effect of GMA4CG_V6 on disease development at 3 dpi. (d) Relative lesion size following the application of GMA4CG_V6 on the stems surface at 3 dpi. Antifungal activities at 7 dpi (e,f). For panel (b,d), each data point represents the average lesion area/size of five samples relative to control (no peptide, 0 µM), with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with p < 0.05. This experiment was repeated three times with similar results.

Figure 3

In planta preventative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555. (a) In-pot assay showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on tobacco leaves sprayed with S. sclerotiorum 555 ground mycelia. (b) Representative pictures (CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on tobacco plants. (c) In-pot assay showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 on soybean leaves sprayed with S. sclerotiorum 555 ground mycelia. (d) Representative pictures (CropReporter) showing the antifungal activity of GMA4CG_V6 against S. sclerotiorum soybean leaves. The mock samples were sprayed only with the 1× SFM without the fungus and the peptide.

Figure 4

In planta curative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555. A suspension of ground mycelium at optical density (OD) 0.5 was used to spray soybean leaves followed by the spray of GMA4CG_V6. The mock samples were sprayed only with the 1× SFM without the fungus and the peptide. (a) Representative pictures of in pots assay showing the curative antifungal activity of GMA4CG_V6 against S. sclerotiorum 555 at 48 h post-infection. (b) Representative pictures (under white light and with CropReporter based on the value of potential photosynthetic efficiency F_V/F_M) taken 48 h post-infection showing the curative antifungal activity of GMA4CG_V6, (Arrow) for treated leaf.

Figure 5

Effect of GMA4CG_V6 on sclerotia production by S. sclerotiorum 555. (a) Representative pictures showing the effect of various GMA4CG_V6 variants against sclerotia production in vitro after 14-day treatment. Arrows point to sclerotia. (b) Picture showing the total number of sclerotia produced 30 days following peptide treatment. Six plates were used for each treatment. (c) Average number of sclerotia per plate. Each data point represents the average of six plates, with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with p < 0.05. (d) Representative pictures showing the antifungal activity of GMA4CG_V6 against sclerotia production on soybean pods fourteen days post-treatment. Arrows point to sclerotia. (e) Average number of sclerotia per pod. Each data point represents the average of five pods, with error bars representing the standard errors of the means between replicates. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with p < 0.05. (f) Confocal microscopy images showing the inhibition of sclerotia development by GMA4CG_V6 after two days of growth in vitro. Arrows showing the early development of sclerotia—growth of hyphal tips and dichotomous branching. The scale bar = 10 µm. (g) Effect of different GMA4CG_V6 variants on the expression level of genes involved in sclerotia production in S. sclerotiorum 555. Results were analyzed using ANOVA, followed by a Tukey’s post hoc test. * Indicates significant differences between data of the control and the treated samples with p < 0.05.

Figure 6

Membrane permeabilization activity and uptake of sub-lethal concentrations of GMA4CG_V6 by fungal cells. (a) Confocal microscopy images and corresponding bright field images of SYTOX Green uptake in S. sclerotiorum 555 hyphae treated with 24 μM GMA4CG_V6 for 30 min. (b) The intracellular localization of 12 µM TMR-labeled GMA4CG_V6 in S. sclerotiorum 555. The confocal microscope images were captured 2–5 min after GMA4CG_V6 challenge. The scale bar = 10 μm.

Figure 7

Effect of topical application of GMA4CG_V6 on the growth of soybean plants. (a) Representative picture showing no toxicity of GMA4CG_V6 treatment on soybean plants at 45 days post-treatment. (b) Above-ground and root biomass of plants treated with water or GMA4CG_V6 at 45-day post-treatment. Each data point represents the average mass of five plants, with error bars representing the standard errors of the means between replicates.

Figure 8

A proposed model illustrating the MoA of GMA4CG_V6 against S. sclerotiorum 555. GMA4CG_V6 sprayed on leaves infected with S. sclerotiorum 555 first binds to the pathogen’s cell walls, and the peptide starts to permeabilize the fungal plasma membrane. GMA4CG_V6 within the fungus can repress the expression of fungal genes involved in sclerotia development. The surface application of GMA4CG_V6 efficiently inhibits fungal growth and significantly reduces sclerotia development and white mold symptoms. Finally, soybean treatment with GMA4CG_V6 does not affect above- or below-ground biomass.