A strain of Trichoderma guiyuan B10 and its application

Abstract

This invention provides a *Trichoderma guiyuan* strain B10 and its applications, relating to the field of agricultural microbial control technology. The *Trichoderma guiyuan* strain B10 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 3.25634. This invention overcomes the shortcomings of existing technologies. This strain can inhibit the pathogenic fungi *Neoplasmosis polychaete*, *Neoplasmosis polychaete*, *Neoplasmosis spp.*, *Botrytis cinerea*, and *Metacarpus* spp. of garlic leaf spot disease, while simultaneously promoting the growth of garlic plants, exhibiting both disease resistance and growth-promoting functions in seedlings.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial control technology, specifically to a strain of Trichoderma guiyuan B10 and its application. Background Technology

[0002] Garlic fruit ( Malania oleifera It belongs to the genus *Garcinia* of the family Olacaceae. Malania The garlic plant (Helianthus chinensis), mainly distributed in Guangnan and Funing counties in southeastern Yunnan Province and western Guangxi Province, is listed as a national second-class protected wild plant. Its seed oil is rich in nervonic acid, which can promote the repair and regeneration of damaged nerve tissue, hence its reputation as "liquid gold" among vegetable oils. Furthermore, the garlic plant is tolerant of poor soil and thrives in limestone mountains, making it a superior native tree species for combating desertification in Guangnan and Funing counties of Yunnan Province.

[0003] In recent years, Yunnan Province, the origin of garlic cloves, has vigorously promoted artificial cultivation. However, the spread and damage caused by leaf spot disease have severely reduced the survival and retention rates of garlic clove seedlings in afforestation. Currently, the pathogenic fungus for leaf spot disease has been identified as *Neoproterozoa*. Neopestalotiopsis saprophytica Polychaete spp. Pestalotiopsis australasiae New Clostridium genus fungi Neofusicoccum parvum Staphylococcus spp. fungi Botryosphaeria fabicerciana and fungi of the genus *Metacarpa* Diaporthe sojae Previous studies have shown that spraying chemical fungicides is effective in controlling disease in three-year-old garlic plants at the early stages of infection, but its effectiveness is less than ideal for plants with lesions covering the entire leaf or for one-year-old seedlings infected with the disease. Furthermore, long-term use of chemical fungicides can easily lead to a series of problems such as increased plant resistance, excessive pesticide residues, and environmental pollution. Therefore, seeking ecological and efficient control methods is an important way to ensure the sustainable and healthy development of the garlic cultivation industry.

[0004] Regarding research on *Trichoderma guiyuanensis*, Chinese patent CN202410660097.2 discloses a strain of *Trichoderma guiyuanensis* 23-2 and its control effect on black spot disease of *Paeonia suffruticosa*. Chinese patent CN202411435350.0 discloses a fungal agent of *Trichoderma guiyuanensis* 15-28 and its application, showing that this fungus can effectively promote the growth and development of *Paeonia suffruticosa* seedlings and improve their resistance. Chinese patent CN202411338680.8 discloses a strain of *Trichoderma guiyuanensis* CGMCC No. 41486 and its application in controlling anthracnose of *Camellia oleifera*. Chinese patent CN201911082075.8 discloses a strain of *Trichoderma guiyuanensis* TC952 and its inhibitory effect on *Botrytis cinerea*, *Fusarium graminearum*, and *Rhizoctonia solani*. Chinese patent CN202311128536.7 discloses a strain of Trichoderma guiyuan CGMCC No.40462 and its application in the treatment of heavy metal pollution in water. This shows that different Trichoderma guiyuan strains have different differences in plant disease resistance and environmental management. Based on this, a strain that can prevent garlic fruit diseases can be screened out, which has broad application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Guizhou Trichoderma B10 strain and its applications. The Guizhou Trichoderma B10 strain can effectively control the damage caused by garlic leaf spot disease, while promoting the growth of garlic plants. It also has both disease resistance and growth-promoting functions for garlic seedlings.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A strain of Trichoderma guiyuanensis B10, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 3.25634.

[0007] The Trichoderma tuftedum strain B27 has the ability to produce indoleacetic acid (IAA).

[0008] The aforementioned *Trichoderma* strain B10 from Guizhou can control garlic fruit diseases and / or promote garlic fruit growth.

[0009] Preferably, the ITS nucleotide sequence of the Guizhou Trichoderma B10 strain is shown in SEQ ID NO: 1; the strain was isolated from the roots of healthy garlic plants in Yangmaba, Dongbao Township, Guangnan County, Yunnan Province, and has strong extracellular hydrolytic enzyme activity, including chitinase, cellulase and lipase.

[0010] The disease affecting garlic cloves is garlic clove leaf spot.

[0011] Preferably, the pathogenic fungus causing the garlic leaf spot disease is *Neoprothiolane* (…). Neopestalotiopsis saprophytica ), Polychaete spp. (Pestalotiopsis australasiae ), genus *Neocysporium* fungi ( Neofusicoccum parvum ), Staphylococcus spp. fungi ( Botryosphaeria fabicerciana ) and fungi of the genus *Metacarpa* ( Diaporthe sojae Any one of them.

[0012] Preferably, the preferred method for controlling garlic fruit diseases is to prepare Trichoderma guiyuan B10 spores with a concentration of 1×10⁻⁶. 6 -1×10 8 The product, with a concentration of 1 unit / mL, is applied by foliar spraying for prevention and control.

[0013] Preferably, the preferred method for promoting the growth of garlic bulbs is to prepare Trichoderma guiyuan B10 spores with a concentration of 1×10⁻⁶. 6 -1×10 9 Products with a concentration of 1 unit / mL promote seedling growth when applied by root irrigation or foliar spraying.

[0014] This invention provides a strain of *Trichoderma guiyuan* B10 and its applications, which have the following advantages compared with existing technologies: This invention provides a strain of *Trichoderma guiyuan* B10, which is effective against the fungus *Neoproteroides*, the pathogen causing leaf spot disease in garlic berries. Neopestalotiopsis saprophytica Polychaete spp. Pestalotiopsis australasiae New Clostridium genus fungi Neofusicoccum parvum Staphylococcus spp. fungi Botryosphaeria fabicerciana and fungi of the genus *Metacarpa* Diaporthe sojae The inhibition rates reached 32.96%, 35.29%, 85.00%, 86.43%, and 81.92%, respectively. Spraying Trichoderma guiyuan B10 spore solution onto garlic clove leaves showed inhibition against the pathogenic fungus. N. saprophytica The control efficacy was 43.41%. Meanwhile, Trichoderma guiyuan B10 has the ability to produce IAA. Drenching the soil around the base of the stem of garlic seedlings with the spore liquid of this fungus can significantly increase the plant height and number of leaves of garlic. Spraying the leaves with the spore liquid of Trichoderma guiyuan B10 can significantly increase the biomass of the seedlings. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the colony and microstructure of Trichoderma guiyuan B10 in Example 1 of the present invention, where a is the colony morphology of B10 cultured in PDA medium at 25℃ for 7 days; b is the colony morphology of B10 cultured in CMD medium at 25℃ for 7 days; c is the colony morphology of B10 cultured in SNA medium at 25℃ for 7 days; d is the conidiophore of B10 cultured in SNA medium at 25℃ for 3 days; and e is the conidia of B10 cultured in SNA medium at 25℃ for 10 days. Figure 2 In Embodiment 1 of the present invention, based on tef1 and rpb2The Bayesian phylogenetic tree constructed by gene co-construction is labeled with support at the nodes of the branches; Bayesian posterior probability <75% and maximum likelihood bootstrap value <75% are marked with -; bold text indicates the sequence that generated Trichoderma guiyuan B10 in this study; Figure 3 The colony growth diameter and growth rate of Trichoderma Guizhouense B10 cultured on PDA medium in Example 1 of this invention were measured after culturing for 2 days at 5℃, 10℃, 15℃, 20℃, 25℃, 30℃ and 35℃. Figure 4 The results of cell wall degrading enzyme assays of *Trichoderma guiyuan* B10 in Example 3 of this invention are shown below. In this table, a represents the result of B10 inoculated on butyrate detection medium; b represents the result of blank medium inoculated on butyrate detection medium; c represents the result of B10 inoculated on cellulase detection medium; d represents the result of blank medium inoculated on cellulase detection medium; e represents the result of B10 inoculated on lipase detection medium; and f represents the result of blank medium inoculated on lipase detection medium. Figure 5 The inhibition rate of *Trichoderma guiyuan* B10 against the main pathogenic fungus of leaf spot disease in Example 3 of this invention is given, where: data are expressed as mean ± standard error, and different lowercase letters indicate significant differences between different treatments. P <0.05); Figure 6 Inhibition of Trichoderma guiyuan B10 N . saprophytica Scanning electron microscopy observation, where a is N . saprophytica Hyphae morphology after 3 days of culture in PDA medium; b represents Trichoderma guiyuan B10 and... N . saprophytica Hyphae morphology after 3 days of co-culturing in PDA medium; white arrows indicate... N . saprophytica Mycelium, the blue arrow indicates the mycelium of B10.

[0016] Figure 7 The results show the color development of Trichoderma Guizhouis B10 spore fermentation broth and Salkowski colorimetric solution. Among them, a is a positive control with an equal volume of IAA standard and Salkowski colorimetric solution, b is a negative control with an equal volume of sterile water and Salkowski colorimetric solution, c is the color development reaction with an equal volume of B10 fermentation broth and Salkowski colorimetric solution, and d is the B10 fermentation broth control.

[0017] Figure 8 The effect of root irrigation inoculation with *Trichoderma guiyuan* B10 on the growth and development of garlic seedlings was investigated; where a represents the increase in plant height; b represents the increase in leaf area; c represents fresh weight; and d represents dry weight. Data are expressed as mean ± standard error. This indicates a significant difference between the two treatments. P <0.05), This indicates a significant difference between the two treatments. P <0.01); Detailed Implementation To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The following *Trichoderma guiyuanensis* B10 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 16, 2025, with accession number CGMCC No. 3.25634, and is classified as *Trichoderma guiyuanensis*. Trichoderma guizhouese .

[0019] Example 1: Identification of Trichoderma guiyuan B10 species: 1. Morphological identification PDA medium formula: 200g potato, 20g glucose, 20g agar powder, bring to a final volume of 1000mL with distilled water, sterilize at 121℃ for 20min, and set aside.

[0020] CMD culture medium formula: 40g corn flour, 20g agar, 20g glucose, bring to a final volume of 1000mL with distilled water, sterilize at 121℃ for 20min, and set aside.

[0021] SNA medium formula: KH2PO4 1.0 g, KNO3 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, agar 20 g, distilled water 1000 mL, sterilize at 121℃ for 20 min, and set aside.

[0022] After culturing *Trichoderma guiyuanensis* B10 in the dark at 25°C for 7 days on PDA medium, edge colonies were collected using a 5mm diameter sterile punch and transferred to the center of PDA, CMD, and SNA media. After 7 days of incubation in the dark at 25°C, the mycelia on PDA were dense and abundant, appearing as white cottony or flocculent structures; on CMD, the mycelia were dense and white; and on SNA, the mycelia were fewer, with green pustular structures, mainly distributed at the colony edges. Each group contained 2–3 phialides, measuring 9.01–11.11 × 2.35–3.53 μm. Conidia were smooth, oval or elliptical, measuring 2.5–3.1 × 2.2–2.5 μm. Figure 1 ).

[0023] 2. Molecular biological identification DNA was extracted from the tested *Trichoderma* samples using urea extraction. The original DNA solution was then analyzed for in vivo transcriptional spacer (ITS) and transcription elongation factor. (tef1 ) and RNA polymerase large subunit ( rpb2 The PCR amplification of the three gene fragments, along with the relevant primers and PCR reaction conditions, are shown in Table 1 below: Table 1 PCR amplification systems were all performed in 25 μL volumes, containing 1 μL of DNA bacterial culture, 0.5 μL each of forward and reverse primers, 12.5 μL of TaqMaster Mix, and 10.5 μL of sterile water. Amplification products were detected by gel electrophoresis and sent to Kunming Shuoqing Biotechnology Co., Ltd. for bidirectional sequencing. Sequencing results were manually assembled and corrected in CExpress, then compared with NCBI, and reference sequences with high similarity were downloaded from literature reviews. tef1 and rpb2 Phylogenetic trees were constructed using the Bayesian method (BI) and the maximum likelihood method (ML) based on gene fragments.

[0024] like Figure 2 As shown, Trichoderma guiyuan B10 and T.guizhouense 30.24.06.3 and T . guizhouense LESF 132 clustered into one branch, with a Bayesian posterior probability of 1.00 and a maximum likelihood bootstrap value of 95%. Based on colony morphology characteristics, this bacterium was identified as... T. guizhouense .

[0025] Furthermore, the ITS sequence of *Trichoderma guiyuan* B10 is shown in SQE ID No. 1: .

[0026] Example 2: Colony growth rate of Trichoderma guiyuan B10 at different temperatures: After culturing the bacterium in the dark at 25°C for 7 days on PDA medium, colonies from the edge of the bacterium were transferred to the center of the PDA medium using a sterile punch with a diameter of 5 mm. These colonies were then incubated in the dark for 2 days at constant temperatures of 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C. The results showed that the bacterium grew fastest at 25-30°C, followed by 20°C, with slower growth at 35°C, and no growth below 10°C. Figure 3 ).

[0027] Example 3: Cell wall degrading enzyme activity of Trichoderma guiyuan B10: 1. Chitinase activity assay The culture medium for determining the chitinase activity of Trichoderma is formulated as follows: 4.5 g / L colloidal chitin, 3.0 g / L (NH4)2SO4, 0.3 g / L MgSO4, 2.62 g / L K2HPO4·3H2O, 1 g / L citric acid hydrate, 15 g / L agar, 0.15 g / L bromocresol purple, 200 μL Tween 80, and pH adjusted to 4.7.

[0028] 2. Trichoderma cellulase activity The culture medium for determining the cellulase activity of Trichoderma is formulated as follows: 3.0 g / L NaNO3, 1.31 g / L K2HPO4·3H2O, 0.5 g / L MgSO4, 0.5 g / L KCl, 0.01 g / L FeSO4·7H2O, 10 g / L carboxymethyl cellulose, and 20.0 g / L agar; adjust the pH to 5.0.

[0029] After inoculation, the plates were incubated in the dark at 28°C for 3 days. 0.1% Congo red solution was poured into the plates for staining. After 15 min, the Congo red staining solution was discarded, and the culture medium was washed with 1 mol / L NaCl for 15 min.

[0030] 3. Trichoderma lipase activity The culture medium for the Trichoderma lipase activity assay consisted of 0.1 g / L phenol red, 20 g / agar, 10 mL of lipid substrate (olive oil), and 1.11 g / L CaCl2. The pH was adjusted to 7.4 using 0.1 mol / L NaOH.

[0031] The mycelial discs of the tested strain B10 were inoculated into the center of the above-mentioned culture medium and incubated at 25±2℃ for 3 days. The color change reaction of the culture medium was then observed. The biological assay was repeated 4 times, with each experiment repeated twice.

[0032] like Figure 4 As shown, the chitinase in *Trichoderma* can decompose colloidal chitin in the culture medium, causing the pH value of the medium to rise. The acid-base indicator bromocresol purple in the chitinase medium has an indication range of 5.2 (yellow) to 6.8 (purple); therefore, the closer the color of the medium is to purple, the stronger the chitinase activity of the fungus. The results showed that the chitinase medium inoculated with *Trichoderma guiyuanensis* B10 turned deep red, indicating strong chitinase activity. Furthermore, *Trichoderma* cellulase can hydrolyze the carbon source cellulose in the culture medium. After staining with Congo red, the unhydrolyzed portion turned red, while the hydrolyzed portion turned orange. The results indicated that the cellulase inoculated with *Trichoderma guiyuanensis* B10 could hydrolyze the medium, turning it orange, indicating strong cellulase activity. In addition, the lipase activity test showed a darkening of the medium color, indicating that the fungus possesses certain lipase activity.

[0033] Example 4: Results of plate confrontation culture assay for the inhibition of Trichoderma guizhouense B10 against the pathogenic fungus causing leaf spot disease of garlic cloves: Trichoderma guiyuan B10 and Trichoderma neonatorum were selected. Neopestalotiopsis saprophytica ( N . saprophytica ), Polychaete spp. Pestalotiopsis australasiae ( P . australasiae ), genus *Neococcus* fungi Neofusicoccum parvum ( N . parvum Staphylococcus spp. fungi Botryosphaeria fabicerciana ( B. fabicerciana ), genus *Metacarpa* fungi Diaporthe sojae ( D . sojae The pathogenic fungus was isolated and identified from typical diseased leaves of 3-year-old artificially cultivated garlic pods in Guangnan and Funing counties, Yunnan Province. The tested strains were cultured in PDA medium for 7 days. 10mm diameter mycelial discs of *Trichoderma* and the pathogen were collected using a sterile punch and inoculated onto the same 90mm diameter PDA medium, with the two discs 5cm apart and aligned on the same straight line in the center of the medium. The media were incubated at 25±2℃ for 7 days, with four replicates per treatment.

[0034] The antagonistic ability of each fungus was observed and measured, expressed as the percentage of radial inhibition of pathogen growth and pathogen growth. The calculation formula is: PIRG(%)=(R1−R2) / R1×100%, where R1 represents the radial growth radius of the pathogen in the control and R2 represents the radial growth radius of the pathogen in the antagonistic test.

[0035] like Figure 5 As shown, *Trichoderma guiyuan* B10 exhibits certain inhibitory effects against different pathogenic fungi causing leaf spot disease in garlic berries, particularly against… B. fabicerciana The inhibitory effect was the strongest, at 86.43%, followed by [the inhibitory effect on]. N. parvum and D. sojae The inhibition rates were 85.00% and 81.92%, respectively. P. australasiae and N. saprophytica The inhibition rates were relatively low, at 35.29% and 32.96% respectively, and Trichoderma guiyuan B10 showed relatively low inhibition rates. B. fabicerciana , N. parvum and D. sojae The inhibition rate of this bacterium is related to its effect on P. australasiae and N. saprophytica The inhibition rates differed significantly.

[0036] Example 5: Trichoderma guiyuan B10 inhibition N . saprophytica Scanning electron microscopy observations: Trichoderma guiyuan B10 and N . saprophytica The strains were cultured separately in PDA medium for 7 days. Mycelial discs were then collected using a 10mm diameter sterile punch and inoculated onto the same PDA plate, with a 50mm gap between the discs. The plates were then incubated in the dark at 25℃ for 3 days, with the mycelial contact area approximately 1cm. 2 After freeze-drying and gold sputtering, the samples were observed using a Zeiss Sigma 300 scanning electron microscope.

[0037] like Figure 6 As shown, pathogens N . saprophytica When cultured alone, the hyphae are plump and smooth. However, after being cultured in opposition to Trichoderma B10 for 3 days, the hyphae of B10 spirally intertwined. N . saprophytica The hyphae destroy the cell walls of pathogenic bacteria by producing cell wall degrading enzymes, thereby absorbing the nutrients from the pathogenic hyphae and causing them to shrivel.

[0038] Example 6: Trichoderma Guizhouis B10 spore suspension against *Neoproterozoa garicus* N . saprophytica The prevention and control effect: 1. Seedling Cultivation: Select garlic clove seeds of uniform size and free from disease symptoms from the current year. Peel off the green skin, wash off the mucus on the seed surface with clean water, soak in 0.5% potassium permanganate for 15 minutes, rinse with clean water, and air dry naturally. Then, place the treated seeds in a sand stratification chamber for 6 months to allow germination. A mixture of red soil (red soil: humus: perlite = 3:2:1) was autoclaved, and the seedling pots were surface-sterilized with 75% ethanol. Germinated garlic clove seeds were transplanted individually into seedling pots, and sterile water was applied regularly. The experiment was conducted when the garlic cloves had grown 6-8 leaves.

[0039] 2. Preparation of test bacterial suspension: Trichoderma guizhouense B10 and Trichoderma neonatorum were used. N.saprophytica The spores were inoculated onto PDA plates and cultured until sporulation occurred. A small amount of sterile water was added to the surface of the culture medium using a pipette. Spores were then scraped off with a sterile spreader and filtered through a sterile funnel to obtain a spore suspension. The spore concentration was counted using a hemocytometer. The Trichoderma guiyang B10 spore suspension was diluted to 1×10⁻⁶. 6 Place the cfu / mL solution in a small spray bottle for later use; N. saprophytica The concentration is 1×10 8 cfu / mL, or Guizhou Trichoderma B10 ( T. guizhouense )and N.saprophytica The mycelia were inoculated into potato dextrose (PD) liquid medium and cultured at 150 rpm and 25°C for 5 days. After filtering the mycelia, 1×10⁻⁶ mycelia were prepared. 6 cfu / mL and 1×108 (spore fermentation broth at CFU / mL).

[0040] 3. The experiment included a treatment group and a control group. In the treatment group, pathogenic spore suspension was inoculated onto the lower epidermis of the leaves using a micro-syringe. Five leaves were selected from each garlic bulb plant, and three random sites were chosen from each leaf. 10 μl of pathogenic spore suspension was injected into each site. After inoculation, *Trichoderma guiyuan* solution was sprayed onto both sides of the leaves, ensuring full contact with the leaves and injection sites. The standard for spraying was measured by the dripping of *Trichoderma guiyuan* solution. Finally, the plant leaves were covered with a plastic bag. If the treatment group used spore suspension, sterile water was sprayed onto the leaves as a control. If the treatment group used spore fermentation broth, PD medium was sprayed onto the leaves. All other procedures were the same as the treatment group. Fifteen garlic bulb plants were inoculated for each treatment. Sixty days later, the disease index and control effect of Trichoderma in the treatment groups inoculated with pathogen and inoculated with Trichoderma were statistically analyzed. The disease grading criteria for leaf spot disease are detailed in Table 2. Disease index = ∑(number of diseased leaves at each level × disease grade) / (total number of leaves investigated × highest grade value) × 100%. Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0041] As shown in Table 3 below, the disease incidence of seedlings was investigated and statistically analyzed 60 days after inoculation with pathogens and Trichoderma spores. Among them, the disease incidence was significantly higher after inoculation with *Trichoderma neonatorum*. N. saprophytica The disease index of garlic seedlings was high, reaching 53.00%, while the disease index of seedlings was significantly reduced to 30.00% by foliar spraying of Trichoderma spore suspension, with a control effect of 43.41%.

[0042] Table 2 Grading Criteria for Leaf Spot Disease Table 3 Disease index and control efficacy of *Trichoderma guizhouense* and *Neoprothiolane neomycetes* inoculation into garlic seedlings. Note: Data are expressed as mean ± standard error.

[0043] Example 7: IAA production capacity determination of Trichoderma guiyuan B10: After activation, the B10 test strain was inoculated into PDB liquid medium and cultured at 180 rpm and 28°C for 12 days. Mycelia were then filtered, and the fermentation broth was collected. The fermentation broth was mixed with Salkowski colorimetric solution in an equal proportion and allowed to stand for 30 minutes. Qualitative analysis was performed based on the color development results; a darker color indicated stronger IAA production capacity, while no change indicated the strain lacked IAA production ability. The OD value of the colored fermentation broth was measured using a UV spectrophotometer after mixing the chromogenic fermentation broth with Salkowski colorimetric solution in an equal proportion. 530 The IAA activity of the tested strains was calculated using a standard curve.

[0044] like Figure 7 As shown, when IAA standard is mixed with an equal volume of Salkowski colorimetric solution, it turns deep red. When sterile water is mixed with an equal volume of Salkowski colorimetric solution, no color is developed. When B10 fermentation broth is mixed with an equal volume of Salkowski colorimetric solution, the color is darker than that of B10 fermentation broth. This indicates that the IAA produced by B10 reacts with the colorimetric solution. Further testing showed that the IAA production activity of this bacterium was 1.65 μg / mL.

[0045] Example 8: The growth-promoting effect of Trichoderma Guizhoue B10 inoculation on garlic seedlings Seedling cultivation: Select garlic bulb seeds of uniform size and free from disease symptoms from the current year's growth. After peeling off the green outer skin, wash the seeds with clean water to remove the mucus on the surface. Then soak the seeds in 0.5% potassium permanganate solution for 15 minutes, rinse with clean water, and air dry naturally. Place the treated seeds in a sand stratification chamber for 6 months until germination. A mixture of red soil (red soil: humus: perlite = 3:2:1) was autoclaved, and the seedling pots were surface-sterilized with 75% ethanol. Germinated garlic bulb seeds were transplanted individually into seedling pots, and sterile water was applied regularly. The experiment was conducted when the garlic bulbs had 6-8 leaves.

[0046] Preparation of the test bacterial suspension: *Trichoderma guiyuanensis* was inoculated onto PDA plates and cultured at 25°C in the dark for 7 days. Mycelial cakes with a diameter of 1 cm were punched at the edge of the colony using a sterile punch, placed in PDA liquid medium, and cultured in the dark for 7 days. Then, *Trichoderma guiyuanensis* spores were scraped off by adding a small amount of sterile water using the plate spreading method. The spore suspension was obtained by filtration through a sterile funnel, filtered through sterile filter paper, and then diluted to 1×10⁻⁶ spore concentration using a hemocytometer. 6 per mL.

[0047] The experiment included treatment and control groups. Each treatment group received 100 mL of the tested bacterial solution via root drenching around the stem base and foliar spraying, respectively. The control group received the same amount of sterile water via the same application method. Each treatment was replicated eight times. Seedling height and leaf number were measured before application and after 30 days of cultivation for each treatment, and the increase in height and leaf number was recorded. The weight of intact seedlings in each treatment group was then measured using an electronic balance with an accuracy of 0.01 g. The samples were then placed in a forced-air drying oven at 105℃ for 30 min to blanch, followed by drying at 65℃ to constant weight.

[0048] like Figure 8As shown, after one month of root drenching with *Trichoderma guiyuanensis* B10, the plant height and leaf number of garlic seedlings increased by 1.98 cm and 2.00 leaves, respectively, representing increases of 43.48% and 70.94% compared to the control, both significantly different from the control. This indicates that inoculation with this fungus effectively promotes the growth of garlic seedlings. Simultaneously, the fresh weight and dry weight of the inoculated seedlings were 30.29 g and 13.63 g, respectively, increasing by 34.20% and 36.85% compared to the control, both significantly different from the control, indicating that inoculation with this fungus can promote the increase of seedling biomass to a certain extent.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Trichoderma guiyuan ( Trichoderma guizhouese strain B10, characterized in that, The Guizhou Trichoderma B10 strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 3.25634.

2. The strain according to claim 1, characterized in that: The Trichoderma tuftini strain B27 has the ability to produce indoleacetic acid.

3. The application of the Guizhou Trichoderma B10 strain as described in claim 1 in the prevention and control of garlic fruit diseases and / or the promotion of garlic fruit growth.

4. The application according to claim 3, characterized in that: The disease affecting garlic cloves is garlic clove leaf spot.

5. The application according to claim 3, characterized in that: The pathogenic fungus causing the leaf spot disease of garlic cloves is *Neoprothiolane*. Neopestalotiopsis saprophytica ), Polychaete spp. ( Pestalotiopsis australasiae ), genus *Neocysporium* fungi ( Neofusicoccum parvum ), Staphylococcus spp. fungi ( Botryosphaeria fabicerciana ) and fungi of the genus *Metacarpa* ( Diaporthe sojae Any one of them.

6. The application according to claim 3, characterized in that: The application method for controlling garlic fruit diseases involves preparing Trichoderma Guizhouense B10 spores at a concentration of 1×10⁻⁶. 6 -1×10 8 The product, with a concentration of [number] cells / mL, is applied by foliar spraying for prevention and control.

7. The application according to claim 3, characterized in that: The application method to promote the growth of garlic cloves involves preparing Trichoderma guiyuan B10 spores at a concentration of 1×10⁻⁶. 6 -1×10 9 Products with a concentration of 1 unit / mL promote seedling growth when applied by root irrigation or foliar spraying.

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