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. 2024 Apr 17:15:1377683.
doi: 10.3389/fmicb.2024.1377683. eCollection 2024.

Antifungal mechanism of cell-free supernatant produced by Trichoderma virens and its efficacy for the control of pear Valsa canker

Yang Zhang  1 , Ying Lu  1 , Zhaoyang Jin  1 , Bo Li  1 , Li Wu  1 , Yujian He  1   2   3
Affiliations

Antifungal mechanism of cell-free supernatant produced by Trichoderma virens and its efficacy for the control of pear Valsa canker

Yang Zhang et al. Front Microbiol. .

Abstract

Introduction: Pear Valsa canker, caused by Valsa pyri (V. pyri), poses a major threat to pear production. We aimed to assess the effectiveness of the cell-free supernatant (CFS) produced by Trichoderma virens (T. virens) to control the development of pear Valsa canker and reveal the inhibitory mechanism against the pathogenic fungi.

Results: Using morphological characteristics and phylogenetic analysis, the pathogen G1H was identified as V. pyri, and the biocontrol fungus WJ561 was identified as Trichoderma virens. CFS derived from WJ561 exhibited strong inhibition of mycelial growth and was capable of reducing the pathogenicity of V. pyri on pear leaves and twigs. Scanning electron microscopy (SEM) observations revealed deformations and shrinkages in the fungal hyphae treated with CFS. The CFS also destroyed the hyphal membranes leading to the leakage of cellular contents and an increase in the malondialdehyde (MDA) content. Additionally, CFS significantly inhibited the activities of catalase (CAT) and superoxide dismutase (SOD), and downregulated the expression of antioxidant defense-related genes in V. pyri, causing the accumulation of reactive oxygen species (ROS). Artesunate, identified as the main component in CFS by liquid chromatograph-mass spectrometry (LC-MS), exhibited antifungal activity against V. pyri.

Conclusion: Our findings demonstrate the promising potential of T. virens and its CFS in controlling pear Valsa canker. The primary inhibitory mechanism of CFS involves multiple processes, including membrane damage and negatively affecting enzymatic detoxification pathways, consequently leading to hyphal oxidative damage of V. pyri. This study lays a theoretical foundation for the utilization of T. virens to control V. pyri in practical production.

Keywords: Trichoderma virens; Valsa pyri; antifungal activity; cell-free supernatant; reactive oxygen species.

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Conflict of interest statement

The 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
Colony appearance and maximum likelihood phylogenetic tree of Valsa pyri G1H. (A) Colony appearance of G1H at 1, 2, and 3 days of incubation from above and below. (B) Phylogenetic tree of G1H based on the combined ITS, EF1α, and Btu genes. Bootstrap support values equal to or greater than 70% are shown at the nodes. Leucostoma persoonii isolate SXYLt and Leucostoma persoonii isolate 32-2w were used as the outgroup. G1H was highlighted in red.
Figure 2
Figure 2
Colony appearance and maximum likelihood phylogenetic tree of Trichoderma virens WJ561. (A) Colony appearance of WJ561 at 1, 3, and 5 days of incubation from above and below. (B) Phylogenetic tree of WJ561 based on the combined ITS, TEF1-α, and RPB2 genes. Bootstrap support values equal to or greater than 70% are shown at the nodes. The tree was rooted in two isolates of Protocrea farinosa strain CBS 121551 and Protocrea farinosa strain TFC 96–85. WJ561 was highlighted in red.
Figure 3
Figure 3
Dual culture assay and volatile organic compound antifungal bioassay of Trichoderma virens WJ561 against V. pyri G1H. (A) Phenotype and (B) inhibition rate of G1H co-cultured with 0-day-old and 1-day-old WJ561 for 3 days in dual culture assay. (C) Phenotype and (D) inhibition rate of G1H affected by VOCs of 0, 1, and 2-day-old WJ561. Error bars indicated standard errors of the means of three repeated experiments. According to Duncan’s multiple range test, columns marked by different letters represented statistically different (p < 0.05).
Figure 4
Figure 4
Inhibition of Valsa pyri G1H mycelial growth by the cell-free supernatant (CFS) of Trichoderma virens WJ561. (A) Colony size and (B) growth inhibition rate of G1H in the presence of CFS for different incubation times. (C) Size of the mycelia ball grown in the presence of different CFS concentrations. (D) mycelial dry weight. The data shown are meant ± standard deviations (n = 3). According to Duncan’s multiple range test, columns marked by different letters are represented as statistically different (p < 0.05).
Figure 5
Figure 5
Effect of different concentrations of Trichoderma virens WJ561 CFS on the pathogenicity of Valsa pyri G1H. (A) Development of lesion on the detached leave infected with G1H after being treated with WJ561 CFS. (B) Development of lesion on the detached twigs infected with G1H after being treated with WJ561 CFS. (C) Canker lesion length of twigs after inoculation of a G1H plug treated with WJ561 CFS. (D) Disease reduction percentage rate caused by G1H on twigs treated with WJ561 CFS. The data shown are meant ± standard deviations (n = 3). According to Duncan’s multiple range test, columns marked by different letters are represented as statistically different (p < 0.05).
Figure 6
Figure 6
Stability of T. virens WJ561 cell-free supernatant. (A) Temperatures. Different letters above the bars indicate significant differences within the temperature treatment group according to the Kruskal–Wallis test (H = 13.876, p < 0.05). (B) pH values. Different letters above the bars indicate significant differences within the pH values treatment group according to the Kruskal–Wallis test (H = 19.479, p < 0.05). (C) Ultraviolet light treatment time. Different letters above the bars indicate significant differences within the ultraviolet light treatment group according to Duncan’s multiple range test (p < 0.05). (D) Storage time. Different letters above the bars indicate significant differences within the different storage time treatment groups according to the Kruskal–Wallis test (H = 9.804, p < 0.05). The experiments of all treatment groups were conducted three times, and the data shown are meant ± standard deviations (n = 3).
Figure 7
Figure 7
Effect of cell-free supernatant treatment on the morphology of Valsa pyri G1H observed by scanning electron microscopy. G1H mycelia treated with (A) 0% CFS, (B) 5% CFS, (C) 10% CFS, and (D) 15% CFS.
Figure 8
Figure 8
Effect of T. virens WJ561 cell-free supernatant (CFS) on hyphal cytomembrane integrity of Valsa pyri G1H. (A) Hyphae were stained with PI and observed with a fluorescence microscope. (B) MDA content. (C) Nucleic acid content. (D) Soluble protein content as measured by A280. Vertical bars represent standard deviations of the means (n = 3). Columns followed by different letters within MDA content and A280 are statistically different according to Duncan’s multiple range test (p < 0.05). Different letters above the bars indicate significant differences within nucleic acid content according to Duncan’s multiple range test (p < 0.05).
Figure 9
Figure 9
Effects of cell-free supernatant (CFS) on ROS accumulation and the enzyme activities involved in scavenging ROS in Valsa pyri. (A) The images of ROS accumulation were recorded using confocal laser microscopy. (B) The activity of ROS scavenging enzymes CAT in hyphal cells. (C) The activities of ROS scavenging enzymes SOD in hyphal cells. (D) Gene expression levels of Cat. (E) Gene expression levels of Sod. According to Duncan’s multiple range test (p < 0.05), different letters above the columns indicate significant differences within each group.
Figure 10
Figure 10
(A) Classification and (B) annotation of metabolites of T. virens WJ561. (C) Mass spectra of artesunate. (D) Antagonistic activity of artesunate against V. pyri G1H.
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References

    1. Abe K., Kotoda N., Kato H., Soejima J. (2007). Resistance sources to Valsa canker (Valsa Ceratosperma) in a germplasm collection of diverse Malus species. Plant Breed. 126, 449–453. doi: 10.1111/j.1439-0523.2007.01379.x - DOI
    1. Arocho A., Chen B., Ladanyi M., Pan Q. (2006). Validation of the 2-∆∆Ct calculation as an alternate method of data analysis for quantitative Pcr of Bcr-Abl P210 transcripts. Diagn. Mol. Pathol. 15, 56–61. doi: 10.1097/00019606-200603000-00009, PMID: - DOI - PubMed
    1. Barman M., Dandasena D., Suresh A., Bhandari V., Kamble S., Singh S., et al. . (2023). Artemisinin derivatives induce oxidative stress leading to DNA damage and caspase-mediated apoptosis in Theileria Annulata-transformed cells. Cell Commun. Signal. 21, 1–15. doi: 10.1186/s12964-023-01067-7, PMID: - DOI - PMC - PubMed
    1. Bertrand S., Azzollini A., Schumpp O., Bohni N., Schrenzel J., Monod M., et al. . (2014). Multi-well fungal co-culture for De novo metabolite-induction in time-series studies based on untargeted metabolomics. Mol. BioSyst. 10, 2289–2298. doi: 10.1039/c4mb00223g, PMID: - DOI - PubMed
    1. Boukaew S., Zhang Z., Prasertsan P., Igarashi Y. (2023). Antifungal and Antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces Philanthi Rl-1-178 on the two Aflatoxigenic Fungi and identification of its active component. J. Appl. Microbiol. 134, 1–9. doi: 10.1093/jambio/lxac091, PMID: - DOI - PubMed

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This project was financially supported by the National Natural Science Foundation of China (Grant No.51972302).