The effectiveness of T. virens WJ561 was tested on detached twigs and leaves in the laboratory. Healthy twigs and leaves from ‘Hongxiao’ pear were treated as previously described (Zang et al., 2007; Zhao et al., 2022). The twigs were prepared by cutting them into 12 cm segments, washing them with tap water, and disinfecting them with 75% alcohol. After rinsing them with sterile water, both ends were sealed with paraffin wax once dry. All twigs were treated with hot iron nail caps (5 mm in diameter) in the neutral position to scald them. On each segment, CFS with different working volume fractions (0, 5, 10, and 15%) was uniformly applied three times and left to air dry for 15 min. Following this, a 6-mm diameter mycelial disk of G1H was inoculated on the scalded area of each twig. These inoculated twigs were placed in a clean container at 25°C and covered with fresh-keeping film. The humidity was regulated using a sterile filter paper. The lesion size was recorded after 5 days and repeated in triplicate.

After surface disinfection, the leaves were repeatedly wiped with 500 μL of WJ561 CFS at concentrations of 0, 5, 10, and 15%. The treated leaves were naturally air-dried and then punctured with sterile stainless needles to form a symmetrically uniform hole (1 mm wide). Then, a G1H plug was inoculated in the pretreated holes. The leaves were then placed on sterilized filter paper to maintain humidity and then incubated at 25°C. Photos of the lesions were recorded after 4 days, and the experiments were repeated twice with three biological replicates.

The stability of CFS was evaluated as described (Ghribi et al., 2012; Zhang et al., 2016; Li et al., 2020). Thermal tolerance was tested by subjecting WJ561 CFS to temperatures of 4, 50, 80, 100, and 121°C for 30 min and then measuring the remaining antifungal activity after cooling to room temperature. Acid–base stability was assessed by adjusting the pH of the filtrates to 1, 3, 5, 7, 9, 11, and 13 with 0.1 M HCl or NaOH for 2 h. Then, the filtrates were readjusted to the original pH values before determining their antifungal activity. Radiation stability was examined by exposing the filtrates to ultraviolet light for 0.5, 1, 2, 3, and 4 h, respectively. To determine the effect of storage time on CFS stability, antifungal assays were performed after the filtrates had been stored at −20°C for 4, 8, and 12 months. Finally, the impact of different CFS treatments on the mycelial growth of G1H on the PDA plate was determined according to step 2.2.3. All experiments were repeated in triplicate, and each treatment contained three plates under the same conditions.

Fresh mycelia of G1H were treated with 0, 5, 10%, or 15% WJ561 CFS at 25°C for 24 h. The treated mycelia were filtered and fixed with 2.5% glutaraldehyde for 24 h. This was followed by dehydration with graded ethanol (30, 50, 70, and 90%, v/v) as described (Fan et al., 2023). The dried samples were coated with gold and subsequently observed using scanning electron microscopy (SEM, Hitachi Regulus 8,220, Japan).

A few treated mycelia obtained in step 2.5 were immersed in 5 mM propidium iodide (PI, Shanghai Acmec Biochemical Technology Co., Ltd., Shanghai, China) for 20 min at 30°C and washed three times with a phosphate-buffered saline (PBS) (0.05 M, pH 7.2–7.4) to remove the dye. The red fluorescence was examined using laser confocal microscopy (Laica STELLARIS 5, Germany) (Zhao et al., 2022). Each group was treated with three samples, and the experiment was repeated three times.

Mycelium samples treated in step 2.5 were homogenized in ice-cold 0.05 M PBS (pH 7.2–7.4) within an ice bath. The MDA content was determined using a commercial thiobarbituric acid (TBA) kit (Bioroyee (Beijing) Biotechnology Co., Ltd., Beijing, China). The filtrated solutions were used for the determination of nucleic acid and soluble protein exudation via the NanoDrop One (Thermo Fisher Scientific, Waltham, MA, United States), as described previously with some modifications (Cai et al., 2015; Fan et al., 2023). Each treatment consisted of three biological replicates, and the experiments were performed three times.

Fresh mycelium samples treated in step 2.5 were resuspended in 0.05 M PBS and stained with 1 μM 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 20 min at 37°C and then rinsed three times with PBS to remove residual dye. Confocal laser microscopy was used for fluorescence observation as descri bed by Kai et al., 2020.

The enzyme activities of catalase and superoxide dismutase were determined using commercial kits (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The total RNA of V. pyri G1H was extracted from the mycelia collected in step 2.5 using a commercial kit (Vazyme, Nanjing, China), and the cDNA was achieved through a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States). RT-qPCR was performed in a Real-Time PCR System (Applied Biosystems). The PCR reaction conditions were as follows: one cycle at 50°C for 2 min and 95°C for 2 min, then 40 cycles of 15 s at 95°C and 1 min at 60°C. The gene expression level was normalized by the internal reference gene actin with the 2^−∆∆Ct method (Arocho et al., 2006; He et al., 2018). Specific primers for gene evaluation are shown in Table 2. Each group consisted of three biological replicates, and the experiments were repeated twice.

To explore the types and abundance of metabolites produced by WJ561, six biological replicates of 4-day-old WJ561 CFS were collected and frozen in liquid nitrogen until used for untargeted metabolomics analysis. In total, 1 mL of the freeze-dried samples was resuspended in 100 μL of pre-chilled 80% methanol by vortexing and incubated on ice for 5 min. The resulting samples were centrifuged at 15,000 g at 4°C for 15 min. The obtained supernatant was diluted with LC–MS grade water to a final concentration containing 53% methanol. The samples were then transferred to a new tube and centrifuged at 15,000 g at 4°C for 15 min. Finally, the supernatant was injected into the LC–MS/MS system for analysis.

LC–MS analyses were performed on a Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, United States), coupled to an Orbitrap A Q Exactive^TM HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States), in Novogene Co., Ltd. (Beijing, China). Samples were injected onto a HypersilGoldcolumn (100 × 2.1 mm, 1.9 μm) using a 12-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The gradient program of eluent B was set as follows: 0–1.5 min for 2%; 1.5–3 min for 2 to 85%; 3–10 min for 85 to 100%; 10–10.1 min for 100 to 2%; 10.1–11 for 2%; 11–12 min for 2%. A Q Exactive^TM HF mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.5 kV, capillary temperature of 320°C, sheath gas flow rate of 35 psi, and aux gas flow rate of 10 L/min, S-lens RF level of 60, and aux gas heater temperature of 350°C.

The raw data were processed using Compound Discoverer 3.3 software (CD 3.3, Thermo Fisher Scientific, Waltham, MA, United States) for baseline filtering, peak alignment, picking, and normalization to produce peak intensities for retention time (RT) and m/z data pairs. The mzCloud,² mzVaultand, and MassList databases were used to identify metabolites. These metabolites were annotated using the HMDB database. Statistical analyses were performed using statistical software R (version 3.4.3), Python (version 2.7.6), and CentOS (CentOS release 6.6).

Antifungal tests were conducted to evaluate the effects on the mycelial growth of V. pyri, as previously described (Wang et al., 2022). To determine the contact effects, artesunate was dispersed in dimethyl sulfoxide (DMSO) at a final concentration of 1% and added to tepid PDA immediately before emptying the Petri dishes. Concentrations of artesunate ranging from 0.03 to 3 mg/mL were tested. The controls received the same quantity of 1% DMSO mixed with PDA. After solidification, a disk cut from the actively growing front of a 3-day-old colony of the desired pathogenic fungi V. pyri G1H was aseptically placed in the center of each treatment plate. Following the same steps, we evaluated the control efficacy of 3 mg/mL lauric acid, arachidonic acid, and linoleic acid against V. pyri. All Petri dishes were then incubated at 25°C and observed after 2 days, to calculate the growth inhibition of fungal hyphae. The experiment was performed in triplicate with three biological replicates.

The statistical analyses were conducted using IBM SPSS 26.0 and Excel 2016. Normality (Shapiro–Wilk test, p > 0.05) and variance homogeneity (Levene’s test, p > 0.05) were checked, and parametric tests were used. Data results were analyzed using a one-way analysis of variance (ANOVA), Welch’s test, or Kruskal–Wallis H test and presented as the mean ± standard error of the least squares means. According to Duncan’s multiple range test, Welch’s test, and the Kruskal–Wallis test, a p-value of <0.05 was accepted as statistically significant. Different letters indicated the significant difference among the treatments.

Pear Valsa canker is a severe disease that can cause significant damage to tree productivity. The symptoms are visually distinct and include cankers on pear trunks, scaffold limbs, and diebacks of twigs. To investigate the pathogens responsible for pear Valsa canker, we isolated fungi from the infected pear bark. Among the isolates, morphological observations revealed that G1H can proliferate normally on PDA medium and form colonies (Figure 1A). The radial growth rate of mycelia was rapid, completely covering the plate within no more than 3 days. Colonies were milk-white, thin, with a dense surface. The medium on the back of the plate was milk-yellow.

The ITS, EF1α, and Btu gene sequences of G1H were used as a query in a BLAST search of the NCBI GenBank database. Therefore, as a next step, a phylogenetic analysis was conducted using a multilocus dataset of concatenated ITS, EF1α, and Btu obtained from 12 samples. Leucostoma persoonii isolate SXYLt and Leucostoma persoonii isolate 32-2w were used as the outgroup. For more detailed information on the specific characteristics of each isolate, please refer to Table 3. The analysis revealed that G1H was in the same clade with Valsa mali var. pyri isolate GSZYpm160, and the clade classification was strongly supported by bootstrap analysis (Figure 1B). Based on the morphology and phylogeny, the pathogenic fungi could be classified into Valsa pyri.

We meticulously examined the colony shape to identify WJ561 (Figures 2A,B). After 1 day of cultivation at 28°C, all strains displayed white villous colonies on the PDA medium. We conducted a thorough analysis of the morphology of the colony to distinguish WJ561 (as shown in Figure 2A). Following 24 h of incubation at 28°C, all the strains exhibited white, hairy colonies on the PDA medium. The back of the colony is yellowish-white. By the 3rd day, light green to dull green sporulation bands emerged in the center of all media, gradually extending toward the surroundings. Colonies were dense and floccose in texture, with a fluffy surface. By the 5th day, green spores were dispersed throughout the entire plate with concentric rings, exhibiting a deep green, grayish green, or chartreuse color.

The phylogenetic trees were constructed using a dataset consisting of 15 sequences derived from 3 gene loci (ITS, TEF1-α, and RPB2) obtained from 15 isolates and 45 related sequences of Trichoderma and Protocrea species (Table 4). In the maximum likelihood phylogenetic tree, WJ561 was clustered in the same clade of Trichoderma virens isolate YZB-1 and Trichoderma virens isolate TB2-7 with 100% bootstrap support (Figure 2B). Based on the morphology and phylogeny, we identified WJ561 as Trichoderma virens and deposited it at the China General Microbiological Culture Collection Center (CGMCC) as CGMCC No. 40848.

As shown in Figures 4A,B, WJ561 CFS had a significantly inhibitory effect on the mycelial growth of V. pyri on PDA in Petri dishes after treatments for 48 h. However, the incubation time is a significant factor influencing the antifungal potency of WJ561 CFS. There were significant differences among WJ561 CFS prepared by different fermentation times in inhibiting mycelial growth of G1H, with the highest inhibitory rate of 100% observed in 4-day-old WJ561 CFS. The results indicated that potent antifungal activity has the potential for further research and development. In addition, we investigated the impact of varied concentrations of WJ561 CFS on the vegetative growth of G1H in a PDB liquid medium. It was observed that the mycelial ball in the CFS treatment group was smaller than the control (Figure 4C). The size of the mycelial ball in the treatment group was negatively associated with the CFS concentration. At 15% of CFS, the dry weight of mycelia was only 4.6% of that in the control group (Figure 4D).

The WJ561 CFS was effective in preventing the expansion of pear Valsa canker lesions on leaves and twigs (Figures 5A,B). Compared to the control group treated with PBD, the lengths of lesions of CFS-treated twigs were observed to be significantly shorter on the 5th day, and there was a 73.5% decrease in lesion expansion rate after being treated with 15% CFS (Figures 5C,D). The CFS treatment was able to reduce hyphal growth in the tissues, thereby decreasing the inoculum density of the pathogen. The results indicated that WJ561 can be an efficient biocontrol agent of pear Valsa canker.

The WJ561 CFS appeared to be insensitive at 100°C and lower but sensitive to higher temperatures with significantly reduced activity at 121°C (Figure 6A). The lowest inhibitory rates were observed on pH 13, and ultraviolet treatment for a longer time decreased the antagonistic effect (Figures 6B,C). No significant difference was observed in the inhibitory effect of WJ561 CFS against V. pyri G1H when stored at 4°C within 4 months. However, the inhibition rate observed at 8 months was slightly lower than that at 12 months, which could be attributed to variations in the batches of CFS. Although the antagonistic effect decreased over time, the inhibition rate remained at approximately 89% even after a year (Figure 6D).

Scanning electron microscopy was applied to evaluate the morphological alteration to confirm whether the mycelial morphology of V. pyri G1H was affected by WJ561 CFS. After incubation at 25°C, a typical morphology with rougher, more sunken, and shriveled surface structure was observed in mycelia treated with CFS compared to the control group (Figure 7). Moreover, a more significant contraction and disruption could be observed when the mycelia were treated with 15% CFS, which might result in membrane permeability and loss of cytoplasmic contents (Figure 7D).

Shriveled hyphae observed in the WJ61 CFS treatment group showed that CFS might cause hyphal cell membrane damage and cell mortality. A DNA-staining fluorescent probe, PI, was used to determine whether cells have lost plasma membrane integrity by detecting red fluorescence. The results revealed that more hyphae exhibited red fluorescence in the treated group than in the control group (Figure 8A). This indicated that the number of hyphae losing plasma membrane integrity increased with the increase in CFS. It was observed that 10% CFS was sufficient to cause significant damage to the cell membrane of hyphae, thereby highlighting the potential of CFS in disrupting cellular membranes.

To delve into the mechanism of CFS-induced membrane damage, we measured the MDA content, which is one of the essential markers when the membrane system suffers from stress. Our findings concluded that the MDA content in the treated group was higher than that of the control group (Figure 8B), demonstrating that CFS promoted the oxidation of hyphae of V. pyri and generated the lipid peroxidation end product MDA. The exudation of cellular contents has been thought to respond to damage to the plasma membrane. To better investigate the oxidative effect of CFS on V. pyri, the nucleic acid and protein leakage of G1H was detected. The results showed that increased contents of nucleic acid and soluble protein were observed in the CFS-treated group (Figures 8C,D). Overall, these results indicated that the WJ561 CFS could disrupt the integrity of the hyphal membrane of V. pyri by promoting the occurrence of peroxidation damage.

Hyphal cells with elevated ROS levels have been shown to generate membrane damage (Zhao et al., 2022). We next wanted to investigate whether WJ561 CFS treatment increases ROS production and oxidative stress in V. pyri. H₂DCFDA, a well-known fluorescence probe, is used to detect intracellular ROS. As shown in Figure 9A, it was normal to observe that the presence of ROS in hyphal cells was negligible in the control group, while much stronger fluorescence was observed in the treated group. Moreover, the fluorescence intensity enhanced significantly with the increase in the concentration of the WJ561 CFS, indicating that CFS could strongly induce cells to produce ROS in a dose-dependent manner. In general, the accumulation of ROS in cells is usually linked with an increase in the scavenging capacity. We next assessed the enzyme activity of catalase and superoxide dismutase and the expression levels of related genes in treated hyphae. However, such a decrease in the enzyme activities of CAT and SOD was detected by CFS treatments (Figures 9B,C). Compared with the control group, the expression levels of these two related genes Cat and Sod were also significantly downregulated by qRT-PCR analysis (Figures 9D,E). These findings indicated CFS-induced ROS accumulation in membrane cells by the inhibition of ROS-scavenging pathways, leading to hyphae membrane peroxidation damage.

To investigate the metabolites through which the WJ561 CFS exerted its effects in controlling V. pyri G1H, we performed an untargeted metabolomic analysis of CFS by LC–MS. In total, 1,142 metabolites were identified, including 798 in the positive mode and 344 in the negative mode. The list of each metabolite in the positive and negative modes is presented in Tables S1 and S2, respectively. At the class 1 level, 761 metabolites were mainly composed of 217 lipids and lipid-like molecules, 192 organic acids and derivatives, 123 organoheterocyclic compounds, 57 organic oxygen compounds, 57 benzenoids, 52 nucleosides, nucleotides, and analogs, 35 phenylpropanoids and polyketides, 16 organic nitrogen compounds, 10 alkaloids and derivatives, 1 hydrocarbon, and 1 organosulfur compound (Figure 10A). Pathway analysis showed that these metabolites were significantly enriched in 10 HMDB pathways, including lipids and lipid-like molecules and organic acids and derivatives (Figure 10B). Based on the metabolite analysis mentioned above, some lipids and lipid-like molecules may be related to reactive oxygen species balance and pathogen inhibition (Roh et al., 2017; Wang et al., 2022). This analysis detected a [M + Na]⁺ peak at m/z 407.16614 and afforded the molecular formula C₁₉H₂₈O₈ (Figure 10C). We conducted a preliminary investigation into the efficacy of metabolites on V. pyri G1H. In the present study, the antifungal test of artesunate at 0.3 mg/mL significantly inhibited the growth of V. pyri (Figure 10D). In addition, 3 mg/mL lauric acid, arachidonic acid, and linoleic acid also slightly inhibited pathogenic fungi (Supplemenatry Figure S1).