Application of Trichoderma monticola GUWM22 in promoting growth and preventing root rot of pepper

Abstract

The application discloses application of Trametes versicolor GUWM22 in promoting growth of peppers and in preventing and treating pepper root rot, and belongs to the technical field of microorganism application. The application proves that the Trametes versicolor GUWM22 has a significant growth-promoting effect and an effect of preventing and treating pepper root rot. The strain can significantly improve the germination rate of pepper seeds, promote elongation of hypocotyls and radicles and growth of seedlings, and increase biomass of plants and development of fibrous roots. R. solani Under the condition of infection, the strain can effectively relieve inhibition of diseases on growth of the peppers. Meanwhile, the GUWM22 significantly improves activities of defense enzymes such as SOD, CAT, POD, PPO and PAL in pepper leaves. The indoor potting control effect is 66.67%, and the field control effect is as high as 84.94%, and the strain shows excellent double functions of growth promotion and biocontrol. In addition, the strain can also inhibit a plurality of pathogenic bacteria. The application provides technical support for application of the Trametes versicolor GUWM22.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to the application of Trichoderma wumeng GUWM22 in promoting pepper growth and preventing pepper root rot. Background Technology

[0002] Plant diseases cause significant economic losses every year. Currently, the control of plant diseases mainly relies on chemical control. However, the continued large-scale use of chemical pesticides leads to increased pesticide resistance in pathogens, pesticide residues in the soil or environment, and the resurgence of pathogenic microorganisms, causing serious harm to the environment and non-target organisms.

[0003] Biological control is one of the important technologies for green pest control in crops. Biological control involves screening and utilizing beneficial microorganisms to produce biological agents for the prevention of plant diseases and pests, thereby achieving the goal of disease control. It represents a new approach and method for green pest control. Microbial products are characterized by safety, sustainability, broad spectrum, and environmental friendliness. The mechanisms of action of microbial agents include antibiotic resistance, competitive action, hyperparasitism, and induction of systemic resistance in plants. *Trichoderma wumeng* (… Trichoderma wumeng GUWM22 has been proven to significantly inhibit anthracnose fungus in peppers and can prevent and control anthracnose in peppers, but it is still unknown whether it has a growth-promoting effect on peppers or whether it has any effect on other diseases of peppers. Summary of the Invention

[0004] The purpose of this invention is to provide the application of *Trichoderma wumeng* GUWM22 in promoting pepper growth and controlling pepper root rot, thereby solving the problems existing in the prior art. This invention confirms that *Trichoderma wumeng* GUWM22 has significant growth-promoting effects and is effective in controlling pepper root rot. Furthermore, this strain can also inhibit various pathogens. This invention provides technical support for the application of *Trichoderma wumeng* GUWM22.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides *Trichoderma wumengense* (… Trichoderma wumeng The application of GUWM22 in promoting chili pepper growth, wherein the preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

[0006] This invention also provides the application of Trichoderma wumeng GUWM22 in the preparation of a fungal agent that promotes the growth of chili peppers, wherein the preservation number of Trichoderma wumeng GUWM22 is CCTCC NO: M20242639.

[0007] This invention also provides the application of Trichoderma wumeng GUWM22 in the prevention and / or treatment of pepper root rot, wherein the preservation number of Trichoderma wumeng GUWM22 is CCTCC NO: M20242639.

[0008] This invention also provides the application of Trichoderma wumeng GUWM22 in the preparation of fungal agents for the prevention and / or treatment of pepper root rot, wherein the preservation number of Trichoderma wumeng GUWM22 is CCTCC NO: M20242639.

[0009] Optionally, the pathogen causing the pepper root rot includes Rhizoctonia solani (…). Rhizoctonia solani ).

[0010] This invention also provides the application of Trichoderma wumeng GUWM22 in inhibiting pathogens, including Fusarium kuganensis (…). Fusarium cugenangense Alternaria microphylla ( ), Alternaria tenuissima ), Eris medusa ( Diaporthe eres ), Water lily anthracnose bacteria ( Colletotrichum nymphaeae ), Apple sclerotium ( Monilinia fructicola ), Rhizoctonia solani, and Rhizoctonia solani ( Helicobasidium mompa Fusarium graminearum ( ), Fusarium graminearum ), Fusarium tumefaciens ( Fusarium asiaticum ), Bean thorny discus ( Colletotrichum lindemuthianum ), Corynebacterium multiflorum ( Corynespora cassiicola Banana anthrax ( Colletotrichum musae ), neatly arranged small sclerotia ( Sclerotium rolfsii Anthracnose (Camellia anthrax) Colletotrichum camelliae ), Ash anthracnose fungus ( Colletotrichum spaethianum ), intercropping of green beans with schizocarps ( Diaporthe phaseolorum ) and / or Fusarium solani ( Fusarium solani ); The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

[0011] This invention also provides the application of Trichoderma wumeng GUWM22 in the preparation of fungal agents that inhibit pathogens, including Fusarium kuganensis, Alternaria spp., Erisia spp., Anthracnose fungus of water lily, Sclerotium spp. of apple, Rhizoctonia solani, Rhizoctonia solani, Fusarium spp. of mulberry, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Colletotrichum spp. of Asian, Colletotrichum spp. of bean, Corynebacterium multiflorum, Anthracnose fungus of banana, Sclerotium spp. of uniformity, Anthracnose fungus of camellia, Anthracnose fungus of ash, Intercropping fungus of bean and / or Fusarium spp. of bark; The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

[0012] The present invention also provides a method for preventing and controlling root rot of pepper, including the step of treating pepper plants with Trichoderma wumeng GUWM22; The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639; The pathogen causing pepper root rot includes Rhizoctonia solani.

[0013] Optionally, the treatment of the chili plants includes drenching the chili plants with water or spraying the chili plants with water and then drenching the chili plants with water.

[0014] The present invention also provides a method for promoting chili pepper growth, including the step of treating chili pepper plants with Trichoderma wumeng GUWM22; The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

[0015] The present invention discloses the following technical effects: This invention confirms that *Trichoderma wumeng* GUWM22 has significant growth-promoting effects and is effective in controlling pepper root rot. This strain can significantly improve pepper seed germination rate, promote hypocotyl and radicle elongation and seedling growth, and increase plant biomass and fibrous root development. R. solani Under infection conditions, it can effectively alleviate the inhibitory effect of diseases on pepper growth. Simultaneously, GUWM22 significantly increased the activity of defense enzymes such as SOD, CAT, POD, PPO, and PAL in pepper leaves. The control efficacy in indoor potted plants was 66.67%, and the field control efficacy reached as high as 84.94%, demonstrating excellent dual functions of promoting growth and biocontrol.

[0016] This invention also tested the relative efficacy of GUWM22 against pepper root rot in continuously cropped soil, finding it to be 65.74%, significantly reducing the disease index and increasing pepper plant height, root length, and biomass. Furthermore, GUWM22 increased soil organic matter, total nitrogen, and hydrolyzable nitrogen content, regulated soil pH to near neutral, maintained stable available potassium, and effectively alleviated the obstacles of continuous pepper cropping.

[0017] This invention further clarifies the effect of GUWM22 on R. solani This strain employs multiple biocontrol mechanisms. It entangles with and disintegrates pathogenic mycelia through hyperparasitism; its volatile substances, non-volatile metabolites, and sterile fermentation broth (with an inhibition rate of 95.33%) disrupt the cell membrane integrity of pathogens, induce nuclear damage and abnormal chitin structure in the cell wall, simultaneously triggering reactive oxygen species bursts, inhibiting catalase and superoxide dismutase activity, increasing malondialdehyde content, and significantly reducing the content of soluble protein and soluble sugar in mycelia, thus interfering with their metabolism and energy supply. It can also exert inhibitory effects on a variety of pathogens.

[0018] In summary, *Trichoderma wumeng* GUWM22 is a biocontrol strain with multiple functions, including highly efficient disease prevention, growth promotion and yield increase, and improvement of soil microecology in continuous cropping. It has significant theoretical value and application prospects in the green control of pepper root rot and the restoration of continuous cropping obstacles. This invention provides technical support for the application of *Trichoderma wumeng* GUWM22. Attached Figure Description

[0019] Figure 1 pathogens F. cugenangense Morphological characteristics; A / B: State after 15 days of culture on PDA (A is the front, B is the back); C: Conidiomata on PDA medium; DE / H: Conidiophores and conidia; F / I: Conidia; G: Chlamydospores; Scale bar = 10 μm; Figure 2 pathogens F. cugenangense Maximum likelihood (ML) tree; Figure 3 For vaccination F. cugenangense Symptoms in the plant 15 days later; Figure 4 pathogens Rhizoctonia solani Morphological characteristics; A / B: State after 3 days of culture on PDA (A is the front, B is the back); CE: Mycelium; F: Sclerotium; Figure 5 pathogens Rhizoctonia solani Maximum likelihood (ML) tree; Figure 6 For vaccination R. solani Symptoms in the plant 15 days later; Figure 7 The growth-promoting effect of GUWM22 on chili peppers; Figure 8 The germination diagrams for each group of seeds, as well as statistical graphs of germination rate, hypocotyl length, and radicle length; Figure 9 To determine the disease control effect of GUWM22 against pepper root rot; Figure 10 Growth of chili peppers (Guizhou Dangwu chili peppers) after 15 days of treatment with GUWM22; Figure 11 The effect of GUWM22 on the prevention and control of pepper root rot; Figure 12 Effects of GUWM22 on the activities of defensive enzymes CAT, PAL, PPO, POD and SOD, and the content of MDA in pepper leaves. Figure 13 Soil pH, available phosphorus, total phosphorus, total potassium, organic matter, total nitrogen, hydrolyzable nitrogen, and available potassium content for CK and GUWM22 treatments; Figure 14 For GUWM22 R. solani Antibacterial effect; A: CK group; B: GUWM22 treatment; C: Colony diameter; Figure 15 For GUWM22 R. solani The parasitic effect; Figure 16For the volatile substances of GUWM22 R. solani Inhibition effect; A: CK group; B: GUWM22 treatment; C: Colony diameter; Figure 17 For the volatile substances of GUWM22 R. solani Inhibition effect; A: CK group; B: GUWM22 treatment; C: Colony diameter; Figure 18 For GUWM 22 aseptic fermentation broth to R. solani Inhibition effect; A: CK group; B: GUWM22 treatment; C: Colony diameter; Figure 19 For the aseptic fermentation broth of GUWM22 P. capsici and F. cugenangense The antibacterial effect; A: CK- P. capsici B: GUWM22 processing - P. capsici C: P. capsici Colony diameter D: CK- F. cugenangense E: GUWM22 processing - F. cugenangense F: F. cugenangense Colony diameter; Figure 20 For the GUWM22 aseptic fermentation broth to R. solani The effect of mycelial biomass; Figure 21 Processing GUWM22 R. solani Effects of mycelial antioxidant enzymes CAT and SOD activity on MDA; Figure 22 Processing GUWM22 R. solani The effect of mycelial soluble protein and soluble sugar content; Figure 23 For the GUWM22 aseptic fermentation broth to R. solani Effects on the cell membrane; Figure 24 For the GUWM22 aseptic fermentation broth to R. solani The influence of the cell nucleus; Figure 25 For the GUWM22 aseptic fermentation broth to R. solani The effect of cell wall; Figure 26 For GUWM22 aseptic fermentation broth to R. solani The effects of reactive oxygen species accumulation; Figure 27 The growth of GUWM22 on plates within 0-72 hours; Figure 28 The dynamic changes in colony diameter (A) and hyphal dry weight (B) of GUWM22; Figure 29To evaluate the protease, chitinase, cellulase, and siderophore production activities of GUWM22; Figure 30 The colony status (A) and colony diameter (B) of GUWM22 on plates at different temperatures; Figure 31 The colony status (A) and colony diameter (B) of GUWM22 under different nutrient substrates; Figure 32 The colony status (A) and colony diameter (B) of GUWM22 at different pH values; Figure 33 The colony state (A) and colony diameter (B) of GUWM22 under different concentrations of NaCl; Figure 34 The colony state (A) and colony diameter (B) of GUWM22 under different concentrations of NaHCO3; Figure 35 The results show that GUWM22 has a broad-spectrum antibacterial activity. Detailed Implementation

[0020] The *Trichoderma wumeng* used in this invention ( Trichoderma wumeng GUWM22 has been disclosed in patent CN120192855B - A strain of Trichoderma wumeng GUWM22 and its application, with accession number CCTCC NO: M20242639.

[0021] Example 1: Isolation and Identification of the Pathogen of Pepper Root Rot Field surveys revealed that in the early stages of the disease, the underground root system showed mild browning, with slight lesions visible on the taproot, while the above-ground parts showed no obvious abnormalities, though the plants exhibited a wilting state resembling water shortage. In the later stages, extensive browning and rotting of the underground root system occurred, with a large number of fibrous roots falling off, resulting in complete loss of root function. The above-ground parts subsequently wilted and yellowed, eventually leading to death. Patches of dead plants were observed in the field, causing severe yield reduction in chili peppers.

[0022] A five-point sampling method was used to systematically collect samples of chili pepper root rot-infected plants from a typical chili pepper planting base in Xiuwen County, Guizhou Province, resulting in 58 samples of diseased plants. At the early stage of the disease, fungal isolation was performed on the collected diseased plant samples using tissue isolation methods. After purification and culture, 41 pure culture fungal strains were obtained.

[0023] 1. Pathogen: Fusarium kugnanense ( Fusarium cugenangense ) Internal transcribed spacer (ITS) sequence alignment analysis (ITS-BLAST) revealed that the eight strains were Fusarium fungi, and the colony morphology of the cultures was consistent across strains. Three representative pure cultures were randomly selected, designated GUCC 25-0179, GUCC 25-0180, and GUCC 25-0181, for morphological identification, molecular biological identification, and pathogenicity testing.

[0024] Morphological observation results show ( Figure 1 This strain, when cultured on PDA medium for 10-15 days (25℃), produces colonies that cover the entire culture dish. The hyphae are cottony, and the colony color gradually transitions from purple at the center to white at the edge, with a filamentous structure at the colony edge. Small conidia are oval, measuring 7.6-13.4 μm × 2.5-6.8 μm (n=30); chlamydospores are spherical to nearly spherical, measuring 9.7-11.1 μm × 9.6-10.7 μm (n=30), terminal, solitary or paired, with a rough cell wall surface; large conidia are sickle-shaped, measuring 45.5-53.2 μm × 5.9-7.6 μm (n=30).

[0025] Molecular biological identification results showed that ( Figure 2 The sequences obtained from sequencing were compared with those obtained from the NCBI database using BLAST. The results showed that the RPB2, TEF1, and TUB2 gene sequences of the three representative strains, GUCC 25-0179, GUCC 25-0180, and GUCC 25-0181, were consistent with those obtained from the NCBI database. F. cugenangense The similarity to (CBS 130304) was 99.89%, 99.69%, and 99.79%, respectively. Multilocus phylogenetic analysis based on maximum likelihood (ML) showed that the above three representative strains were similar to *Fusarium kuganensis* (CBS 130304). F. cugenangense The standard strains (InaCC F984ᵀ and CBS 130304) clustered together with a support rate of 98%, indicating that they belong to the same phylogenetic species. In summary, based on morphological and phylogenetic identification results, GUCC 25-0179, GUCC 25-0180, and GUCC 25-0181 are all... F. cugenangense .

[0026] Pathogenicity test results showed ( Figure 3 A fungal culture medium representing strain GUCC 25-0179 was inoculated onto the roots of healthy pepper plants for pathogenicity testing. Results showed that 5 days after inoculation, the lower leaves began to wilt; 10 days after inoculation, the upper leaves gradually curled, and all lower leaves wilted and some died; 15 days after inoculation, the entire pepper plant died completely. (Uninoculated plants...) F. cugenangense The control pepper plants showed no symptoms. Root examination revealed that the roots of the inoculated peppers were browned and rotten, with most lateral roots dead and the taproot rotten; the stem base showed a distinct brown to dark brown color, with slight constriction and thinning of the stem base tissue, and some areas exhibiting slight wrinkling. In contrast, the uninoculated peppers... F. cugenangense The control plants showed no abnormalities in their roots.

[0027] 2. Pathogen: Rhizoctonia solani ( Rhizoctonia solani AG-4 HG-1 subspecies ITS-BLAST analysis revealed that 18 of the 41 pure culture fungal strains tested belonged to the genus Rhizoctonia. Rhizoctonia Furthermore, the colony morphology of the cultures of each strain remained consistent. To clarify the specific taxonomic position and pathogenicity of the strains, three representative pure cultures (numbered GUCC 25-0182, GUCC 25-0183, and GUCC25-0184) were randomly selected, and morphological identification, molecular biological identification, and pathogenicity determination were systematically carried out.

[0028] Morphological observation results show that ( Figure 4 This strain grows rapidly on PDA medium, covering an entire 90 mm petri dish within 3 days. Its hyphae and colony characteristics are as follows: colonies are gray with abundant aerial hyphae; after 3-4 days of culture, all strains form sclerotia, initially white and powdery, gradually turning brown, and are scattered. Hyphae branching angles are acute to right angles, with significant constriction at branch points, and a septum near each branch; in the later stages of culture, some hyphal cells swell into ellipsoids to tubes, each containing multiple nuclei. These characteristics are similar to those of *Rhizoctonia solani* (…). R. solani It matches the typical morphological characteristics of ).

[0029] Molecular biological identification results showed that ( Figure 5 The ITS sequences of three representative strains were submitted to the NCBI database for BLAST comparison. It was found that the ITS gene sequences of GUCC 25-0182, GUCC 25-0183, and GUCC 25-0184 were similar to those of... R. solani The similarity between AG-4 and HG-1 was 100%. A phylogenetic tree constructed based on the rDNA-ITS gene showed that the three representative strains were similar to... R. solani AG-4 and HG-1 clustered into the same clade with a clade support of 100%, indicating that they belong to the same species phylogenetically. Based on the combined morphological identification and phylogenetic analysis, GUCC 25-0182, GUCC 25-0183, and GUCC 25-0184 were determined to be... R. solaniAG-4 HG-1.

[0030] Pathogenicity test results showed ( Figure 6 ), a fungal cake representing strain GUCC 25-0182 was inoculated onto the roots of healthy pepper plants for pathogenicity testing. Three days after inoculation, the lower leaves began to wilt; seven days after inoculation, the upper leaves gradually curled, and all the lower leaves wilted and partially died; fifteen days after inoculation, all the leaves on the entire plant died. (Uninoculated plants...) R. solani The control pepper plants showed no symptoms of disease throughout the entire process and grew normally. Root examination of the inoculated plants revealed... R. solani The roots of the chili pepper plants in the control group were noticeably brown and rotten, with all lateral roots dead and the main root completely rotten, losing its normal absorption function. The base of the stem was noticeably brown, and the tissue at the base of the stem showed obvious signs of slight constriction and thinning, with some areas showing obvious wrinkling. In contrast, the roots of the control plants were normal in color and showed no signs of rot. The lateral and main roots were growing well and showed no abnormalities.

[0031] In summary, this invention is the first to identify... F. cugenangense and Rhizoctonia solani ( Rhizoctonia solani The AG-4 HG-1 subspecies can infect peppers and cause pepper root rot.

[0032] Example 2: Growth-promoting effect of Trichoderma wumeng GUWM22 and its biocontrol effect on pepper root rot 1. Growth-promoting effect of Trichoderma Umengense GUWM22 1.1 Determination of growth-promoting properties of chili peppers Place the chili seeds in a petri dish lined with moist filter paper to germinate (28℃, in darkness). Once the seeds show signs of germination, transplant them into plastic pots filled with substrate soil. After the chili peppers have grown two cotyledons, select seedlings of uniform growth and transplant them into containers filled with substrate soil, planting one seedling per pot.

[0033] To prepare a suspension of Trichoderma wumengense GUWM22, the activated GUWM22 strain was inoculated onto PDA plates and cultured at 28°C in the dark for 7 days. The spores on the plates were washed with sterile water, filtered through sterile gauze, and the spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 8 CFU / mL, for later use.

[0034] The experiment consisted of two groups: the treatment group received 50 mL of GUWM22 bacterial suspension per seedling as rhizosphere irrigation; the control group received an equal volume of water as irrigation. Growth indicators of the two groups of peppers were measured 30 days after treatment.

[0035] 1.2 Effects on the germination rate of chili seeds Select plump, uniformly sized, and disease- and pest-free chili seeds, and use the soaking method to place the seeds in a 1×10 8 Seeds were soaked in CFU / mL GUWM22 bacterial suspension for 12 h; the control group was treated with sterile water under the same conditions. The standard paper germination method was used, with 50 seeds evenly placed in petri dishes lined with double layers of moist filter paper, three replicates per dish. The petri dishes were incubated at 25℃ in a constant temperature and light incubator with 12 h / 12 ​​h light / dark cycles. Seed germination rate was recorded on day 7, and plumule and radicle lengths were measured.

[0036] Germination rate (%) = (final number of germinated seeds / total number of seeds tested) × 100.

[0037] 2. Biocontrol effect of Trichoderma Umengense GUWM22 against pepper root rot The *Trichoderma harzianum* and *Bacillus subtilis* used in this embodiment were purchased from Bayer AG. The chemical agent was a mixture of 0.01% 28-epibrassinolide, 10.5% abamectin-pyridaben, and 30% pyraclostrobin. The brand of the 0.01% 28-epibrassinolide was Taqianzhan, the brand of the 10.5% abamectin-pyridaben was Suohong, the brand of the 30% pyraclostrobin was Nongfuxian, and the brand of the 50% thiophanate-methyl was Bato. The compound Bacillus agent is disclosed in the literature: Zhou Zhicheng. Preliminary Study on the Biocontrol Mechanism of *Bacillus sicca* GUWM35 and Development of Compound Agent [D]. Guizhou University, 2023.

[0038] 2.1 Indoor potted plant control efficacy The root rot pathogen used in this embodiment is Rhizoctonia solani (… Rhizoctonia solani The AG-4 HG-1 subspecies (provided by the Plant Pathology Laboratory of Guizhou University) was used to test the indoor control efficacy against pepper root rot using a potted root drenching method. The cultivation substrate was sterilized soil. The experiment consisted of 5 treatments, with 3 replicates per group. Details of each treatment and application method are shown in Table 1.

[0039] Table 1. Drugs and application methods for each treatment First, pepper seedlings were pretreated using a bacterial suspension drenching method for 7 consecutive days. Seven days after pretreatment, pathogen inoculation was performed using the diseased soil method: sterilized and cooled nutrient soil was evenly spread on a sterile plate, and pre-cultured fresh pathogenic fungal cakes were evenly sown onto the soil surface. The mixture was thoroughly stirred using sterile tools to ensure uniform mixing of the pathogen and soil. The inoculation amount was controlled at 5-7 g of fresh fungal cakes per 100 g of soil. After stirring, the experimental diseased soil was prepared. A blank control group was prepared using nutrient soil from the same batch, treated with the same sterilization process, but without any pathogen inoculation. Thirty days after inoculation, the disease incidence was investigated. The number of diseased plants at each level was recorded according to the disease severity grading standard (Table 2), and the incidence rate, disease index, and control effect were calculated.

[0040] Table 2 Grading Criteria for the Incidence Severity of Pepper Root Rot Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100; Disease index = [∑(number of diseased plants at each level × representative value of the corresponding disease level) / (total number of plants surveyed × representative value of the highest disease level)] × 100; Prevention and control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100.

[0041] 2.2 Field efficacy The experiment was conducted at the chili pepper experimental base in Xifeng County, Guiyang City, Guizhou Province. The field experiment included 8 treatments, with 4 replicates per treatment (30 chili pepper plants per plot). Details of the pesticides applied and the application methods for each treatment are shown in Table 3. Specifically, pesticides T6 and T7 were mixed at a 1:1 volume ratio, sprayed on the leaves, and the root drenching dosage was 20 mL.

[0042] Table 3. Drugs and application methods for each treatment The field trial involved four applications of pesticide and disease surveys, with an interval of 7 days between applications. Before the pesticide spraying and root drenching, the number of diseased plants in each treatment group was surveyed and the disease index was calculated. Five days after the last pesticide spraying and root drenching, the number of diseased plants was surveyed again, and the disease index and relative control efficacy were calculated.

[0043] 3. Results and Analysis 3.1 Growth-promoting effect of GUWM22 on chili peppers After treatment with GUWM22, the plant height, fresh weight, and dry weight of the pepper plants were all higher than those of the control plants treated with water. Figure 7 (Tables 4-5). Specifically, the plant height of peppers in the GUWM22 treatment group increased significantly, and the fibrous root development was more vigorous. This indicates that GUWM22 has a significant growth-promoting effect.

[0044] Table 4 Effects of GUWM22 on agronomic traits of chili peppers Note: Different lowercase letters in the same column indicate significant differences at the p<0.05 level using Duncan's test; the same applies to the following table.

[0045] Table 5 Effects of GUWM22 on the biomass of chili peppers 3.2 Effect of GUWM22 on the germination rate of pepper seeds Compared with the control (CK), the GUWM22 treatment significantly improved the seed germination rate. Figure 8 The GUWM22 treatment significantly promoted the elongation of the hypocotyl and radicle in seedlings, with statistically significant differences between groups. Morphological observation showed that the seeds in the GUWM22 treatment group germinated more uniformly, and the seedlings grew more vigorously. Overall, GUWM22 has a positive regulatory effect on seed germination and early seedling morphogenesis.

[0046] 3.3 The potted control efficacy of GUWM22 against pepper root rot Pot experiment results showed that GUWM22 treatment effectively reduced the incidence of pepper root rot. (Inoculation alone...) R. solani The disease index of the first group was 68.52. After treatment with GUWM22, the disease index dropped to 22.84, achieving a control effect of 66.67%. This control effect was comparable to that of 50% thiophanate-methyl chemical agents and significantly superior to commercially available Trichoderma harzianum microbial agents. Figure 9 (See Table 6). In summary, GUWM22 has excellent control effects against pepper root rot.

[0047] The results of chili pepper growth index testing showed that inoculation R. solani Subsequently, the GUWM22 treatment group showed significantly higher plant height, root length, fresh weight, and dry weight compared to the other treatment groups, and also showed significantly higher growth indicators compared to the uninoculated group. R. solani There was no significant difference between the control group and the control group (water). Figure 9 (Tables 7-8) further confirm that GUWM22 also has a growth-promoting effect.

[0048] Table 6. Results of the control efficacy of GUWM22 against pepper root rot. Table 7 Effects of GUWM22 on agronomic traits of chili peppers Table 8 Effects of GUWM22 on the biomass of chili peppers 3.4 Field control efficacy of GUWM22 against pepper root rot Before application of the pesticide, the incidence rate and disease index of pepper root rot in each treatment were statistically analyzed. The results showed (Table 9) that there were no significant differences in the initial incidence rate and disease index among different treatments. p >0.05) indicates that the initial disease level of the experimental materials is uniform and consistent, the experimental grouping is scientific and reasonable, and the groups are highly comparable.

[0049] The control effects of different treatments on pepper root rot after pesticide application showed significant differentiation, with obvious differences in incidence rate, disease index, and relative control efficacy (Table 10). Among them, treatments T2 and T5 had significantly lower incidence rates and disease indices than other treatments, with control efficiencies of 84.17% and 84.94%, respectively, showing the best control effects; treatments T6 and T7 also had control efficiencies above 80%, demonstrating good disease inhibition capabilities; treatments T3, T4, and T8 had control efficiencies between 55% and 77%, showing only moderate inhibition of disease development; the control group T1 had the highest incidence rate and disease index, indicating the most severe disease occurrence.

[0050] Comparison of disease changes before and after pesticide application revealed that, influenced by natural disease occurrence in the field, both the incidence and disease index increased after pesticide application in all treatments, but the magnitude of the increase varied significantly among different treatments. The control group T1 showed the largest increase in both disease index and incidence, indicating rapid disease spread and a sharp increase in the severity of damage. In contrast, the highly effective treatment groups T2 and T5 showed slower disease development, with significantly lower increases in incidence and disease index compared to the control group, effectively curbing the spread of pepper root rot.

[0051] Table 9. Incidence and disease index of pepper root rot in the field before pesticide application. Table 10. Disease control effect of pesticide application on root rot of peppers in the field. Example 3: The effect of GUWM22 on the control of root rot of peppers in continuously cropped pepper fields and its impact on soil fertility. 1. The control effect of GUWM22 on root rot of pepper in continuously cropped fields and its impact on pepper growth. Soil samples from typical chili pepper root rot-infected fields in Xiuwen County, Guizhou Province, were collected as the diseased soil substrate. Huaxi Dangwu chili pepper (susceptible variety) and Dongjinfu chili pepper (resistant variety) were selected as test materials. A control group (CK) and a GUWM22 treatment group were established for root drenching. Each treatment consisted of 15 pots, replicated three times. The root rot incidence rate, disease index, plant height, stem diameter, root fresh weight, and aboveground biomass were investigated to evaluate the control effect and growth-promoting effect of GUWM22 on root rot of different resistant chili pepper varieties in the diseased soil. 2. Effects on the activity of chili pepper's defensive enzymes and malondialdehyde content After 30 days of treatment, pepper leaves from both the control and treatment groups were collected, placed in sterile 2 mL EP tubes, flash-frozen in liquid nitrogen, and then stored at -80°C for subsequent determination of peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL) activities, and malondialdehyde (MDA) content (using a detection kit from Beijing Solarbio Science & Technology Co., Ltd.). Each treatment was performed in triplicate.

[0052] 3. Effects of GUWM22 on the physicochemical characteristics of rhizosphere soil in continuous cropping of chili peppers A pot experiment was conducted, with a control group (CK) and a GUWM22 treatment group. After 50 days of cultivation, rhizosphere soil samples were collected from continuously cropped peppers. Soil pH, organic matter, available nitrogen, available phosphorus, available potassium, and other physicochemical indicators were measured. The differences between the two groups were compared through statistical analysis to analyze the effect of GUWM22 on the physicochemical properties of rhizosphere soil from continuously cropped peppers.

[0053] 4. Results and Analysis 4.1 The control effect of GUWM22 on root rot of pepper in continuously cropped fields and its impact on pepper growth Typical soil samples of chili root rot disease were collected from Xiuwen County, Guizhou Province. Pot experiments showed that the diseased soil had strong pathogenicity (the mortality rate of infected chili varieties reached over 65% after 15 days of planting). Figure 10 A pot experiment was conducted using a disease-resistant chili pepper variety (Shandong Jinfu). A control group and a GUWM22 treatment group were set up (treatment was performed once every 6-7 days, for a total of 6 times). After 50 days of planting, the disease index of the GUWM22 treatment group decreased significantly from 80.00 to 27.41, with a control effect of 65.74% (Table 11), indicating that GUWM22 has a significant control effect on chili pepper root rot.

[0054] Further measurements of plant growth indicators revealed that the GUWM22 treatment group showed significantly better results than the CK group in key growth indicators such as plant height, root length, fresh weight, and dry weight. Figure 11 (Tables 12-13) These results not only further confirm that GUWM22 has a significant growth-promoting function, but also clarify its application potential in the green control of pepper root rot, providing experimental basis for the subsequent development of GUWM22 into a biocontrol agent.

[0055] Table 11. Control effect of GUWM22 on pepper root rot Table 12 Results of GUWM22's effect on the growth promotion of chili peppers Table 13 Effects of GUWM22 on the biomass of chili peppers 4.2 Effects of GUWM22 on the activity of defensive enzymes and malondialdehyde (MDA) content in chili peppers GUWM22 treatment significantly increased the activities of PPO, CAT, PAL, POD and SOD in pepper leaves. Figure 12 Among the treatments, the increases in SOD and CAT activities were particularly significant (P<0.05), indicating that GUWM22 can efficiently activate the plant's antioxidant enzyme system, synergistically scavenging reactive oxygen species and alleviating oxidative stress. The increased activities of PAL and PPO promoted the synthesis of disease-resistant substances such as phytoalexins and lignin, strengthening structural and chemical defense capabilities. Simultaneously, the MDA content also increased under GUWM22 treatment. Combined with the significant increase in defense enzyme activity, this accumulation may not be due to excessive membrane lipid peroxidation damage, but rather a controlled oxidative stress signal induced by Trichoderma, which can further trigger downstream disease-resistant defense responses, ultimately enhancing the disease resistance of pepper leaves.

[0056] 4.3 Effects of GUWM22 on the physicochemical characteristics of rhizosphere soil in continuous cropping of chili peppers Soil physicochemical property analysis showed that, compared with the control group, the GUWM22 treatment significantly improved the core soil fertility indicators ( Figure 13 The treatment group showed a significant increase in soil organic matter content, along with a marked increase in total nitrogen and hydrolyzable nitrogen content, effectively enhancing the accumulation of soil organic carbon and nitrogen supply capacity. Regarding pH, the soil in the treatment group shifted significantly from slightly alkaline to neutral, optimizing the soil micro-ecological environment and promoting microbial activity and nutrient activation. In terms of mineral nutrients, the available potassium content remained stable, while the total potassium, total phosphorus, and available phosphorus contents decreased slightly, possibly due to the treatment promoting nutrient absorption and utilization or form transformation. In summary, the GUWM22 treatment achieved overall improvement in soil fertility by increasing soil organic matter and available nitrogen and optimizing pH, demonstrating a good regulatory effect.

[0057] Example 4: Trichoderma wumeng GUWM22 against R. solani Antibacterial mechanism The culture medium and its composition used in this embodiment are shown in Table 14.

[0058] Table 14 Culture medium formulation 1. GUWM22 R. solani Antibacterial activity 1.1 GUWM22 pair R. solani Antibacterial activity and reparasitic effect The plate confrontation method was used to determine the pair of GUWM22. R. solani The antibacterial effect of GUWM22 was observed periodically during the confrontation culture. R. solaniInteractions after hyphal contact. A small amount of hyphae were collected using a sterile dissecting needle from the contact area between the two hyphae and the area covered by GUWM22 pathogens to prepare temporary slides. The interaction between GUWM22 and pathogens was observed under an optical microscope. R. solani The phenomenon of hyperparasitism was recorded, including morphological characteristics such as hyphal entanglement, penetration, and disintegration.

[0059] 1.2 Effects of GUWM22 volatile substances on R. solani Inhibition assay Using the plate-on-plate culture method, biocontrol bacteria GUWM22 mycelial discs (7 mm in diameter) were inoculated into the center of PDA medium and incubated at 28°C for 3 days. R. solani The mycelium was inoculated in the center of another PDA medium. The two culture dishes were then removed from their caps and inverted together, with the GUWM22 inoculated dish placed at the bottom. R. solani The inoculated petri dishes were placed on top and sealed with sealing film to form the treatment group. PDA medium containing uninoculated GUWM22 mycelial cakes was mixed with... R. solani Inoculated petri dishes were placed face down as a control group, and each treatment was replicated three times. All petri dishes were incubated in the dark at 28°C, and the growth of pathogen colonies was observed periodically. After 3 days of incubation, the diameter of pathogen colonies was determined using the cross-sectional method. The inhibition rate was calculated using the following formula: Inhibition rate = [(C-7) - (T-7)] / (C-7) × 100%, where C is the average growth diameter of pathogens in the control group and T is the average growth diameter of pathogens in the treatment group.

[0060] 1.3 GUWM22 non-volatile substances on R. solani Inhibition assay The cellophane method was used for assaying. Sterile cellophane was placed on the surface of PDA medium, and a GUWM22 bacterial pellet was inoculated into the center of the cellophane. The medium was then incubated in the dark at 28°C for 3 days, allowing the strain to grow only on the cellophane and infiltrate its non-volatile metabolites into the lower medium. After incubation, the cellophane and bacterial cells were removed, and the medium was inoculated into the center of the culture medium. R. solani Mycelial colonies. A control group was prepared by inoculating sterile PDA plates with sterile cellophane covering them; each treatment was replicated three times. The plates were incubated at 28°C for 2 days, and the diameter of the pathogenic bacterial colonies was measured using the cross-hatching method.

[0061] 1.4 The effect of aseptic fermentation broth of GUWM22 on R. solani Antibacterial assay The activated GUWM22 was perforated using a sterile punch, and the resulting bacterial pellet was inoculated into PDB medium. The mixture was then incubated at 28°C and 180 rpm for 72 h to obtain the GUWM22 fermentation broth. The fermentation broth was centrifuged at 4°C and 8000 rpm for 15 min, and the supernatant was collected. This supernatant was then sterilized by vacuum filtration through a 0.22 μm sterile filter to obtain sterile GUWM22 fermentation broth. This sterile broth was thoroughly mixed with sterile PDB medium at a 2:1 volume ratio to prepare a composite plate containing metabolites. The center of this plate was then inoculated with the culture medium. R. solani Mycelial pellets. An equal proportion of sterile PDB was added to the PDA in the control group, with each treatment repeated three times. The pellets were incubated at 28°C for 3 days, and the diameter of the pathogenic bacterial colonies was measured using the cross-sectional method.

[0062] 2. The aseptic fermentation broth of GUWM22 on... P. capsici and F. cugenangense Antibacterial assay According to "1.4 GUWM22 aseptic fermentation broth for..." R. solani The procedure in the "Antimicrobial Assay" was to determine the antimicrobial activity of the aseptic fermentation broth of Trichoderma wumeng GUWM22 against Phytophthora capsici (a type of fungus). Phytophthora capsici , P. capsici ) and Fusarium kuganensis ( F. cugenangense (It has an antibacterial effect.)

[0063] 3. The effects of GUWM22 aseptic fermentation broth on... R. solani Effects of mycelial biomass and antioxidant enzyme activity According to "1.4 GUWM22 aseptic fermentation broth for..." R. solani The sterile fermentation broth was prepared according to the procedure in the "antibacterial assay". R. solani Inoculate onto PDA medium and incubate at 28℃ for 3 days. Take 7 mm diameter mycelial discs and inoculate them onto PDB liquid medium containing GUWM22 metabolites (metabolite to PDB volume ratio 2:1). Incubate at 28℃ and 150 r / min with shaking for 5 days. After incubation, filter and collect the mycelium, wash three times with distilled water, and weigh the fresh weight of the mycelium. Then, absorb excess water from the mycelium, weigh 0.1 g into a pre-chilled centrifuge tube, add 1 mL of pre-chilled buffer, and homogenize in an ice bath. Centrifuge at 4℃ and 12000 r / min for 20 min. Collect the supernatant as crude enzyme solution for determining the contents of SOD, CAT, and MDA (kit, Beijing Solarbio Science & Technology Co., Ltd.).

[0064] 4. The effects of GUWM22 aseptic fermentation broth on... R. solani Effects of soluble proteins and soluble sugars Collect “3. GUWM22 aseptic fermentation broth on” R. solaniThe mycelia used in the study of "Effects on Mycelial Biomass and Antioxidant Enzyme Activity" were weighed at 0.1 g and placed in a pre-cooled centrifuge tube. The tubes were then flash-frozen in liquid nitrogen and stored at -80°C for later use. Subsequently, the contents of soluble protein and soluble sugar were measured and analyzed according to the kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0065] 5. The effects of GUWM22 aseptic fermentation broth on... R. solani Effects on hyphal cell membrane integrity According to "1.4 GUWM22 aseptic fermentation broth for..." R. solani The procedure for "antibacterial assay" involves preparing GUWM22 sterile fermentation broth, mixing it with PDA medium at a volume ratio of 2:1 to form a drug-containing plate, and using a PDA plate without sterile fermentation broth as a control. R. solani Inoculate the colony in the center of the above-mentioned plate, incubate upside down at 28°C for 2 days, then pick fresh hyphae from the edge of the colony, rinse twice with sterile water, add 10 μg / mL propidium iodide (PI) staining solution, stain at room temperature in the dark for 15 min, wash away excess dye with sterile water, and observe under a fluorescence microscope (excitation wavelength 535 nm, emission wavelength 615 nm). Each treatment was repeated in 3 replicates, and 5 fields of view were randomly selected for observation and photographic recording.

[0066] 6. The effects of GUWM22 aseptic fermentation broth on... R. solani The influence of hyphae on cell nuclei According to "1.4 GUWM22 aseptic fermentation broth for..." R. solani In the "antibacterial assay", the procedure involves preparing a drug-containing plate and placing it... R. solani After inoculation, the cells were incubated upside down at 28°C for 2 days. Fresh hyphae from the colony edges were picked, rinsed twice with sterile water, and stained with an appropriate amount of DAPI staining solution (final concentration 1 μg / mL) at room temperature in the dark for 10 min. After washing away excess dye with sterile water, the cells were observed under a fluorescence microscope (excitation wavelength 340 nm, emission wavelength 488 nm). Each treatment was repeated in triplicate, and five fields of view were randomly selected for observation and photographic recording.

[0067] 7. The effects of GUWM22 aseptic fermentation broth on... R. solani The influence of hyphal cell walls Collect “6. GUWM22 aseptic fermentation broth for” R. solani The hyphae in the section "Influence of Hyphae on Cell Nuclei" were rinsed twice with sterile water, placed on a glass slide, and a suitable amount of fluorescent whitening agent (CFW) staining solution (final concentration 0.1 mg / mL) was added. Staining was performed at room temperature in the dark for 5 min. After rinsing off excess dye with sterile water, a coverslip was placed on the slide, and the samples were observed under a fluorescence microscope (excitation wavelength 365 nm, emission wavelength 435 nm). Each treatment was performed in triplicate, and five fields of view were randomly selected for observation and photographic recording.

[0068] 8. The effects of GUWM22 aseptic fermentation broth on... R. solani The effect of reactive oxygen accumulation in mycelia Collect “6. GUWM22 aseptic fermentation broth for” R. solani The hyphae in the section "Influence of Hyphae on Cell Nuclei" were rinsed twice with sterile water, placed on a glass slide, and a suitable amount of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining solution (final concentration 10 μmol / L) was added. Staining was performed at room temperature in the dark for 20 min. After washing away excess dye with sterile water, a coverslip was placed, and the samples were observed under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). Each treatment was performed in triplicate, and five fields of view were randomly selected for observation and photographic recording.

[0069] 9. Determination of growth rate and mycelial dry weight of strain GUWM22 Growth rate determination of strain: Activated GUWM22 strain was used to create mycelial cakes at the edge of colonies using a sterile punch with a diameter of 7 mm. These cakes were then inoculated into the center of PDA plates and incubated upside down in a 28℃ incubator. Colony diameter was measured every 8 hours using the cross-hatching method, with each measurement repeated three times until the colonies completely covered the plate.

[0070] Mycelial dry weight determination: Activated GUWM22 strain was used to collect mycelial cakes from the colony edge using a sterile punch with a diameter of 7 mm. Six mycelial cakes were placed in each Erlenmeyer flask containing 100 mL of PDB liquid medium and incubated at 28℃ with shaking at 150 r / min. Every 8 h, a sample was collected, and mycelia were collected by filtering with sterile gauze. The mycelia were rinsed three times with sterile water and dried in a 60℃ oven until constant weight. The mycelial dry weight (g) was measured. Each time point was repeated in triplicate.

[0071] 10. Biological characteristics of GUWM22 Cellulase assay: Inoculate the GUWM22 strain in the center of a cellulase medium, incubate upside down at 28°C for 2-5 days, repeat 3 times, and observe whether a clear zone appears around the colony. Protease assay: Inoculate the GUWM22 strain in the center of a protease medium, incubate at 25°C for 2-5 days, repeat 3 times, and observe whether a clear zone appears around the colony. Chitinase assay: Inoculate the GUWM22 strain in the center of a chitinase medium, incubate at 28°C for 3-7 days, repeat 3 times, and observe whether a clear zone appears around the colony. Siderophore assay: Inoculate the strain in the center of a CAS plate, incubate at 30°C for 2-5 days, repeat 3 times, and observe whether an orange-yellow halo appears around the colony.

[0072] 11. Effect of temperature on the mycelial growth of GUWM22 After activation, the GUWM22 strain was used to create mycelial cakes at the edge of colonies using a sterile punch with a diameter of 7 mm. These cakes were then inoculated into the center of PDA plates and incubated upside down for 2 days at temperature gradients of 15, 20, 25, 28, 30, 35, and 40°C, with each treatment having three biological replicates. During incubation, the colony diameter was measured timed using the cross-hatching method to determine the optimal growth temperature for the GUWM22 strain.

[0073] 12. Effects of nutrient substrate on the mycelial growth of GUWM22 Using a 7 mm diameter sterile punch, mycelial cakes were collected from the edge of activated GUWM22 colonies and inoculated onto the center of plates containing different nutrient substrates, including PDA, SNA, MEA, OA, TOA, CMA, WA, and Bengal Red agar. The plates were incubated upside down at 28°C for 2 days, with each treatment having three biological replicates. Colony diameter was determined using the cross-cross method at specific times to evaluate the environmental adaptability of the GUWM22 strain under different nutrient conditions.

[0074] 13. Effect of pH on the mycelial growth of GUWM22 Mycelial cakes were collected from the edges of activated GUWM22 colonies using a 7 mm diameter sterile punch. PDA medium was adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 with 1 mol / L NaOH and 1 mol / L HCl, respectively, with unadjusted PDA medium serving as a control (CK). Mycelial cakes were inoculated into the center of PDA plates at the different pH values ​​and incubated upside down in the dark at 28°C for 2 days. Each treatment was performed in triplicate, and colony diameter was determined using the cross-hatching method.

[0075] 14. Effects of salt stress on the mycelial growth of GUWM22 Using a 7 mm diameter sterile punch, mycelial cakes were collected from the edge of activated GUWM22 colonies and inoculated into the center of PDA plates with different NaCl concentrations (1%-10%). The plates were incubated upside down at 28°C for 2 days, with each treatment having three biological replicates. Colony diameter was determined using the cross-crossing method, and mycelial growth rate was calculated.

[0076] 15. Effects of Alkali Stress on Mycelial Growth of GUWM22 Using a 7 mm diameter sterile punch, mycelial cakes were collected from the edge of activated GUWM22 colonies and inoculated into the center of PDA plates with different NaHCO2 concentrations (0.1%-1%). The plates were incubated upside down at 28 ℃ for 2 days, with each treatment having three biological replicates. Colony diameter was determined using the cross-crossing method.

[0077] 16. Determination of the antibacterial spectrum of GUWM22 The antagonistic effect of the pathogen strains listed in Table 15 (all pathogen strains were provided by the Plant Pathology Laboratory of Guizhou University) on the pathogens was determined using the plate confrontation culture method. Activated strain GUWM22 and pathogenic bacterial pellets (5 mm in diameter) were inoculated on both sides of a PDA plate, with a distance of 4 cm between them. A PDA plate inoculated only with the pathogen served as a control. The plates were incubated at a constant temperature of 25℃, and the colony diameter was recorded after 7 days. The antibacterial effect of strain GUWM22 was evaluated by calculating its inhibition rate against the pathogens. The inhibition rate was calculated using the formula: Inhibition rate = [(CT) / C] × 100%. Where C and T represent the average growth radius of the tested pathogens in the control and treatment groups, respectively.

[0078] Table 15 Main strains 17. Results 17.1 GUWM22 pairs R. solani Antibacterial activity and its reparasitic effect GUWM22 against R. solani The results of its antibacterial activity and reparasitic effect are shown in Figure 14 and Figure 15 GUWM22 against R. solani GUWM22 has a significant inhibitory effect on the growth of bacterial colonies. R. solani GUWM22 significantly inhibited the growth of bacterial colonies. After 3 days of culture, GUWM22 showed a significant inhibitory effect on... R. solani The colony growth inhibition rate reached 76.34%. Further observation of the interaction between the hyphae of the two organisms revealed that after 5 days of cultivation, GUWM22 and... R. solani The contact area showed obvious R. solani Hyphae disintegration phenomenon. Microscopic observation of the contact area between the hyphae of the two organisms revealed that GUWM22 hyphae could entangle and reparasitize on the surface. R. solani The surface of the hyphae indicates that GUWM22 is affected by... R. solani It has the ability to regenerate.

[0079] 17.2 The effect of volatile substances in GUWM22 on R. solani Inhibition effect The results of the plate-to-plate culture method showed that ( Figure 16 ), vaccination R. solani 3 days later, the CK group R. solani The cells were completely confluent in 90 mm culture dishes, while the GUWM22 treatment group showed improvement. R. solani It has a significant inhibitory effect on colony growth. According to the colony diameter measurement, the colony diameter of the GUWM22 treatment group was significantly lower than that of the control group, which was specifically manifested as a significant slowdown in the mycelial extension rate of the pathogen, a reduction in the amount of aerial mycelia, and a decrease in the number of sclerotia.

[0080] 17.3 GUWM22 non-volatile substances on R. solani Inhibition effect The effects of non-volatile metabolites of GUWM22 on the body were determined using the cellophane culture method. R. solani The antibacterial activity of GUWM22 was observed. After 3 days of culture, the results showed that the non-volatile metabolites of GUWM22 significantly inhibited... R. solan i. Mycelial growth ( Figure 17 Specifically, this manifests as a significant slowdown in the mycelial extension rate of pathogens, a marked decrease in aerial mycelial biomass, and a substantial reduction in the number of sclerotia formed.

[0081] 17.4 Metabolites of GUWM22 R. solani Antibacterial assay The sterile fermentation broth from GUWM22 shake-flask fermentation for 3 days was mixed with PDA medium to prepare drug-containing plates. Results after 3 days of incubation showed (…). Figure 18 GUWM22 aseptic fermentation broth can significantly inhibit R. solani Mycelial growth showed an antibacterial rate of 95.33%.

[0082] 17.5 GUWM22 aseptic fermentation broth on P. capsici and F. cugenangense Antibacterial effect result( Figure 19 It was found that the aseptic fermentation broth of GUWM22 could significantly inhibit P. capsici and F. cugenangense Mycelial growth showed inhibition rates of 66.94% and 92.49%, respectively.

[0083] 17.6 GUWM22 aseptic fermentation broth on R. solani Effects of mycelial biomass and antioxidant enzyme activity GUWM22 aseptic fermentation broth on R. solani The inhibitory effect on mycelial growth is shown in [reference needed]. Figure 20 Compared with the control group (CK), the mycelial growth of the pathogen was significantly inhibited in the GUWM22 treatment group, with loose mycelial clumps and a significant reduction in biomass. The mycelial fresh weight statistics also showed a significant reduction in mycelial biomass in the GUWM22 treatment group. These results indicate that GUWM22 can effectively inhibit the vegetative growth of the pathogen, providing direct evidence for its biocontrol function.

[0084] After treatment with GUWM22 aseptic fermentation broth R. solani The activities of CAT and SOD were significantly reduced compared with those of CK, while the content of MDA was significantly increased. Figure 21 This result confirms that GUWM22 can inhibit the antioxidant defense system of pathogens, disrupt their reactive oxygen species metabolic balance, and induce membrane lipid peroxidation and cell membrane damage, thereby achieving the effect of... R. solani Effective inhibition of growth.

[0085] 17.7 GUWM22 aseptic fermentation broth on R. solani Effects of soluble proteins and soluble sugars GUWM22 aseptic fermentation broth on R. solani The effect of mycelial soluble protein and soluble sugar content is shown in [reference needed]. Figure 22 Compared to CK, the GUWM22 treatment group R. solani The contents of soluble protein and soluble sugar in the hyphae both showed a highly significant decreasing trend. Soluble protein and soluble sugar are the core material basis for fungal hyphal growth and metabolism, participating in key physiological processes such as structural construction, enzyme catalysis and energy supply, and osmotic regulation, respectively. The simultaneous significant decrease in the contents of both indicates that GUWM22 can interfere with... R. solani It disrupts the metabolism of proteins and carbohydrates, damaging their normal material synthesis and energy supply systems, ultimately inhibiting the growth and reproduction of pathogens.

[0086] 17.8 GUWM22 aseptic fermentation broth on R. solani Effects on hyphal cell membrane integrity The effects of GUWM22 aseptic fermentation broth on the effects of PI (propidium iodide) staining on the body were detected. R. solani The effect on hyphal cell membrane integrity, the results are as follows Figure 23 As shown, under bright field conditions, there was no significant difference in hyphal morphology between the control group and the treatment group. After PI staining, observation under a fluorescence microscope revealed that the hyphae in the control group only showed weak background fluorescence, with no obvious red fluorescence, indicating that their cell membrane structure was intact and PI could not enter the cell.

[0087] After processing by GUWM22, R. solani The hyphae exhibited strong red fluorescence, with a significantly higher fluorescence intensity than the control group, and the fluorescence distribution was widespread, indicating that the cell membrane integrity of the hyphae in the treated group was severely disrupted, allowing PI to enter the cells in large quantities and bind to DNA. In conclusion, GUWM22 can significantly disrupt the cell membrane integrity of Rhizoctonia solani hyphae, which may be one of the important mechanisms by which it exerts its antibacterial effect.

[0088] 17.9 GUWM22 aseptic fermentation broth on R. solani The influence of hyphae on cell nuclei The effects of DAPI fluorescence staining on the effects of GUWM22 aseptic fermentation broth on [unclear - possibly related to environmental factors] were detected using the DAPI fluorescence staining method. R. solani The effect of hyphae on cell nuclei, the results are as follows Figure 24 As shown, under bright field conditions, there was no significant difference in hyphal morphology between the control group and the treatment group. After DAPI staining, observation under a fluorescence microscope revealed that the cell nuclei of the control group hyphae were regular in morphology, evenly distributed, and had normal fluorescence intensity, indicating that the cell nuclei were structurally intact and functionally normal.

[0089] After processing by GUWM22, R. solani Significant abnormalities were observed in the mycelial cell nuclei. In the treated group, the mycelial cell nuclei exhibited chromatin condensation, irregular nuclear morphology, and increased or decreased fluorescence intensity. Some mycelial cell nuclei showed fragmentation or disappearance, indicating that GUWM22 caused severe damage to the mycelial cell nuclear structure. In conclusion, GUWM22 can significantly induce nuclear damage in *Rhizoctonia solani* mycelial cells, disrupting nuclear structure and function, which may be one of the important mechanisms by which it exerts its antibacterial effect.

[0090] 17.10 GUWM22 aseptic fermentation broth on R. solani The influence of hyphal cell walls The effects of CFW (calcium fluorescent white) staining on the effects of GUWM22 aseptic fermentation broth on [the following text is incomplete and requires further context: "the CFW (calcium fluorescent white) staining method was used to detect the effects of GUWM22 aseptic fermentation broth on the [the following text is incomplete and requires further context: ""] R. solani The effect of hyphal cell walls, the results are as follows Figure 25 As shown, under bright field conditions, there was no significant difference in hyphal morphology between the control group and the treatment group. After CFW staining, observation under a fluorescence microscope revealed that the hyphae in the control group exhibited uniform and continuous green fluorescence with consistent fluorescence intensity and regular hyphal morphology, indicating that their cell wall structure was intact and chitin was normally distributed.

[0091] After treatment with GUWM22, the CFW staining fluorescence of Rhizoctonia solani hyphae showed significant abnormalities. The fluorescence intensity of the treated hyphae was significantly reduced, the fluorescence distribution was uneven, and some areas showed fluorescence loss or breakage, indicating that the chitin structure in the cell wall was damaged and the cell wall integrity was impaired. In conclusion, GUWM22 can significantly disrupt the cell wall structure of Rhizoctonia solani hyphae, which may be one of the important mechanisms by which it exerts its antibacterial effect.

[0092] 17.11 Effect of GUWM22 aseptic fermentation broth on reactive oxygen species accumulation in R. solani mycelia Detection using DCF fluorescence staining method R. solani The levels of reactive oxygen species within the hyphae were as follows: Figure 26 As shown, under bright field conditions, no significant difference was observed in the hyphal morphology between the control group and the treatment group. After DCF staining, observation under a fluorescence microscope revealed that the hyphae in the control group only exhibited weak green fluorescence, indicating a low level of reactive oxygen species (ROS); while the hyphae in the GUWM22 metabolite-treated group showed strong green fluorescence, with a significantly higher fluorescence intensity than the control group, indicating that the GUWM22 metabolites induced [oxidative stress]. R. solani Excessive accumulation of reactive oxygen species (ROS) within the hyphae. In summary, GUWM22 metabolites can significantly induce an ROS burst within the hyphae, disrupting cellular redox homeostasis and thus exerting an antibacterial effect.

[0093] 17.12 Determination of growth rate and mycelial dry weight of strain GUWM22 The GUWM22 strain was cultured on PDA plates at 28°C, and the colony diameter gradually increased with the extension of the culture time. Figure 27 The rapid growth phase lasted from 0 to 32 hours, during which the colony diameter increased rapidly. After 32 hours, the growth rate gradually slowed down, and the colony diameter tended to stabilize between 48 and 72 hours. Figure 28 (A) indicates that the colonies have fully colonized the plate. The differences in colony diameter between different incubation time points are statistically significant, indicating that strain GUWM22 has a good growth rate under solid culture conditions.

[0094] The GUWM22 strain was cultured in PDB liquid medium at 28°C and 150 r / min with shaking. The dry weight of the mycelium gradually increased with the extension of the culture time. Figure 28 (B) During the 0-16 h culture period, the mycelial dry weight increased slowly; from 16-48 h, the logarithmic growth phase began, and mycelial biomass accumulated rapidly; after 48 h, growth slowed down, and from 64-72 h, the growth essentially reached a stable phase. The differences in mycelial dry weight between different culture time points were statistically significant, indicating that strain GUWM22 has a good biomass accumulation capacity under liquid culture conditions.

[0095] 17.13 Biological characteristics of GUWM22 The GUWM22 strain showed positive reactions in cellulase, protease, and chitinase assays, and produced siderophore halos on CAS plates, indicating that this strain possesses multiple hydrolytic enzyme activities and the ability to produce siderophores. Figure 29 This provides a potential biochemical basis for its application in the biological control of plant diseases and soil ecological adaptation.

[0096] 17.14 Effect of Temperature on the Mycelial Growth of GUWM22 Temperature is a key environmental factor regulating the mycelial growth of GUWM22. GUWM22 can maintain growth within the range of 15–35℃, with an optimal growth temperature of 28℃ and a suitable growth range of 20–30℃. High temperatures of 40℃ completely inhibit mycelial growth, while growth is significantly inhibited at 35℃. Low temperatures of 15℃ only slow down the growth rate without causing growth cessation. Figure 30 ).

[0097] 17.15 Effects of nutrient substrate on the mycelial growth of GUWM22 The effects of different culture media on the mycelial growth of GUWM22 were highly significant. Figure 31This strain grows best in nutrient-rich media, with PDA being the optimal growth medium, and TOA and MEA being suitable growth media. Nutrient-poor media (WA, OA) can maintain the growth of the strain, but the growth is significantly weakened. SNA and CMA have a significant inhibitory effect on mycelial growth, and Bengal red medium (RBA) can significantly inhibit the mycelial growth of GUWM22.

[0098] 17.16 Effect of pH on the mycelial growth of GUWM22 The effects of pH on the mycelial growth of GUWM22 were significantly different. Figure 32 This strain has an extremely wide pH range and can grow normally in pH 3 to 10. Among them, pH 3 is the optimal growth pH, ​​and pH 3 to 7 is the suitable growth range. Within this range, the growth rate of the strain is not significantly different from that of the blank control. Under pH 8 to 10 conditions, the growth rate of the strain only decreased slightly and was not completely inhibited, but was only accompanied by changes in colony morphology and culture medium color.

[0099] 17.17 Effects of Salt Stress on Mycelial Growth of GUWM22 NaCl stress has a highly significant inhibitory effect on the mycelial growth of GUWM22. Figure 33 Furthermore, the degree of inhibition increased with increasing NaCl concentration. This strain was only slightly inhibited at low concentrations (1%) of NaCl, and could maintain a certain growth capacity; as the concentration increased, mycelial growth was gradually inhibited, and growth was significantly limited at 6%~7% NaCl; NaCl concentrations of 8% and above could completely inhibit the mycelial growth of GUWM22, thus clarifying its salt tolerance threshold.

[0100] 17.18 Effects of Alkali Stress on Mycelial Growth of GUWM22 NaHCO3 stress has a highly significant inhibitory effect on the mycelial growth of GUWM22. Figure 34 The inhibition level increased continuously with increasing NaHCO3 concentration. This strain did not completely stop growing in the range of 0.1% to 1% NaHCO3 concentration, but significant growth inhibition was observed at low concentrations (0.1%), and growth height was limited at 1% concentration. Compared with neutral salt NaCl, GUWM22 showed weaker tolerance to alkaline salt NaHCO3, clarifying its sensitivity to alkaline salt stress.

[0101] 17.19 Determination of the antibacterial spectrum of GUWM22 To clarify the broad-spectrum antifungal activity of GUWM22, its inhibition rate against the mycelial growth of various crop pathogenic fungi was determined. GUWM22 showed varying degrees of antifungal activity against all 25 tested pathogenic fungi. Figure 35 Among them, the pathogen causing passion fruit stem rot (… F. solaniThe inhibition rate of ) was 65.23%, and the inhibition rate of the root rot fungus of Codonopsis pilosula ( ) was 65.23%. R. solani The inhibition rate was 71.90%, and the inhibition rate against *Tobacco Target Spot Bacteria* (… R. solani The inhibition rate was 78.97%. (This is in contrast to the kiwifruit soft rot pathogen (…). A. alternata , P. lithocarpus , B. dothidea ), brown rot fungus of honey plum ( D. eres , M. fructicola , C. nymphaeae Sunshine Rose rot fungus ( A. alternata, A. tenuissima Banana anthracnose bacteria ( C. musae ), Konjac white mold pathogen ( S. rolfsii ), Codonopsis pilosula root rot fungus ( A. alternata , A. tenuissima, H. mompa ), tea leaf spot pathogen ( F. graminearum , F. asiaticum ), bean leaf spot disease ( C. lindemuthianum , C. cassicola ) Leaf spot fungus of Solomon's seal ( C. camelliae ), Stemona leaf spot fungus ( C. spaethianum ), Prickly pear leaf spot pathogen ( D. beans Tobacco black shank bacterium ( P. nicotianae ), pepper anthracnose bacteria ( C. truncatum The mycelial growth inhibition rate of the pathogenic fungi was higher than 80%.

[0102] The above results indicate that Trichoderma wumeng GUWM22 has significant antifungal activity against a variety of crop pathogenic fungi, with a broad antifungal spectrum, and has the potential to be developed as a broad-spectrum biocontrol strain.

[0103] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Trichoderma wumeng ( Trichoderma wumeng The application of GUWM22 in promoting chili pepper growth is characterized by, The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

2. The application of Trichoderma wumeng GUWM22 in the preparation of a fungal agent to promote pepper growth, characterized in that, The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

3. The application of Trichoderma wumeng GUWM22 in the prevention and / or treatment of pepper root rot, characterized in that, The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

4. The application of *Trichoderma wumeng* GUWM22 in the preparation of fungal agents for the prevention and / or treatment of pepper root rot, characterized in that... The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

5. The application as described in claim 3 or 4, characterized in that, The pathogens causing pepper root rot include Rhizoctonia solani (… Rhizoctonia solani ).

6. The application of Trichoderma wumeng GUWM22 in inhibiting pathogenic fungi, characterized in that, The pathogens include Fusarium kuganensis (… Fusarium cugenangense Alternaria microphylla ( ), Alternaria tenuissima ), Eris medusa ( Diaporthe eres ), Water lily anthracnose bacteria ( Colletotrichum nymphaeae ), Apple sclerotium ( Monilinia fructicola ), Rhizoctonia solani, and Rhizoctonia solani ( Helicobasidium mompa Fusarium graminearum ( ), Fusarium graminearum ), Fusarium tumefaciens ( Fusarium asiaticum ), Bean thorny discus ( Colletotrichum lindemuthianum ), Corynebacterium multiflorum ( Corynespora cassiicola Banana anthrax ( Colletotrichum musae ), neatly arranged small sclerotia ( Sclerotium rolfsii Anthracnose (Camellia anthrax) Colletotrichum camelliae ), Ash anthracnose fungus ( Colletotrichum spaethianum ), intercropping of green beans with schizocarps ( Diaporthe phaseolorum ) and / or Fusarium solani ( Fusarium solani ); The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

7. The application of *Trichoderma wumeng* GUWM22 in the preparation of fungal agents for inhibiting pathogenic fungi, characterized in that... The pathogens include Fusarium kuganensis, Alternaria spp., Erisia spp., Anthracnose fungus, Leymus chinensis, Rhizoctonia solani, Rhizoctonia solani, Rhizoctonia solani, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Colletotrichum spp., Corynebacterium multiflorum, Anthracnose fungus, Sclerotium spp., Anthracnose fungus, Anthracnose fungus, Anthracnose fungus, Alternaria spp., and / or Fusarium solani. The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

8. A method for preventing and controlling root rot in chili peppers, characterized in that, This includes the steps of treating pepper plants with Trichoderma wumengense GUWM22; The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639; The pathogen causing pepper root rot includes Rhizoctonia solani.

9. The method as described in claim 8, characterized in that, The treatment of the chili pepper plants includes drenching the roots of the chili pepper plants, or spraying the chili pepper plants and then drenching the roots of the chili pepper plants.

10. A method for promoting chili pepper growth, characterized in that, This includes the steps of treating pepper plants with Trichoderma wumengense GUWM22; The preservation number of the *Trichoderma wumeng* GUWM22 is CCTCC NO: M20242639.

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