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. 2024 May 30:15:1388669.
doi: 10.3389/fmicb.2024.1388669. eCollection 2024.

Biocontrol potential of endophytic Bacillus subtilis A9 against rot disease of Morchella esculenta

Xue Chen #  1 , Yin Zhang #  1   2   3 , ShengQian Chao #  1   2   3 , LiLi Song  1   2   3 , GuoGan Wu  1   2   3 , Yu Sun  1   2   3 , YiFan Chen  1   2   3 , BeiBei Lv  1   2   3   4   5
Affiliations

Biocontrol potential of endophytic Bacillus subtilis A9 against rot disease of Morchella esculenta

Xue Chen et al. Front Microbiol. .

Abstract

Introduction: Morchella esculenta is a popular edible fungus with high economic and nutritional value. However, the rot disease caused by Lecanicillium aphanocladii, pose a serious threat to the quality and yield of M. esculenta. Biological control is one of the effective ways to control fungal diseases.

Methods and results: In this study, an effective endophytic B. subtilis A9 for the control of M. esculenta rot disease was screened, and its biocontrol mechanism was studied by transcriptome analysis. In total, 122 strains of endophytic bacteria from M. esculenta, of which the antagonistic effect of Bacillus subtilis A9 on L. aphanocladii G1 reached 72.2% in vitro tests. Biological characteristics and genomic features of B. subtilis A9 were analyzed, and key antibiotic gene clusters were detected. Scanning electron microscope (SEM) observation showed that B. subtilis A9 affected the mycelium and spores of L. aphanocladii G1. In field experiments, the biological control effect of B. subtilis A9 reached to 62.5%. Furthermore, the transcritome profiling provides evidence of B. subtilis A9 bicontrol at the molecular level. A total of 1,246 differentially expressed genes (DEGs) were identified between the treatment and control group. Gene Ontology (GO) enrichment analysis showed that a large number of DEGs were related to antioxidant activity related. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the main pathways were Nitrogen metabolism, Pentose Phosphate Pathway (PPP) and Mitogen-Activated Protein Kinases (MAPK) signal pathway. Among them, some important genes such as carbonic anhydrase CA (H6S33_007248), catalase CAT (H6S33_001409), tRNA dihydrouridine synthase DusB (H6S33_001297) and NAD(P)-binding protein NAD(P) BP (H6S33_000823) were found. Furthermore, B. subtilis A9 considerably enhanced the M. esculenta activity of Polyphenol oxidase (POD), Superoxide dismutase (SOD), Phenylal anineammonia lyase (PAL) and Catalase (CAT).

Conclusion: This study presents the innovative utilization of B. subtilis A9, for effectively controlling M. esculenta rot disease. This will lay a foundation for biological control in Morchella, which may lead to the improvement of new biocontrol agents for production.

Keywords: Lecanicillium aphanocladii; Morchella esculenta; biological control; fungal disease; transcriptome analysis.

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Conflict of interest statement

BL was employed by Shanghai Co-Elite Agricultural Sci-Tech (Group) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) Identity of endophytic bacteria. The morphological characteristics are shown, and 25 different bacterial species were identified. (B) Without inoculation of B. subtilis A9, Lecanicillium aphanocladii overgrew the PDA. In the presence of B. subtilis A9, the radial growth of L. aphanocladii was significantly inhibited, showing a clear inhibition zone between the fungal colony and the bacterial colony. (C) Observation of biocontrol bacteria B. subtilis A9 under SEM (TM4000 20 kV 12.0 mm X8.00k Mix M), a rod-shaped bacteria of 10 μ. (D) Four M. esculenta endophytes, showing stronger antagonistic activity in vitro against L. aphanocladii.
Figure 2
Figure 2
A flow chart of the pot and field assay.
Figure 3
Figure 3
Sequencing of the whole genome of B. subtilis A9. (A) Phylogenetic tree. (B) GO annotations distribute bar charts. The horizontal axis is the secondary classification of GO, and the vertical axis is the number of genes in the classification (right). Percentage indicates is the total number of annotated genes (left). Different colors represent different orthologs. (C) KEGG pathway classification bar chart. The horizontal axis is the name of the metabolic pathway involved, and the vertical axis is the number of genes annotated to that pathway. (D) Genomic circos map; displayed in the circle map are GC content, sequencing depth, gene element display, and COG function display, from outside to inside.
Figure 4
Figure 4
Observation of B. subtilis A9 with L. aphanocladii G1 using SEM. In (A), the hyphae of the CK group were intact and uniformly distributed, and there were noticeable spores; in (B), the hyphae were clearly broken and entangled after B. subtilis A9 treatment, and the number of spores was clearly less. (C–F) Pot assay observations. (C) Morchella esculenta inoculated with L. aphanocladii G1. (D) B. subtilis A9 was administered before L. aphanocladii G1. (E) M. esculenta inoculated only with B. subtilis A9. (F) Noninoculated control. (G–J) Field assay observation. (G) M. esculenta inoculated with pathogen G1. (H) B. subtilis A9 administered before L. aphanocladii G1. (I) M. esculenta inoculated only with pathogen L. aphanocladii G1. (J) Noninoculated control.
Figure 5
Figure 5
Transcriptome analysis of B. subtilis A9 on M. esculenta. (A) Differentially expressed gene analysis. (B,C) RT-qPCR verification of upregulated (B) and downregulated differentially expressed genes (C).
Figure 6
Figure 6
(A) GO enrichment. Abscissa is GO term, and ordinate is GO term enriched-log10 (p-value). (B) KEGG enrichment. The abscissa is the rich factor (the number of differential genes annotated to a pathway/the total number of genes annotated to the pathway), and the ordinate is the pathway. The size of the point in the map indicates the number of genes annotated in the corresponding pathway (upregulated or downregulated, related to the gene set selected at the time of analysis), and the color indicates the level of significance.
Figure 7
Figure 7
Antioxidant enzyme analysis: (A) catalase (CAT), (B) polyphenol oxidase (PPO), (C) phenylalanine ammonia lyase (PAL), and (D) superoxide dismutase (SOD). All data were presented as means of three replicates ± SD, and error bars represent SD for three replicates. Means with asterisk have significant differences(∗∗p < 0.01 and ∗∗∗∗p < 0.0001).
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