Application of trichoderma harzianum in cooperation with cellulase to rotten straw and growth promotion

Abstract

The application relates to the field of agricultural microorganisms, in particular to the application of Trichoderma harzianum and cellulase in the decomposition of straw and the promotion of growth. The application uses Trichoderma harzianum IVF1 microbial agent and cellulase in cooperation, can significantly accelerate the decomposition process of tomato straw, improve the straw degradation rate and the release amount of reducing sugar; meanwhile, the straw decomposition leachate can significantly promote the growth of cucumber and other plants.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to the application of Trichoderma harzianum and cellulase in the synergistic effect of composting straw and promoting growth. Background Technology

[0002] Tomato straw has a dense lignocellulose structure, and its degradation cycle under natural conditions is as long as 30-60 days. This results in slow decomposition, incomplete degradation, and susceptibility to pathogens, severely hindering its large-scale application. (Trichoderma genus) Trichoderma Fungi are recognized as excellent microbial resources with functions of biocontrol, growth promotion, and straw degradation. However, single Trichoderma strains have drawbacks such as weak environmental adaptability, insufficient secretion of key enzymes (such as cellulase), and low initial degradation efficiency of crystalline cellulose. Using cellulase preparations alone is problematic due to high cost, poor stability, and inability to sustain proliferation in the field. Therefore, developing a composite technology that can rapidly initiate and sustainably and efficiently degrade straw, with degradation products effectively promoting crop growth, is of great significance for solving the current problem of straw return to the field. Summary of the Invention

[0003] The present invention aims to solve the problems of slow straw decomposition, incomplete degradation, and lack of synergistic growth promotion function in the prior art.

[0004] The purpose of this invention is to provide the application of Trichoderma harzianum IVF1 and cellulase for the degradation of crop straw.

[0005] Another object of the present invention is to provide the application of Trichoderma harzianum IVF1 and cellulase in the preparation of decomposed plant straw that promotes plant growth.

[0006] According to the technical solution of this application, Trichoderma harzianum ( Trichoderma harzianum The application of IVF1 and cellulase in the degradation of crop straw, wherein the Trichoderma harzianum ( Trichoderma harzianum The accession number for IVF1 is CGMCC No. 42324.

[0007] According to the technical solution of this application, the crop straw is tomato straw.

[0008] According to the technical solution of this application, the volume ratio of the Trichoderma harzianum IVF1 inoculant to the cellulase preparation is 1:2 to 2:1.

[0009] According to the technical solution of this application, the volume ratio of the Trichoderma harzianum IVF1 inoculant to the cellulase preparation is 1:1.

[0010] According to the technical solution of this application, Trichoderma harzianum ( Trichoderma harzianumThe application of IVF1 and cellulase in the preparation of decomposed plant straw that promotes plant growth, wherein the *Trichoderma harzianum* ( Trichoderma harzianum The accession number for IVF1 is CGMCC No. 42324.

[0011] According to the technical solution of this application, the crop straw is tomato straw.

[0012] According to the technical solution of this application, the plant is a cucumber.

[0013] According to the technical solution of this application, the decomposed plant straw is used to increase the fresh weight and root length of cucumber plants.

[0014] The mechanism of action of this invention lies in the fact that the exogenously added cellulase preparation can rapidly initiate the hydrolysis of the surface cellulose of straw, destroying its dense structure and creating favorable conditions for the rapid colonization and reproduction of Trichoderma harzianum IVF1. Subsequently, the colonized Trichoderma harzianum IVF1 strain can continuously secrete various hydrolytic enzymes such as endonuclease and filter paper enzyme, achieving deep degradation of straw lignocellulose. This synergistic mechanism of "rapid initiation of exogenous enzymes + continuous enzyme production by the strain" not only significantly improves the degradation efficiency of straw and the release of small molecule nutrients such as reducing sugars, but its composting products, rich in beneficial microorganisms and active substances, can also significantly promote root development and plant growth.

[0015] The Trichoderma harzianum IVF1 and its synergistic composition disclosed in this invention have the following beneficial effects: 1. Excellent strain performance: The *Trichoderma harzianum* IVF1 strain screened in this invention is an excellent strain with high cellulase production. Its filter paper enzyme activity and endonuclease activity are the best among the tested strains, with an enzyme activity of 13.275 U / mL. The *Trichoderma harzianum* strain of this invention can effectively inhibit cucumber gray mold and cowpea wilt. Compared with the registered *Trichoderma harzianum* LTR-2, the *Trichoderma harzianum* strain of this application has a better inhibitory effect on pathogenic fungi. Further investigation revealed that under inactivated gray mold induction conditions, differentially expressed genes were significantly enriched in pathways related to secondary metabolite biosynthesis, antibiotic synthesis, and amino acid synthesis. The antibacterial active substances produced by the *Trichoderma harzianum* strain of this invention are enhanced, further strengthening the control efficacy. Metabolomics results showed a significant increase in the production of important biologically active organic acids such as citric acid, benzene ring compound emodin, organic heterocyclic compound 6-methylnicotinic acid, and trichoderma diol.

[0016] 2. Significant Synergistic Effect: This invention combines a high-enzyme-producing Trichoderma strain with exogenous cellulase. Through the mechanism of "rapid activation of exogenous enzyme + continuous enzyme production by the strain," a synergistic effect of 1+1>2 is achieved. Experiments show that the degradation rate of tomato straw by this composition is significantly higher than that of single strains, single enzymes, and commercially available composting agents. The degradation rate of Trichoderma harzianum IVF1 combined with exogenous cellulase reaches 86.6%.

[0017] 3. Integrated Functionality: This invention achieves an integrated function of "rapid straw decomposition—efficient nutrient conversion—significant crop growth promotion." The reducing sugars and other active substances released during the decomposition process make the leachate itself an excellent plant growth promoter.

[0018] 4. Broad application prospects: The synergistic composition provided by this invention is suitable for various scenarios such as open-field in-situ return to the field. It has the advantages of accelerating straw decomposition, high degradation efficiency, and environmental friendliness, providing an efficient technical path for the resource utilization of agricultural waste.

[0019] The present invention (Trichoderma harzianum) Trichoderma harzianum IVF1, the preservation number of this bacterium is CGMCC No. 42324, and its classification name is... Trichoderma harzianum, The deposit date is November 21, 2025. The depositary institution is the China General Microbiological Culture Collection Center, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0020] Figure 1 This is a diagram showing the morphology of the transparent zone of Trichoderma on a CMC-Na plate; Figure 2 Displaying the filter paper enzyme activity of different Trichoderma strains; Figure 3 The activity of endocellulase in different Trichoderma strains was shown; Figure 4 A phylogenetic tree of Trichoderma harzianum IVF1 constructed based on the ITS gene sequence; Figure 5 A comparison chart of reducing sugar content in straw leachate from different treatments in a food preservation box experiment; Figure 6 This is a comparison of the growth-promoting effects of different treatments of straw leachate on cucumber plants in a food preservation box experiment. Figure 7 A comparative diagram showing the colonization ability of Trichoderma harzianum IVF1 strains expressing GFP on tomato straw; Figure 8 This is a time series diagram showing the effects of different treatments on the degradation forms of tomato straw in an open-field experiment. Figure 9This is a comparative graph showing the effects of different treatments on the degradation rate of tomato straw in an open-field experiment. Detailed Implementation

[0021] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention. Unless otherwise specified, the raw materials, reagents, strains, etc. used in the present invention can be obtained through commercial channels or conventional methods. Example 1: Initial screening of high cellulase-producing strain IVF1

[0022] Soil samples collected in September 2024 from Qingyuan County, Lishui City, Zhejiang Province were analyzed using the dilution plating method. The culture medium was serially diluted to 10⁻⁶ with sterile water. -4 ~10 -7 For each concentration, 200 μL of the diluted solution was spread onto PDA solid medium and then incubated upside down in a 30°C constant temperature incubator. The colonies growing on the medium were observed. Based on the size, color, and surface pattern of the colonies, larger single colonies were selected and repeatedly streaked on PDA solid medium for 2-3 generations of purification until single colonies appeared. The Trichoderma strain was isolated and purified and named IVF1.

[0023] Based on the preserved *Trichoderma* strains, the cultures were incubated on PDA plates at 28°C for 5 days. Subsequently, 0.5 cm diameter mycelial discs were prepared at the colony edges using a sterile punch and incubated on CMC-Na plates (formula: CMC-Na 10g, NaNO3 1g, K2HPO4 0.5g, KCl 0.5g, MgSO4·7H2O 0.5g, yeast extract 0.5g, agar 18g, distilled water 1L) at 28°C for 4 days. The culture was then stained with 0.1% Congo red for 20 minutes and eluted with 1 mol / L NaCl. The ratio of the clear zone diameter (H) to the colony diameter (C) (H / C) was measured and calculated. The results showed that, using *Trichoderma reesei* TU-6 as a reference strain, 19 *Trichoderma* strains exhibited significant cellulase inhibition zones. Among them, strains IVF1, IVF2, IVF3, and IVF4 had significantly higher H / C ratios than other tested strains, achieving a 100% inhibition rate, indicating their extremely strong cellulose-decomposing ability. Figure 1 ). Example 2: Secondary screening of high cellulase-producing strain IVF1

[0024] The high-cellulase-producing Trichoderma strains IVF1-IVF19 and the reference strain Trichoderma reesei TU-6 were obtained from the initial screening and were used in 1x10⁻¹¹ samples. 8CFU / mL biomass was inoculated into Avicel induction liquid medium (formulation: 20 g Avicel, 0.5 g ammonium sulfate, 1.5 g potassium dihydrogen phosphate, 0.06 g magnesium sulfate, 0.06 g calcium chloride, tap water to natural pH, 1 L distilled water) and cultured at 28 °C with shaking at 180 rpm for 7 days. Filter paper enzyme activity (FPA) and endonuclease activity (CMCase) of the fermentation broth were determined using the DNS method.

[0025] Filter paper enzyme activity (FPA) assay: Whatman No. 1 filter paper was punched into 0.5 mm diameter discs. Two discs were placed at the bottom of a test tube, and 900 μL of acetate-sodium acetate buffer (0.05 M, pH 4.8) was added. After equilibration in a 50°C water bath, 100 μL of appropriately diluted enzyme solution was added, mixed thoroughly, and incubated in a 50°C water bath for 1 hour. At the end of the reaction, 1.5 mL of DNS reagent was added to each test tube, and 100 μL of enzyme solution was added to the blank, and mixed thoroughly. The mixture was boiled in boiling water for 5 min, rapidly cooled, and the absorbance at 540 nm was measured. The amount of enzyme required to hydrolyze the filter paper substrate to produce 1 μmol of reducing sugar (calculated as glucose) per hour by 1 mL of liquid enzyme at 50°C and pH 4.8 is defined as one unit of enzyme activity (U). Results are as follows: Figure 2 As shown, the filter paper enzyme activity of strain IVF1 is 13.3 U / mL.

[0026] Sodium carboxymethyl cellulose (CMC-Na) was used as a substrate to determine endonuclease activity. A 1.5% CMC-Na solution was prepared using citrate-disodium hydrogen phosphate buffer (0.05 M, pH 5.0). 100 µL of the appropriately diluted enzyme solution was added to 900 µL of CMC-Na substrate, vortexed to mix thoroughly, and incubated at 50°C for 30 min. The reaction was terminated by adding 1.5 mL of DNS reagent, and then 100 µL of enzyme solution was added to a blank tube and mixed thoroughly. The tube was boiled in water for 5 min, rapidly cooled, and the absorbance was measured at 540 nm using a microplate reader. One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose per minute to produce 1 μmol of reducing sugar (calculated as glucose) at 50°C and pH 5.0. Results are as follows: Figure 3 As shown, the endonuclease activity of strain IVF1 was the highest among all tested strains, at 3.6 U / mL. Example 3 Identification of high-cellulase-producing Trichoderma strain IVF1

[0027] Genomic DNA was extracted from *Trichoderma* IVF1 using a fungal genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal primers for fungal ITS. After passing 1% agarose gel electrophoresis, the amplified products were purified and sequenced. The obtained ITS sequences were analyzed for homology using the Basic Local Alignment Search (BLAST) tool in the National Center for Biotechnology Information (NCBI) database to preliminarily verify sequence accuracy. After confirming sequence accuracy, reference sequences of *Trichoderma harzianum* and its closely related species were downloaded from NCBI. Multiple alignments of all sequences were performed using BioEdit software to ensure sequence locus consistency. Finally, a phylogenetic tree was constructed using the maximum likelihood method with MEGA 11.0 software, and the confidence level of each branch was assessed using a bootstrap test with 1000 replicates. The phylogenetic analysis results are shown below. Figure 4 As shown, the strain IVF1 identified in this application clusters with the standard strain of *Trichoderma harzianum* within a high-support evolutionary branch and is clearly separated from other *Trichoderma* species. This result definitively identifies strain IVF1 as *Trichoderma harzianum*. According to the *Trichoderma harzianum* of the present invention... Trichoderma harzianum The preservation number of this bacterium is CGMCC No.42324. Example 4: Synergistic degradation of tomato straw by Trichoderma harzianum IVF1 and cellulase

[0028] The prepared Trichoderma harzianum ( Trichoderma harzianum IVF1 strain was inoculated onto PDA medium and cultured for 5 days to promote conidia production. After sufficient conidia were produced, the conidia were gently collected by scraping with a sterile cotton swab. After serial dilution and plate counting, the conidia suspension was quantified until the effective viable count was not less than 1 × 10⁻⁶. 8 CFU / mL. The experiment included single Trichoderma harzianum IVF1 treatment, single cellulase treatment, and treatments with different ratios of Trichoderma harzianum IVF1 and cellulase. The test substrate was dried tomato straw, pulverized to 3–5 cm pieces. 110 g of each sample was accurately weighed and placed in a preservation box. The corresponding inoculum and enzyme preparations were added to each treatment group according to the designed ratio, mixed thoroughly, covered with plastic wrap with several ventilation holes punched in the wrap, and incubated at 28 ℃. The following treatments were set up: Table 1. Treatment groups of Trichoderma harzianum IVF1 and cellulase synergistic degradation of tomato straw .

[0029] After 30 days of composting, the observation results showed that the straw in the T4 treatment group exhibited the most outstanding performance in terms of softening, breakage, and black rot at all observation time points, with a significantly better composting effect than other groups. Table 2 shows that the straw degradation rate of the T4 group was significantly higher than that of the T1, T2, T3, and T7 groups. As shown in Figure 5, the reducing sugar content in the straw leachate of the T4 group reached 594.1 mg, significantly higher than the other treatment groups. As shown in Figure 6, with the water treatment (T1 group) as the control, the corresponding cucumber plant fresh weight was only 1.81 g; while the cucumber plants treated with the straw leachate of the T4 group showed significantly better fresh weight and main root length than other treatment groups, with the plant fresh weight reaching 2.76 g, an increase of 52.5% compared to the water control. The above results indicate that the synergistic composition of bacteria and enzymes provided by this invention can significantly accelerate the straw decomposition process, effectively promote the release of nutrients in straw, and show obvious growth-promoting and efficiency-enhancing effects on crop growth, demonstrating good application results.

[0030] Table 2. Degradation rate of tomato straw treated with synergistic degradation of Trichoderma harzianum IVF1 and cellulase. . Example 5: Colonization experiment of Trichoderma harzianum IVF1

[0031] This embodiment uses endogenous Trichoderma harzianum. pdc1 Startup driver eGFP Gene expression was performed, and a recombinant plasmid named pPdc1-GFP was constructed and introduced into *Trichoderma harzianum* IVF1 strain via protoplast transformation. After culture, transformants on the culture plates were observed using a handheld fluorescent lamp and fluorescent glasses to screen for positive transformants that successfully expressed the eGFP gene. These positive transformants were then subjected to conidial culture. After sufficient conidia were generated from the positive transformants, the conidia were gently scraped off with a sterile cotton swab, serially diluted, and quantified using a plate count method. The spore suspension was adjusted to a viable count of at least 1 × 10⁻⁶ cells / mL. 8 CFU / mL, for later use.

[0032] Three parallel experimental treatments were set up: Group 1 (single Trichoderma harzianum IVF1 treatment), Group 2 (single cellulase treatment), and Group 3 (Trichoderma harzianum IVF1+GFP strain and cellulase mixed at a volume ratio of 1:1). The test substrate was dried tomato straw, pulverized into 3–5 cm pieces. 110 g of each piece was accurately weighed and placed in sterile containers. According to the experimental design proportions for each group, the corresponding bacterial agent and enzyme preparation were added to the corresponding containers, thoroughly mixed, covered with sterile plastic wrap, and several ventilation holes were punched in the plastic wrap. All containers were placed at a constant temperature of 28 ℃ for incubation. After 3 days of incubation, samples were taken for fluorescence observation. Figure 7 As shown, the tomato straw in the water-free blank control group showed no fluorescence signal; the straw treated with Trichoderma harzianum IVF1+GFP strain in group 1 showed a weak fluorescence signal, indicating that Trichoderma harzianum IVF1+GFP strain could successfully colonize the surface of tomato straw; the straw treated with Trichoderma harzianum IVF1+GFP strain and cellulase at a volume ratio of 1:1 in group 3 showed a significant fluorescence signal, indicating that cellulase pretreatment can significantly promote the colonization of Trichoderma harzianum IVF1+GFP strain on the surface of tomato straw, thereby exerting a more efficient straw degradation effect. Example 6: Verification of the effect of in-situ return of tomato straw to the field

[0033] To further verify the synergistic effect of Trichoderma harzianum preparations and cellulase on the in-situ degradation of tomato straw in the complex environment of actual farmland, and to clarify its feasibility and advantages in field application, a field verification experiment on straw return to the field was conducted at the experimental base in Pinggu District, Beijing. The entire process strictly followed field trial specifications to ensure the authenticity, reliability, and repeatability of the experimental results. The tomato straw was mechanically pulverized, and uniformly sized segments of 3–5 cm were obtained. To accurately control the treatment dosage and facilitate sampling and observation, the pulverized tomato straw was precisely weighed at 110 g per portion and placed into well-ventilated mesh bags to ensure full contact between the straw and the experimental reagents without leakage. Subsequently, according to the experimental design shown in Table 3, corresponding doses of Trichoderma harzianum preparations, cellulase, the Trichoderma harzianum-cellulase synergistic combination, and a commercially available inoculant (control group) were added to each mesh bag. Specific treatment settings, reagent dosages, and application methods are detailed in Table 3.

[0034] Table 3. Verification of the effect of in-situ tomato straw return to the field: treatment groups .

[0035] The experiment employed a randomized block design, with three replicates for each treatment. Field management practices were consistent and conformed to local conventional cultivation management standards. Sampling was conducted at fixed points on days 5, 20, 35, and 50 after straw return to the field to observe and record the apparent morphology of the straw and measure the straw degradation rate at the corresponding times. As shown in Figure 8, throughout the entire experimental period, the T4 treatment group exhibited significantly better straw breakage, black rot, and decomposition compared to other treatment groups. Five days after return to the field, the tomato leaves in the T4 group showed obvious signs of decay and disintegration, indicating a significantly faster degradation initiation rate than the control. Figure 9 further illustrates that the synergistic composition demonstrated a significant advantage in the early stages of degradation. Five days after return to the field, the degradation rate of the T4 group reached 60.8%, while the water control T1 group only achieved 31.8%, and maintained a stable and efficient degradation trend throughout the subsequent period. By day 55 after return to the field, the final straw degradation rate of the T4 group reached 86.6%, significantly higher than the other treatment groups. The above field trial results fully demonstrate that the Trichoderma harzianum-cellulase synergistic composition provided by the present invention can still maintain a high efficiency and stable straw degradation capacity under complex open field conditions. The overall composting effect and degradation efficiency are significantly better than conventional treatments and commercially available related products, and it has excellent potential for field application and promotion value. Example 7: Determination of the antifungal effect of Trichoderma harzianum IVF1 metabolites on pathogenic fungi

[0036] Fermentation filtrate preparation: The *Trichoderma harzianum* IVF1 strain of this application was inoculated onto PDA plates and cultured at 28±1℃ for 7 days. Spores were collected and a spore suspension was prepared with sterile water, and the concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 7 CFU / mL. A spore suspension was inoculated into PDB liquid medium at an inoculum of 2% (v / v) and cultured at 28°C with shaking at 180 rpm for 7 days to obtain the fermentation broth. The fermentation broth was then filtered through quantitative filter paper and a 0.22 μm microporous membrane to obtain sterile fermentation filtrate.

[0037] Preparation of drug-containing plates: The above fermentation filtrate was mixed with sterilized PDA medium cooled to approximately 50°C at a ratio of 1:9, and poured into sterile petri dishes to prepare drug-containing plates. The control group consisted of PDA plates with an equal volume of sterile PDB added.

[0038] Antibacterial test: Fresh, activated *Botrytis cinerea* and *Fusarium oxysporum* were used to create 5 mm diameter mycelial discs, which were then inoculated into the center of drug-containing and control plates, respectively. Each treatment was repeated three times and incubated at 28°C for four days.

[0039] Inhibition rate calculation: The diameter of pathogenic bacterial colonies was measured using the cross-multiplication method, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = [(Coronary diameter of control group – Coronary diameter of treatment group) / (Coronary diameter of control group – 5mm)] × 100% Plate confrontation culture revealed that the fermentation broth of Trichoderma harzianum IVF1 inhibited the two pathogenic fungi, Botrytis cinerea and Fusarium oxysporum, by 61.2% and 59.8%, respectively. Example 8: Efficacy of Trichoderma harzianum IVF1 in controlling cucumber gray mold

[0040] 8.1 Preparation of suspensions of pathogens and Trichoderma Botrytis cinerea suspension: After activating Botrytis cinerea on a PDA plate, take 5 mycelial cakes with a diameter of 5 mm and inoculate them into 250 mL of PDB medium. Incubate at 25℃ for 7 days, and then break up the fermentation broth before use.

[0041] Trichoderma harzianum IVF1 suspension: Five Trichoderma mycelial discs with a diameter of 5 mm were inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 7 days. After the culture was completed, the mycelium was broken up using a tissue homogenizer to prepare a homogeneous mycelial fragment suspension for later use, which was then diluted 500 times.

[0042] 8.2 Plant Induction Treatment and Design Sow cucumber seeds in flowerpots, keeping 12 seedlings of uniform growth in each pot. When the seedlings reach the two-leaf-one-heart stage, inoculate them: inoculate the cucumber leaves with a suspension of *Botrytis cinerea*, then place the plants in a culture room at 20-25℃ and 80-90% humidity for 24 hours to promote disease development. After humidity control, dilute the *Trichoderma* fermentation broth and spray it on the leaves, applying 50 mL of the suspension to every 12 cucumber seedlings.

[0043] 8.3 Disease Investigation and Data Analysis Disease severity was assessed 6 days after inoculation. The severity of disease on each leaf was recorded according to the following grading criteria: Grade 0: No lesions; Grade 1: Lesions cover ≤ 5% of the leaf area; Grade 3: Lesions cover 6% to 15% of the leaf area; Level 5: Lesions cover 16% to 25% of the leaf area; Level 7: Lesions cover 26% to 50% of the leaf area; Level 9: The area of ​​lesions accounts for ≥ 51% of the leaf area.

[0044] Based on the survey results, the disease index and prevention and control effectiveness were calculated using the following formula: Disease index = [∑(number of diseased leaves at each level × representative value of that level) / (total number of leaves surveyed × 9)] × 100; Prevention and control effect (%) = [(disease index of blank control group - disease index of treatment group) / disease index of blank control group] × 100.

[0045] Results of different Trichoderma species controlling gray mold in cucumber: Trichoderma harzianum IVF1 spores, diluted 1×10 7 CFU / ml, disease index 11.5, efficacy 78.5%; Trichoderma harzianum IVF1 fermentation broth, diluted 500×, showed a disease index of 10.4 and a control efficacy of 80.7%. Trichoderma harzianum LTR-2 spores, diluted 1×10 7 CFU / ml, disease index 18.2, efficacy 66.1%; Trichoderma harzianum LTR-2 fermentation broth, diluted 500×, showed a disease index of 16.9 and a control efficacy of 68.4%. The disease index was 53.7 after only being vaccinated with Botrytis cinerea. Iprodione 50% wettable powder, diluted 500×, disease index 1.85, control efficacy 90.55%.

[0046] Trichoderma harzianum IVF1 spores and fermentation broth are more effective against gray mold in cucumbers than the mainstream Trichoderma harzianum LTR-2 on the market. Example 9: Efficacy of Trichoderma harzianum IVF1 spores against cowpea wilt disease

[0047] 9.1 Preparation of suspensions of pathogens and Trichoderma Suspension of Fusarium oxysporum pathogen for cowpea wilt: After activating Fusarium oxysporum on PDA plates, five mycelial cakes with a diameter of 5 mm were inoculated into 250 mL of PDB medium and cultured at 25℃ for 7 days. The fermentation broth was then broken up before use.

[0048] Trichoderma suspension: The concentration of Trichoderma harzianum IVF1 spores was adjusted to 1×10⁻⁶ with sterile water. 7 CFU / mL available for use.

[0049] 9.2 Plant Induction Treatment and Design Sow cowpea seeds in pots containing substrate, keeping 12 seedlings of uniform growth per pot. When the seedlings have fully developed their first pair of true leaves, inoculate them using the root-drenching method, applying 10 mL of the prepared pathogen spore suspension to each seedling. After inoculation, place the plants in a greenhouse at 25-28℃ and 70%-85% humidity for normal management to promote disease development. Twenty-four hours after pathogen inoculation, treat with Trichoderma. Apply a diluted Trichoderma solution to the cowpea seedlings via root drenching, applying 10 mL of the fungal suspension to each seedling.

[0050] 9.3 Disease Investigation and Data Analysis Disease severity was assessed starting 14 days after inoculation. The severity of disease on each cowpea plant was recorded according to the following grading criteria: Grade 0: The plant shows no symptoms and is growing normally; Grade 1: Cotyledons or true leaves are slightly yellowed, but the plant does not wilt significantly; Grade 3: One or more true leaves are obviously yellowed and drooping, or the leaf margins show necrosis; Level 5: The plant is wilted overall, and the vascular bundles in the stem show slight browning; Level 7: The plant is severely wilted, growth is stunted, and the vascular bundles in the stem show obvious browning; Level 9: Plant dies.

[0051] Similarly, referring to the investigation method for the control effect of cucumber gray mold, the disease index and control effect of wilt were calculated. Specifically, the control effect of Trichoderma harzianum IVF1 spores on cowpea wilt reached 56.0%. Example 10: Determination of the antifungal effect of Trichoderma harzianum IVF1 metabolites on Botrytis cinerea induced by inactivated gray mold.

[0052] 10.1 Preparation of inducers After activation of *Botrytis cinerea* on PDA medium, it was transferred to PDB liquid medium and cultured at 25°C and 150 rpm for 7 days with shaking. After culture, the mycelium was collected by filtration through a sterile filter membrane and rinsed three times with sterile water. The washed mycelium was then freeze-dried under vacuum to obtain inactivated *Botrytis cinerea* mycelium, which was then sealed and stored at 4°C for later use.

[0053] 10.2 Induced fermentation and metabolite preparation Spore suspensions of Trichoderma harzianum strains IVF1 and LTR-2 (1×10⁻⁶) 7 CFU / mL) were inoculated into the following two fermentation media respectively: Experimental group (induction group): 1% (w / v) of the above-mentioned inactivated Botrytis cinerea mycelium was added to PDB medium; Control group (non-induction group): PDB medium without any added inducers was used.

[0054] Both groups were cultured at 28°C and 180 rpm with shaking for 7 days. After fermentation, the fermentation broth was centrifuged, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain sterile "induced metabolite" and "non-induced metabolite" filtrates, respectively. The antibacterial activity of the metabolite filtrates against *Botrytis cinerea* was evaluated using the mycelial growth rate method. Specifically, the induced metabolite and non-induced metabolite filtrates were mixed with PDA medium at a ratio of 1:9 (v / v) and poured into plates. Plates containing equal volumes of sterile PDB and PDA served as blank controls. Fresh *Botrytis cinerea* mycelial cakes with a diameter of 5 mm were inoculated in the center of each plate and incubated at 25°C for 5 days. The colony diameter was measured using the cross-cross method, and the inhibition rate was calculated using the formula:

[0055] Inhibition rate (%) = [(Coronavirus diameter of control group - Coronavirus diameter of experimental group) / (Coronavirus diameter of control group)] ×100%.

[0056] The results showed that the *Trichoderma harzianum* IVF1-induced metabolites prepared by the method of this invention exhibited significant antibacterial effects against *Botrytis cinerea*, with an inhibition rate as high as 82.1%, representing an 18.1% improvement compared to the non-induced metabolites (64.0% inhibition rate). However, *Trichoderma harzianum* LTR-2 showed no change in its antibacterial effect against *Botrytis cinerea* under inactivated *Botrytis cinerea* induction conditions. These results fully demonstrate that using inactivated *Botrytis cinerea* as an inducer can effectively stimulate *Trichoderma harzianum* IVF1 to produce metabolites with stronger antibacterial activity. Furthermore, different *Trichoderma harzianum* strains do not all produce enhanced antibacterial substances under inactivated *Botrytis cinerea* induction conditions.

[0057] The above embodiments are only used to understand the technical solution of this application and do not limit the scope of protection of this application.

Claims

1. Trichoderma harzianum ( Trichoderma harzianum The application of IVF1 and cellulase in the degradation of crop straw is characterized by, The Trichoderma harzianum ( Trichoderma harzianum The accession number for IVF1 is CGMCC No. 42324.

2. The application according to claim 1, characterized in that, The crop straw mentioned is tomato straw.

3. The application according to claim 1, characterized in that, The volume ratio of Trichoderma harzianum IVF1 to cellulase is 1:2 to 2:

1.

4. The application according to claim 1, characterized in that, The volume ratio of Trichoderma harzianum IVF1 to cellulase is 1:

1.

5. Trichoderma harzianum ( Trichoderma harzianum The application of IVF1 and cellulase in the preparation of decomposed plant straw that promotes plant growth is characterized by, The Trichoderma harzianum ( Trichoderma harzianum The accession number for IVF1 is CGMCC No. 42324.

6. The application according to claim 5, characterized in that, The crop straw mentioned is tomato straw.

7. The application according to claim 5, characterized in that, The plant in question is a cucumber.

8. The application according to claim 7, characterized in that, The decomposed plant straw is used to increase the fresh weight and root length of cucumber plants.

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