Trichoderma atroviride SH-L1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to trichoderma atroviride SH-L1 and application thereof. The preservation number of the trichoderma atroviride SH-L1 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 41751. The invention also provides a microbial agent, which comprises trichoderma atroviride SH-L1 and / or fermentation liquor or metabolite of the trichoderma atroviride SH-L1. The trichoderma atroviride SH-L1 obtained by separation and screening can inhibit the growth of aspergillus flavus, inhibit the generation of aflatoxin or reduce the content of aflatoxin, promote the growth of peanut seedlings, improve the peanut yield and significantly inhibit peanut soil-borne bacteria, and has the advantages of being green, environmentally friendly, high in specificity, environmentally friendly and the like; peanut yield and quality safety can be guaranteed, and healthy development of the peanut industry is promoted.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a biocontrol strain, specifically a Trichoderma viride SH-L1 strain and its application. Background Technology

[0002] In recent years, due to continuous planting in major peanut-producing areas, soil-borne diseases such as peanut root rot, black rot, white mold, and fruit rot have occurred frequently, with the damage increasing year by year, severely affecting peanut yield and quality and causing huge losses. Soil-borne peanut diseases have become a major obstacle to the healthy and sustainable development of my country's peanut industry. Currently, the control of peanut soil-borne diseases mainly relies on the application of chemical agents combined with agricultural measures, but the effects are unsatisfactory. Moreover, the large amount of pesticides used in chemical control leads to excessively high control costs and serious pollution, with a considerable portion of peanuts and their products having seriously excessive pesticide residues. Therefore, environmentally friendly biological control measures are receiving increasing attention.

[0003] In addition to the damage caused by soil-borne diseases, peanuts are also highly susceptible to Aspergillus flavus during growth, harvesting, storage, and transportation. Aspergillus flavus Contamination by Aspergillus flavus. Aflatoxin, produced by Aspergillus flavus, is a class of highly toxic, carcinogenic, teratogenic, and mutagenic secondary metabolites. Peanuts are among the crops most susceptible to Aspergillus flavus infection, and aflatoxin has a very high affinity for peanuts. Aspergillus flavus infection and aflatoxin contamination begin before peanut harvest. Aspergillus flavus is widely present in the soil and can infect peanut kernels before harvest. High soil temperatures and drought can lead to the production of aflatoxin and contamination of peanut kernels. Kernels with low maturity or damage are far more likely to be contaminated with aflatoxin than well-matured, intact kernels. Among existing aflatoxin removal technologies, physical methods such as microwave radiation and ultrasonic treatment can degrade 60%-80% of AFB1 (aflatoxin B1) under laboratory conditions, but they are costly and have unstable effects, and efforts are still needed to adapt them to industrial production. Traditional chemical methods use drugs and oxidants (such as ozone and hydrogen peroxide), which can destroy the toxin structure, but there is a risk of chemical residues, which may harm the ecological balance and food safety. Biological methods, on the other hand, have obvious advantages.

[0004] Screening and isolating biocontrol strains that can inhibit soil-borne pathogens in peanuts, have a significant inhibitory effect on Aspergillus flavus, and efficiently degrade aflatoxin through biological control methods is of great theoretical and practical significance for ensuring peanut quality and safety and promoting the healthy development of the peanut industry. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems existing in the prior art, and to propose a Trichoderma viride SH-L1 strain and its application. This strain is a biocontrol strain that can significantly inhibit the main soil-borne pathogens of peanut, efficiently degrade aflatoxin, and promote peanut growth, thereby ensuring the quality and yield of the peanut industry.

[0006] The technical solution of this invention is: This invention provides a strain of Trichoderma viride ( Trichoderma atroviride The strain SH-L1 has the accession number CGMCC No. 41751.

[0007] The present invention also provides a microbial inoculant comprising the aforementioned Trichoderma viride SH-L1 and / or the fermentation broth or metabolites of the aforementioned Trichoderma viride SH-L1.

[0008] This invention provides the application of the aforementioned Trichoderma SH-L1 or the aforementioned microbial agent in inhibiting plant pathogens.

[0009] Furthermore, the plant is peanut, and the pathogens include Fusarium oxysporum, Cyclospora parasiticum, Sclerotium sclerotiorum, Neocaridia rubra, and Aspergillus flavus.

[0010] The present invention also provides the application of the aforementioned Trichoderma viride SH-L1 in the prevention and control of peanut root rot caused by Fusarium oxysporum, wherein Trichoderma viride SH-L1 can inhibit the sporulation of Fusarium oxysporum.

[0011] The present invention further provides the application of the aforementioned Trichoderma SH-L1 or the aforementioned microbial agent in inhibiting the formation of aflatoxin or reducing the content of aflatoxin.

[0012] This invention provides the application of the aforementioned Trichoderma viride SH-L1 or the aforementioned microbial inoculant in improving peanut yield.

[0013] Furthermore, the application methods include any one or more of the following: seed soaking, root irrigation, and spraying.

[0014] This invention provides the application of the aforementioned Trichoderma SH-L1 or the aforementioned microbial agent in promoting peanut growth.

[0015] Furthermore, the application methods include any one or more of the following: seed soaking, root irrigation, and spraying.

[0016] Information on the preservation of biological material samples: Trichoderma SH-L1, classified and named Trichoderma atrovirideIt was deposited on December 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41751.

[0017] The beneficial effects of this invention are: This invention isolates and screens biocontrol strains with significant antibacterial and growth-promoting effects. Simultaneously, it specifically screens biocontrol strains that have significant inhibitory effects on Aspergillus flavus and can efficiently degrade aflatoxin. The resulting Trichoderma viride SH-L1 can inhibit the growth of Aspergillus flavus, degrade existing aflatoxin, promote peanut seedling growth, and significantly inhibit soil-borne pathogens in peanuts. It has advantages such as being green and environmentally friendly, highly specific, and environmentally friendly, and meets the requirements of sustainable agricultural development. Attached Figure Description

[0018] Figure 1 Morphological observation of strain SH-L1; In the figure, SH-D3 is the morphology of SH-L1 colony after 3 days of growth, SH-D4 is the morphology of SH-L1 colony after 4 days of growth, SH-D5 is the morphology of SH-L1 colony after 5 days of growth, and SH-D14 is the morphology of SH-L1 colony after 14 days of growth.

[0019] Figure 2 This is a phylogenetic tree of strain SH-L1 constructed based on the rDNA-ITS sequence.

[0020] Figure 3 This study investigated the growth-promoting effect of strain SH-L1 on peanut seedlings. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0022] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0023] Example 1: Isolation and Screening of Biocontrol Trichoderma (1) Soil sample collection Peanut and root soil samples were collected from peanut fields in Kashgar Prefecture, Xinjiang Uygur Autonomous Region. The samples were then stored at 4℃.

[0024] (2) Test pathogens Five pathogens were used, as shown in Table 1 below. All pathogen strains used were preserved by the Disease Control Research Group of the Shandong Peanut Research Institute. Among them, the international standard Aspergillus flavus strain NRRL3357 was used.

[0025] Table 1. Five pathogens used and the resulting flower diseases.

[0026] (3) Isolation of biocontrol Trichoderma Fungi were isolated using the soil dilution method. A small amount of soil sample was taken and ground finely in a mortar. 10 g of the finely ground sample was weighed out and dissolved in 90 mL of sterile water. The mixture was shaken at 120 rpm for 10 min and allowed to stand for 5 min. Then, under aseptic conditions, a serial dilution was performed. 1 mL of the stock solution was transferred to a sterile test tube, 9 mL of sterile water was added, and the solution was shaken well to obtain 10 g of the stock solution. -1 Diluent, and so on, to obtain 10 -2 10 -3 10 -4 10 -5 Diluent. Using a pipette, 200 μL of soil suspensions of different concentrations were pipetted onto Martin's-Bangladesh Red agar (3 mL of 1% Bangladesh Red aqueous solution, 0.5 g of magnesium sulfate heptahydrate, 1.0 g of potassium dihydrogen phosphate, 5.0 g of peptone, and 10.0 g of glucose were thoroughly dissolved, then diluted to 1000 mL with distilled water, and 20 g of agar was added and heated to boiling. Before use, 0.3 mL of 1% streptomycin solution was added to every 100 mL of medium). The mixture was spread evenly, and each group was repeated three times. After standing for 20 min, the cultures were incubated upside down in a 25°C incubator. After 2 days, single colonies were picked for isolation and purification. Preliminary screening was performed based on morphological characteristics, and the isolated strains were stored at 4°C.

[0027] (4) Preliminary screening of biocontrol Trichoderma The isolated Trichoderma strains were initially screened using the plate confrontation method.

[0028] Pathogen and Trichoderma mycelial discs were inoculated onto PDA plates, with the pathogen discs placed in the center and the Trichoderma mycelial discs placed on either side, approximately 2 cm from the edge, with the hyphae facing downwards. This was repeated three times. A control group with pathogen inoculated in the center served as the control. The plates were incubated in the dark at 25°C. After 5 days, the colony radius of the pathogen was measured, and antagonistic strains with higher inhibition rates were selected for further research. The colony radius of the treatment groups was calculated using the relative radii of the two colonies.

[0029] Confrontational inhibition rate = (control colony radius - treated colony radius) / control colony radius × 100%.

[0030] Through preliminary screening, an antagonistic bacterium that showed good resistance to four major soil-borne diseases of peanut (root rot, black rot, white mold, and fruit rot) and Aspergillus flavus was isolated from soil samples and named SH-L1.

[0031] Example 2 Identification of biocontrol Trichoderma 1. Morphological observation of colonies The Trichoderma SH-L1 strain, which passed the initial screening, was activated and transferred to PDA solid medium. After being incubated in a constant temperature incubator at 25°C for 48 hours, the colony characteristics of the strains cultured on the solid medium were observed daily, including shape, size, color, texture, and spore morphology.

[0032] The results are as follows Figure 1 As shown, SH-L1 colonies are initially white, round, and cotton-like. SH-L1 grows rapidly, quickly producing pale green spores from the center of the colony. The center then turns green, and eventually the entire colony becomes green. Concentric rings often form on the surface of the colony.

[0033] 2. Molecular biological identification of Trichoderma (1) DNA extraction using CTAB After activating strain SH-L1 on a plate for 3 days, it was re-inoculated onto a PDA. The mycelia cultured for 7 days were collected and placed in a sterilized mortar (the mortar was pre-cooled before use). Liquid nitrogen was quickly poured in and the mixture was ground into powder. The powder was then transferred to a 1.5 mL centrifuge tube and 700 μL of 2% CTAB extraction solution was quickly added. Place the centrifuge tubes in a 65°C water bath and invert the centrifuge tubes every 5 minutes or so (a total of 5 times) until the solution is completely dissolved. After dissolving, add 700 μL of chloroform, shake vigorously to mix, centrifuge at 12000 rpm for 15 min, and transfer the supernatant to a new 1.5 mL centrifuge tube; dissolve again, add chloroform, and repeat the step. Add an equal volume of isopropanol to a new 1.5 mL centrifuge tube, gently shake up and down until DNA flocculent precipitate forms, and place it in a -20°C freezer for 20 min. After removing the flocculent precipitate, centrifuge at 12000 rpm for 15 min at room temperature and discard the supernatant. Wash twice with 500 μL of 70% ethanol, centrifuge at 7500 rpm for 5 min and discard the supernatant. Air dry the DNA. After air drying, the DNA precipitate will be milky white. Add 35 μL of preheated ddH2O at 65℃ to dissolve completely, and measure the concentration using a DNA concentration analyzer. Once the concentration reaches the target, store in a -20℃ freezer.

[0034] (2) PCR amplification After extracting total DNA from strain SH-L1, PCR amplification was performed using universal primers ITS1 and ITS4, and the amplified products were sequenced. The specific procedures are as follows: The PCR amplification primers were ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') (SEQ ID No. 2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (SEQ ID No. 3).

[0035] The PCR reaction system consisted of a 20 μl system, including 10 μL of 2×Taq Master Mix, 1 μL of ITS1, 1 μL of ITS4, 1 μL of DNA, and 7 μL of ddH2O. Before running the PCR instrument, the samples were centrifuged in a small centrifuge to mix and aggregate the samples. The PCR amplification program was as follows: pre-denaturation, 95℃ for 5 min; denaturation, 95℃ for 30 sec; annealing, 56℃ for 30 sec; extension, 72℃ for 40 sec; final extension, 72℃ for 10 min; and incubation at 4℃, cycling.

[0036] (3) Agarose gel electrophoresis detection and sequencing 5 μL of PCR product was placed in a 1% agarose gel for electrophoresis. Compared with Marker2000, the electrophoresis voltage was 120V, and the results were viewed in a gel imaging system after 20 min. The PCR product with bright, single bands was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID No. 1.

[0037] The obtained sequences were aligned using BLAST on the NCBI website and a phylogenetic tree was constructed, such as... Figure 2 As shown. The results indicate that strain SH-L1 is related to Trichoderma viride (…). Trichoderma atroviride The nucleotide sequences of ) are highly homologous, and the results of phylogenetic tree construction and homology analysis show that ( Figure 2 ), strain SH-L1 and Trichoderma viride ( Trichoderma atroviride These species form a separate branch and are the most closely related in evolution, reflecting their closest phylogenetic relationship. Based on the morphological characteristics of strain SH-L1, strain SH-L1 was identified as *Trichoderma viride*. Trichoderma atroviride .

[0038] Trichoderma SH-L1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41751.

[0039] Example 3: Biocontrol effect of Trichoderma viride SH-L1 against pathogens Plate confrontation assay of Trichoderma viride SH-L1 with five pathogens: The plate confrontation method was used. Using a punch, 0.5 cm diameter mycelial discs were taken from the culture media of *Trichoderma viride* SH-L1 activated for 3 days and five pathogens (*Fusarium oxysporum*, *Cyclocarya parasiticum*, *Sclerotium sclerotiorum*, *Neococcus spp.*, and *Aspergillus flavus*). The two types of mycelial discs were placed at opposite ends of a straight line connecting the centers of the two PDA plates, 5 cm apart. A control was used where only pathogens were placed at the center of the petri dish. The plates were incubated at 25°C. Each treatment was repeated three times. The antagonistic and inhibitory effects of strain SH-L1 on the five pathogens were observed daily. After 5 days, the colony diameter of the pathogens was measured, and the inhibition rate of mycelial growth by strain SH-L1 on the five pathogens was calculated.

[0040] Plate confrontation tests showed that SH-L1 had significant inhibitory effects on all five pathogens, as shown in Table 2. Analysis of inhibition rates revealed that SH-L1 exhibited the best inhibitory effect against *Fusarium oxysporum*, reaching 75.38%, followed by *Cyclophorus parasiticus* with an inhibition rate of 75.00%. The inhibition rates against *Sclerotium sclerotiorum* and *Neococcus rubrum* were also above 70%, and the inhibition rate against *Aspergillus flavus* reached 68.66%.

[0041] Table 2. Inhibitory effect of plate-confrontation SH-L1 on five pathogens.

[0042] Example 4: Inhibitory effect of Trichoderma viride SH-L1 on sporulation of Fusarium oxysporum Six SH-L1 mycelial cakes (0.5 cm in diameter) were picked up with a toothpick and transferred to sterilized PDB culture medium under aseptic conditions. After incubation at 25°C and 180 r / min for 5 days in a constant temperature shaking incubator, the mycelium was first filtered off using eight layers of gauze. The resulting filtrate was then centrifuged at 4000 r / min for 10 min at 8°C. The supernatant was filtered through a cell filter to obtain the SH-L1 fermentation broth. The SH-L1 fermentation broth and PDB culture medium were mixed at ratios of 40%, 60%, 80%, and 100% (stock solution) to prepare different concentrations of SH-L1 fermentation broth in small conical flasks. A control group containing only PDB culture medium was used. Each group had two replicates. Equal amounts of *Fusarium oxysporum* mycelial cakes were then added to the different concentrations of fermentation broth and incubated at 25°C and 180 r / min for 5 days in a constant temperature shaking incubator to obtain *Fusarium oxysporum* culture. The mycelium was filtered out using eight layers of gauze to obtain Fusarium oxysporum spore solutions of different concentrations. Samples were taken and counted using a hemocytometer to calculate the sporulation inhibition rate.

[0043] Sporulation inhibition rate = (Sporulation yield in control group - Sporulation yield in treatment group) / Sporulation yield in control group × 100% The inhibitory effect of *Trichoderma viride* SH-L1 on sporulation of *Fusarium oxysporum* is shown in Table 3. Compared with *Fusarium oxysporum* cultured in PDB medium, high concentrations of fermentation broth significantly inhibited sporulation of *Fusarium oxysporum*, with the undiluted solution showing the most significant inhibitory effect. The inhibitory effect decreased with decreasing concentration. Table 3 shows that the undiluted solution (100%) had the most significant inhibitory effect on sporulation of *Fusarium oxysporum*, with an inhibition rate of 97.64%. The 80%, 60%, and 40% undiluted solutions showed inhibitory effects of 88.85%, 41.55%, and 29.28% on sporulation of *Fusarium oxysporum*, respectively.

[0044] Table 3. Inhibitory effect of strain SH-L1 on sporulation of Fusarium oxysporum

[0045] Example 5: Biocontrol effect of Trichoderma viride SH-L1 on soil-borne diseases of peanut in pots and field disease nurseries. 1. Pot experiment: Six mycelial cakes (0.5 cm in diameter) of Trichoderma SH-L1 were picked up with toothpicks and transferred to sterilized PDB culture medium under aseptic conditions. The culture stock solution was prepared by shaking in a constant temperature shaking incubator at 25℃ and 180 r / min for 5 days.

[0046] The soil was air-dried and sterilized under high temperature and pressure to obtain sterile soil. Perlite was added and mixed thoroughly before being divided into flowerpots. Healthy peanut seeds of uniform size (e.g., Huayu 33) were sown in the flowerpots, with a soil covering thickness of about 2-3 cm, 3 seeds per pot. After sowing, the pots were placed in a greenhouse and watered every 3 days. Five days after germination, peanuts of uniform size and growth were selected from each treatment. Five oat grains infested with peanut root rot fungi, black rot fungi, white mold fungi, and fruit rot fungi were inoculated around each peanut plant. Simultaneously, 30 mL of a 10-fold dilution of *Trichoderma* was poured into each peanut pot. A 50% carbendazim solution at 800 times dilution was used as a control, and water was used as a blank control. Four pots were used for each treatment, with three plants per pot. After 25 days of infection, the number of infected peanut plants and the disease index were counted, and the control effect of the biocontrol *Trichoderma* fungus was calculated.

[0047] 2. Field Trial: Diseased ponds for peanut root rot, black rot, white mold, and fruit rot were conducted at the Laixi Experimental Farm of the Shandong Peanut Research Institute. Each pond was 1.5 m long and 0.8 m wide, with a row spacing of 40 cm and a plant spacing of 16.7 cm. Trichoderma viride was used in triplicate. A 50% carbendazim solution diluted 800 times was used as a control, and water was used as a blank control. Root drenching with Trichoderma viride SH-L1 fermentation liquid was applied at sowing time and 30 days after sowing, with approximately 30 mL of a 10-fold diluted fermentation liquid applied per plant each time. Other field management practices were the same as normal production. Disease incidence was assessed after 75 days.

[0048] Peanut strains treated with fermentation broth of *Trichoderma viride* SH-L1 showed significant control effects against peanut root rot, black rot, white mold, and fruit rot in both indoor pot and field trials. SH-L1 achieved a control efficacy of 63.42% against root rot in indoor potted peanuts and 58.82% in the field (Table 4); 61.90% against black rot in indoor potted peanuts and 61.73% in the field (Table 5); 52.27% against white mold in indoor potted peanuts and 57.44% in the field (Table 6); and 62.79% against fruit rot in indoor potted peanuts and 58.06% in the field (Table 7). The control efficacy of SH-L1 against peanut root rot, black rot, white mold, and fruit rot was higher than that of the control fungicide, carbendazim.

[0049] Table 4. Biocontrol effect of strain SH-L1 against peanut root rot

[0050] Table 5. Biocontrol effect of strain SH-L1 against peanut black rot

[0051] Table 6. Biocontrol effect of strain SH-L1 against peanut white mold disease

[0052]

[0053] Table 7. Biocontrol effect of strain SH-L1 against peanut fruit rot.

[0054] Example 6: Relative control effect of Trichoderma viride SH-L1 on Aspergillus flavus infection and toxin content in peanuts. Pond planting experiment: Six SH-L1 mycelial cakes (0.5 cm in diameter) were picked up with toothpicks and transferred to sterilized PDB culture medium under aseptic conditions. The culture stock solution was prepared by shaking in a constant temperature shaking incubator at 25°C and 180 r / min for 5 days.

[0055] The experiment was conducted in a peanut aflatoxin-infected pond at the Laixi Experimental Farm of the Shandong Peanut Research Institute. The pond was 1.5 m long and 0.8 m wide, with a row spacing of 40 cm and a plant spacing of 16.7 cm. The experiment was repeated three times, with water as a blank control. SH-L1 fermented liquid was applied to the roots at sowing time and 30 days after sowing, with approximately 30 mL of a 10-fold diluted fermented liquid applied to each plant each time. Other field management practices were the same as in normal production. After peanut harvest, the infection rate of Aspergillus flavus in peanut shells and kernels, as well as the aflatoxin content in peanut kernels, were investigated to calculate the relative control effect.

[0056] (1) Infection rate of Aspergillus flavus in peanut shells and kernels Twenty peanut pods with two kernels were taken from each plot. The pods were broken open, and half of the shell and one peanut kernel were taken. The shells and kernels were disinfected with 1% sodium hypochlorite for 3 minutes and then inoculated onto PDA medium. The samples were incubated in the dark at 28°C for 6 days. The samples in which Aspergillus flavus was isolated were then investigated. The infection rate of Aspergillus flavus in peanut shells and kernels was the percentage of samples in each treatment in which Aspergillus flavus could be isolated from the total samples.

[0057] (2) Aflatoxin content in peanut kernels Peanut kernels were crushed to pass through a 20-mesh sieve. Aflatoxin content was detected using an indirect enzyme-linked immunosorbent assay (ELISA) method, following the instructions for the Huaan Maike Aflatoxin Total Reagent Kit.

[0058] The specific operating steps are as follows: Grind the peanuts in a grinder for 2 minutes until they can pass through a 20-mesh sieve. Weigh 5 g of the peanuts using a balance and place them in a 50 mL stoppered conical flask. Add 40% methanol to 25 mL of the flask and mix thoroughly. After shaking for 15 min at 28℃ and 200 r / min in an incubator, centrifuge at 5000 r / min for 4 min, take 2 mL of the supernatant, add 10 mL of deionized water, and shake well by hand. The aflatoxin content in the samples was detected using the Total Aflatoxin ELISA Kit (Beijing Huaan Maike Biotechnology Co., Ltd.): The kit was removed from the 4℃ refrigerator and left at room temperature for 30 min. Before use, the kit was thoroughly shaken to avoid air bubbles. An appropriate amount of microplate was taken based on the quantity of peanut extract. The remaining microplates were stored in an aluminum foil bag with desiccant at 4℃. The standards and peanut extract samples were numbered and recorded according to the microplate number. 50 µL of peanut extract sample or reagent standard, 50 µL each of aflatoxin enzyme-labeled reagent and aflatoxin anti-aflatoxin reagent were added sequentially. The mixture was gently shaken to ensure homogeneity. The cover was then placed at 25℃ in the dark for 30 min. The cover was opened, the reagent in the wells was patted dry, and 300 µL of washing buffer was added. The wells were washed four to five times, with a 10-second interval between each wash. After patting dry, ensuring there were no residual air bubbles in the microplate, 50 µL each of substrate solution A and substrate solution B were added, gently mixed, and the cover was placed at 25℃ in the dark for 25 minutes. min; after the time is up, open the cover membrane and add 50µL of stop solution.

[0059] The OD value of each well in the microplate was measured using a dual-wavelength microplate reader (iMark™, ​​BIO-RAD, USA) at a wavelength of 450 nm and a reference wavelength of 630 nm. Data were analyzed using Ridasoft Win.NET software, and the concentration of aflatoxin in each peanut extract sample was calculated based on the standard curve.

[0060] Table 8 shows the inhibitory effects of treating peanuts with fermentation broth of Trichoderma viride SH-L1 on Aspergillus flavus infection and toxin content. The infection rate of peanut shells treated with SH-L1 was 63.33%, while the infection rate of the control shells was 100%, indicating a relative control effect of 36.67% against Aspergillus flavus infection. The infection rate of peanut kernels treated with SH-L1 was 18.33%, while the infection rate of the control kernels was 53.33%, indicating a relative control effect of 65.63% against Aspergillus flavus infection. The aflatoxin content in peanut kernels treated with SH-L1 was 11.2 μg / kg, while the aflatoxin content in the control kernels was 57.6 μg / kg, indicating a relative control effect of 80.56% against Aspergillus flavus infection.

[0061] Table 8. Inhibitory effect of Trichoderma viride SH-L1 on Aspergillus flavus infection and toxin content in peanuts.

[0062] Example 7: Growth-promoting effect of Trichoderma viride SH-L1 on peanut seedlings Six SH-L1 mycelial cakes (0.5 cm in diameter) were picked up with a toothpick and transferred to sterilized PDB culture medium under aseptic conditions. The culture stock solution was prepared by shaking in a constant temperature shaking incubator at 25°C and 180 r / min for 5 days.

[0063] The soil was air-dried and sterilized under high temperature and pressure to obtain sterile soil. Perlite was added and mixed thoroughly before being divided into flowerpots. Healthy, uniform-sized peanut seeds (Huayu 33 variety) were sown in the flowerpots, covered with soil to a thickness of about 2-3 cm, with 3 seeds per pot. After sowing, the pots were placed in a greenhouse and watered every 3 days. Five days after germination, peanuts of uniform size and growth were selected from each treatment, and 30 mL of a 10-fold dilution of shrub mold fermentation solution was poured into each pot. A control group was prepared by watering only with an equal volume of sterilized PDB culture solution. Each treatment had 4 pots, with 3 plants per pot.

[0064] After 30 days of continued cultivation, the fresh and dry weights of each plant were measured to investigate the growth-promoting effect of SH-L1. The growth-promoting effect was expressed as the growth-promoting rate.

[0065] Growth stimulating rate (%) = (Treatment group weight - Control group weight) / Control group weight × 100% The effects of strain SH-L1 on peanut seedling growth from Figure 3 It can be seen that, compared with the blank control, peanuts treated with the fermentation broth of strain SH-L1 showed a significant growth advantage. As shown in Table 9, after 30 days of irrigation with the fermentation broth, the SH-L1 treatment had a significant growth-promoting effect on both dry and fresh weight of peanut seedlings, with growth promotion rates of 70.00% and 67.24%, respectively; in addition, the number of rhizobia in the roots of peanut seedlings treated with SH-L1 fermentation broth increased significantly.

[0066] Table 9. Growth-promoting effect of strain SH-L1 on peanut seedlings

[0067] Example 8: Effect of Trichoderma viride SH-L1 on peanut yield The experiment was conducted at the Laixi Experimental Farm of the Shandong Peanut Research Institute. A randomized block design was used, with each peanut plot measuring 50 m². 2 The experiment was repeated four times, with water serving as a blank control. Root irrigation with SH-L1 fermentation broth was performed at sowing time and 30 days after sowing, with each plant receiving approximately 30 mL of the 10-fold diluted fermentation broth. Other field management practices were the same as in normal production.

[0068] Before peanut harvest, 20 m³ of samples were taken from each treatment. 2 The samples were dried, weighed, and the yield was calculated. The experimental results are shown in Table 10. The average yield of the SH-L1 treatment was converted to a yield of 236.83 kg per mu (approximately 0.067 hectares), which was 16.28% higher than that of the water control (203.67 kg per mu).

[0069] Table 10 Effects of SH-L1 on peanut yield in the field

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dark green Trichoderma ( Trichoderma atroviride ) bacterium SH-L1, characterized in that, and has a preservation number of CGMCC No. 41751.

2. A microbial inoculant, characterized in that, The fermentation broth or metabolite of the Trichoderma atroviride SH-L1 of claim 1.

3. The Trichoderma atroviride SH-L1 of claim 1 or the microbial inoculant of claim 2 for use in inhibiting plant pathogenic fungi.

4. Use according to claim 3, characterized in that, The plant is peanut, and the pathogenic fungi include Fusarium oxysporum, Sarocladium parasiticum, Sclerotium rolfsii, Neocosmospora vasinfecta, and Aspergillus flavus.

5. The use of Trichoderma atroviride SH-L1 according to claim 1 for the control of peanut root rot caused by Fusarium oxysporum, characterized in that, The Trichoderma atroviride SH-L1 can inhibit the sporulation of Fusarium oxysporum.

6. The Trichoderma atroviride SH-L1 of claim 1 or the microbial inoculant of claim 2 for use in inhibiting the production of aflatoxins or reducing the content of aflatoxins.

7. The Trichoderma atroviride SH-L1 of claim 1 or the microbial inoculant of claim 2 for use in increasing the yield of peanuts.

8. Use according to claim 7, characterized in that, The application methods include any one or several of seed soaking, root irrigation, and spraying.

9. The Trichoderma atroviride SH-L1 of claim 1 or the microbial inoculant of claim 2 for use in promoting the growth of peanuts.

10. Use according to claim 9, characterized in that, The application methods include any one or several of seed soaking, root irrigation, and spraying.

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