Biocontrol and Action Mechanism of Bacillus subtilis Lipopeptides' Fengycins Against Alternaria solani in Potato as Assessed by a Transcriptome Analysis

Abstract

Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01's fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01's secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins' biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.

Keywords: Alternaria solani; Bacillus subtilis; cell wall and membrane; conidia; fengycins; transcriptome.

FIGURE 1

Biocontrol effects of B. subtilis ZD01 fermentation broth on the potato early blight in a pot assay under greenhouse conditions. (A) Effects of 5 × 10³, 5 × 10^5, and 5 × 10⁷ CFU/mL of B. subtilis ZD01 fermentation broth on the development of early blight symptoms of potato leaves. (B) The diseased leaf rate and lesion areas of potato leaves inoculated with A. solani HWC-168 with or without the ZD01 fermentation broth treatment. (C) Chlorophyll α fluorescence induction of a diseased potato leaf (kept in darkness for 20 min before the measurement). Data are presented as means of three replicates ± SDs, and error bars represent the SDs for three replicates. Means with different letters have significant differences (p < 0.05).

FIGURE 2

Bacillus subtilis ZD01 significantly reduced the disease severity caused by A. solani HWC-168 on potato leaves in vivo. (A) Evidence of disease development on potato leaves treated with the ZD01supernatant, bacteria and fermentation broth prior to A. solani HWC-168 inoculation. (B) The lesion areas of potato leaves of potato inoculated with A. solani HWC-168 and co-cultured with ZD01 supernatant, bacteria and fermentation broth. (C) Quantitative detection by qPCR of A. solani HWC-168 growth on potato leaves inoculated with A. solani HWC-168 and co-cultured with the ZD01 supernatant, bacteria and fermentation broth. Data are presented as means of three replicates ± SDs, and error bars represent the SDs for three replicates. Means with different letters are significantly different (p < 0.05).

FIGURE 3

Supernatant extracted from the ZD01 fermentation broth exhibited inhibitory effects on A. solani HWC-168 mycelial growth and conidial germination. (A) Effects of the supernatant produced by B. subtilis ZD01 on A. solani mycelial growth. (B) Optical and scanning electron micrographs of A. solani co-cultured with the ZD01 supernatant. (C,D) Reduction in conidial germination of A. solani treated with the ZD01 supernatant. Data are presented as means of three replicates ± SDs, and error bars represent the SDs for three replicates. Means with different letters are significantly different (p < 0.05).

FIGURE 4

Lipopeptides produced by ZD01 show antagonistic effects against the growth of A. solani HWC-168. (A) Colony morphology of A. solani co-cultured with ZD01 lipopeptides. (B) Optical and scanning electron micrographs of A. solani co-cultured with ZD01 lipopeptides. (C) Identification of ZD01 lipopeptides by MALDI-TOF-MS. (D) Colony morphology of A. solani co-cultured with fengycins and surfactins for the analysis of the active ingredients against A. solani. (E) Determination of the MIC value of fengycins against A. solani using the microdilution method. The red arrows indicate wrinkled surfaces hyphal cells treated with lipopeptides; the yellow arrows indicate the vacuolation of hyphae after exposure to lipopeptides.

FIGURE 5

Volcano plots, Venn diagrams, a heatmap and the GO functional classification. (A) The differences in the distribution and density distribution of gene expression in CK_2h, CK_6h, FenT_2h, and FenT_6h. (B) Venn diagrams showing the numbers of common DEGs that are shared in comparisons among CK_2h, CK_6h, FenT_2h, and FenT_6h. (C) Heatmap of the common DEGs in the three treatment groups. (D) GO functional classification results of DEGs shared by the three treatment groups. The DEGs were assigned to three categories: cellular component, molecular function and biological process.

FIGURE 6

Effects of fengycins on A. solani hyphal cell wall integrity and cell membrane permeability. (A) Scanning and transmission electron micrographs of A. solani hyphae co-cultured with fengycins. (B) Chitin contents of A. solani hyphae co-cultured with fengycins and a control groups. (C) Fluorescence microscope imaging of A. solani hyphae treated with fengycins and a control groups. The phase channel shows all the fungal cells in bright-field images, and the Sytox Green channel shows cells attached or inserted with Sytox Green labeling; the merged channel shows the proportion of Sytox Green-labeled cells. (D) Effects of fengycins on ATP release from A. solani. The changes in the extracellular ATP levels of A. solani represent cell membrane damage. The results are presented as the means ± SDs (n = 3).

FIGURE 7

Effects of fengycins on conidial germination and structure. (A,B) Reduction in the conidial germination of A. solani after treatment with fengycins. (C) Transmission electron micrographs of A. solani co-cultured with fengycins. Data are presented as means of three replicates ± SDs, and error bars represent the SDs for three replicates. Means with different letters are significantly different (p < 0.05).

FIGURE 8

A model for the mode of action of fengycins produced by ZD01 against A. solani. Fengycins produced by ZD01 mediate A. solani mycelial growth and conidial germination. Fengycins damage the cell wall integrity, change mycelial cell membrane permeability and alter conidial structures, which subsequently leads to the suppression of fungal growth, mycelial penetration, conidial vitality and germination. Therefore, A. solani fails to infect potato leaves.