Application of trichoderma asperellum in inhibition of mulberry anthracnose bacteria and prevention and treatment of mulberry anthracnose

Abstract

The invention discloses application of trichoderma asperellum to inhibition of mulberry anthracnose bacteria and prevention and treatment of mulberry anthracnose, the trichoderma asperellum is trichoderma asperellum TR41, is identified as trichoderma asperellum, is preserved in the Guangdong Microbial Culture Collection Center, has the preservation number of GDMCC No.64887, and is preserved for July 18, 2024. The invention further discloses a preparation method of the trichoderma asperellum, and the application of the trichoderma asperellum to the inhibition of mulberry anthracnose bacteria and the prevention and treatment of mulberry anthracnose. The trichoderma asperellum TR41 has good inhibition and antagonism effects on mulberry anthracnose, and can be used for preventing and treating mulberry anthracnose and promoting growth of mulberry. In addition, the trichoderma asperellum TR41 has a growth promoting effect on the growth of mulberry seedlings, and the root activity and the chlorophyll content can be improved. An efficient and environment-friendly microbial resource is provided for biological prevention and control of mulberry anthracnose, traditional chemical pesticides can be replaced, and environmental pollution is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microbial control, and in particular to application of Trichoderma aspergillus in inhibiting mulberry anthracnose pathogens and preventing and controlling mulberry anthracnose. Background Art

[0002] Mulberry trees are an important economic crop and ecological species, playing a vital role in agricultural production and ecosystems. Their leaves are not only the primary food source for silkworms but also serve as a high-quality feed resource for livestock. Mulberry leaves are also rich in bioactive substances such as flavonoids, which are beneficial to the human body. They exhibit potential applications in biomedicine, including antioxidant, hypoglycemic, and immunomodulatory properties. Anthracnose is a major fungal disease in mulberry production. The pathogen typically invades from the underside of young leaves, then germinates, forming bud tubes that invade the epidermis of mulberry leaves. This disease causes symptoms in mature leaves, disrupting the physiological metabolism of the tree and ultimately leading to premature aging and abnormal leaf shedding. Severe cases can cause systemic plant wilting, resulting in a 28-70% decrease in mulberry leaf yield, directly impacting both yield and quality. High temperatures and high humidity favor the occurrence and spread of the disease, with rainfall and humidity being key factors influencing its prevalence. Therefore, the occurrence of anthrax severely impacts the economic benefits of the sericulture industry.

[0003] Currently, the control of mulberry anthracnose mainly relies on chemical pesticides. Long-term use causes severe environmental pollution, excessive pesticide residues in soil, and increased pesticide resistance, seriously impacting the economic benefits of the mulberry industry. Therefore, it is imperative to explore biological and eco-friendly pest control technologies. With increasing attention to environmental protection and food safety, the drawbacks of traditional chemical control methods have become more prominent. Biological control is currently a popular control method. While it can effectively prevent and control the disease, it can also effectively reduce pesticide residues, reducing the difficulty of control caused by disease resistance. Therefore, it is necessary to use environmentally friendly antagonistic microorganisms to replace pesticides to control mulberry anthracnose.

[0004] Trichoderma spp., belonging to the subphylum Deuteromycotinae, class Hypomycetes, orders Acanthosporales, family Acanthosporaceae, is one of the most widely used and important biocontrol fungi. As a common commercial biocontrol agent, numerous studies have demonstrated that Trichoderma can induce systemic resistance in plants, exerting competitive, antibiotic, and hyperparasitic effects on pathogens, effectively combating major plant diseases such as root rot and gray mold. Trichoderma grows rapidly and is highly adaptable to diverse environments. It can compete with pathogens for space in the plant rhizosphere, controlling their infestation. Trichoderma's hyperparasitic effect on pathogens is its most direct biocontrol agent, producing a large number of secondary metabolites that parasitize and kill pathogens, enhancing plant disease resistance. In the prior art, Trichoderma harzianum, T. koningiopsis, T. longibrachiatum, and T. asperellum are commonly used as biological control agents for various crops, fruits, and vegetables. However, the use of Trichoderma asperellum for the control of mulberry anthracnose has not been reported. Therefore, exploring the antibacterial effect of Trichoderma against mulberry anthracnose and its mechanism of inhibition could provide new insights into the development of biocontrol agents for the control of mulberry anthracnose in production. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a use of Trichoderma aspergillus in inhibiting mulberry anthracnose pathogens and preventing and controlling mulberry anthracnose. The present invention provides Trichoderma aspergillus TR41, which has a broad-spectrum antibacterial activity. The Trichoderma aspergillus TR41 strain has a good antagonistic effect on the mulberry anthracnose pathogen (Colletotrichum fructicola), with an inhibition rate of more than 78.5047%. It can be used for the prevention and control of mulberry anthracnose and has broad application prospects.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses an application of Trichoderma asperellum in inhibiting anthrax pathogens. The Trichoderma asperellum is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum. It has been deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 64887. The deposit date is July 18, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, No. 59, Compound 100, Xianlie Middle Road, Guangzhou, Postal Code: 510070.

[0007] Wherein, the anthracnose pathogen is mulberry anthracnose pathogen (Colletotrichum fructicola) or strawberry anthracnose pathogen (Colletotrichum siamense).

[0008] Among them, the Trichoderma aspergillus TR41 is used in inhibiting or antagonizing the pathogen of mulberry anthracnose.

[0009] Among them, the Trichoderma aspergillus TR41 can not only significantly inhibit the growth of anthrax fungi, but also significantly inhibit the biological enzyme and protein activities of anthrax fungi.

[0010] The Trichoderma aspergillus TR41 inhibits the hyphae growth of anthrax fungi and inhibits the activities of peroxidase (POD), superoxide dismutase (T-SOD) and chitinase in anthrax fungi.

[0011] Among them, the fermentation product or bacterial agent of Trichoderma aspergillus TR41 is used to inhibit or antagonize the pathogen of mulberry anthracnose.

[0012] Among them, the preparation method of the Trichoderma aspergillus TR41 fermentation product or bacterial agent comprises activating the Trichoderma strain TR41 in PDA culture medium, culturing it upside down in a constant temperature incubator, preparing a conidia suspension, fermenting it in PDB culture medium, and filtering the fermentation liquid to obtain the filtrate to obtain active Trichoderma fermentation liquid.

[0013] The application of Trichoderma asperellum in the prevention and treatment of mulberry anthracnose of the present invention is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum and has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No. 64887 and a deposit date of July 18, 2024.

[0014] The application of Trichoderma asperellum TR41 in promoting the growth of mulberry trees described in the present invention is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum (Trichoderma asperellum), has been deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No. 64887, and a preservation time of July 18, 2024.

[0015] Wherein, the promoting of mulberry tree growth includes increasing root activity and chlorophyll content.

[0016] The Trichoderma TR41 strain described in the present invention is isolated from the rhizosphere soil of a healthy mulberry tree, and is identified as Trichoderma asperellum TR41 through morphological and molecular biological identification.

[0017] Furthermore, the Trichoderma aspergillus TR41 is used to inhibit or antagonize the pathogen of mulberry anthracnose. The Trichoderma aspergillus TR41 inhibits the growth of anthracnose hyphae and slows down its growth rate.

[0018] Furthermore, through the plate confrontation antibacterial test, it was measured that the inhibition rate of Trichoderma astragali TR41 against mulberry anthracnose was as high as 78.5047%. Its active fermentation liquid can not only significantly inhibit the growth of anthracnose, but also significantly inhibit the biological enzyme and protein activity of anthracnose.

[0019] The invention provides a highly effective biocontrol Trichoderma aspergillus strain TR41 for mulberry anthracnose, which has no effect on the growth of mulberry seedlings, has strong antibacterial activity, and is a safe, highly effective and excellent biocontrol microbial resource.

[0020] This study isolated and purified a Trichoderma strain from the root secretions of healthy mulberry trees using a dilution coating method. Morphological and molecular biological identification revealed that the strain, named TR41, was identified. A plate-based antibacterial assay demonstrated that the strain exhibited significant inhibitory activity against Colletotrichum mulberry. Furthermore, by measuring the effects of TR41's active fermentation broth on the enzyme and protein activities of Colletotrichum mulberry, the authors conducted a preliminary investigation into the TR41 strain's antibacterial mechanism. This study aims to elucidate the TR41 strain's antibacterial mechanism and provide a scientific basis for the development of eco-friendly microbial-derived agents for the prevention and treatment of mulberry anthracnose.

[0021] The method for isolating and identifying Trichoderma aspergillus TR41 described in the present invention comprises the following steps:

[0022] (1) Samples were taken from the root soil of healthy mulberry trees, and the strains in the rhizosphere soil were isolated and purified using the dilution coating method.

[0023] (2) The genome of TR41 was extracted using the CTAB method, and a phylogenetic analysis was constructed based on the ITS gene sequence using molecular biological methods.

[0024] (3) In the present invention, the morphological characteristics of the Trichoderma spinulosa TR41 are: the colony is green, the central spores are densely mature, the edges are light green with white velvety hyphae, the aerial hyphae are well developed, the hyphae grow rapidly, are creeping, and flat. After culturing at a constant temperature of 28°C for 3 days, the hyphae are distributed throughout the plate. The colony is large, nearly round, green, and has a dense carpet-like surface. A large number of conidia are produced. The conidia are smooth, round or narrow oval conidia, and are light or green. The conidiophores have septa, vertical opposite branches, and the tips of the terminal branches are thin and slightly curved, with conidial clusters at the tips.

[0025] The present invention also includes the antibacterial mechanism of Trichoderma aspergillus TR41 against mulberry anthrax, which includes the plate standoff antibacterial effect of TR41 on anthrax, the influence of active fermentation liquid on the growth of anthrax, and the influence on enzyme activity and protein activity in anthrax.

[0026] As a feasible biocontrol agent, the Trichoderma aspergillus TR41 described in the present invention has no inhibitory effect on seed germination, plant growth rate, plant seedling root activity and chlorophyll content in plant seedling leaves, and has a certain growth-promoting effect.

[0027] The present invention discovered a strain of Trichoderma, TR41, that exhibits excellent antagonistic effects against mulberry anthracnose. Morphological and molecular biological identification confirmed its membership in the genus Trichoderma aspergillus. TR41 also demonstrated an antibacterial activity of 78.5047% against the fungus and inhibited the activity of enzymes and proteins within the fungus, demonstrating its antibacterial mechanism. Furthermore, TR41 does not affect the growth of mulberry trees, ensuring its safety as a biocontrol strain.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] The present invention isolated and screened Trichoderma aspergillus TR41, which is first reported to have a strong inhibitory and antagonistic effect on mulberry anthracnose, and can be used to prevent and treat mulberry anthracnose. The present invention's Trichoderma aspergillus TR41 has an inhibition rate of up to 78.5047% against mulberry anthracnose. Its active fermentation broth not only significantly inhibits the growth of mulberry anthracnose, but also inhibits the growth and activity of strawberry anthracnose, significantly reducing the activity of biological enzymes and proteins.

[0030] The Trichoderma aspergillus TR41 of the present invention can be used to prepare a novel anthrax biocontrol agent. It does not inhibit seed germination or plant growth rate, effectively eliminating the soil pollution caused by traditional anthrax chemical agents, and is highly effective and environmentally friendly. The fermentation preparation or inoculum of the Trichoderma aspergillus TR41 of the present invention can be produced using fermentation equipment commonly used in the fermentation industry, and has the advantages of low production cost, ease of use, and good antibacterial and control effects. The present invention is of great significance for reducing chemical pesticide residues and protecting the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the morphological characteristics of the Trichoderma spinulosa TR41 strain on PDA culture medium in the present invention, wherein A represents the colony morphology, B and C represent the microstructure, wherein B represents conidia and C represents conidiophores.

[0032] Figure 2 This is a gel image of ITS gene sequence amplification in one embodiment of the present invention.

[0033] Figure 3 This is a phylogenetic tree of the TR41 strain constructed based on the ITS gene sequence in one of the embodiments of the present invention.

[0034] Figure 4This is a diagram showing the antagonistic effect of Trichoderma aspergillus TR41 on mulberry anthracnose fungus Cm-ZJ-1 and an inhibition rate curve in one of the examples of the present invention.

[0035] Figure 5 This is the growth rate determination of Trichoderma aspergillus TR41 and Colletotrichum mulberrye Cm-ZJ-1 in one of the examples of the present invention.

[0036] Figure 6 This is the effect of Trichoderma aspergillus TR41 on the peroxidase (POD) activity of Colletotrichum mulberrye Cm-ZJ-1 in one of the examples of the present invention.

[0037] Figure 7 This is a curve showing the effect of Trichoderma aspergillus TR41 on the total superoxide dismutase (T-SOD) activity of Colletotrichum mulberryii Cm-ZJ-1 in one of the examples of the present invention.

[0038] Figure 8 This is the effect of Trichoderma aspergillus TR41 on the chitinase activity of Colletotrichum mulberrye Cm-ZJ-1 in one of the examples of the present invention.

[0039] Figure 9 This is a curve showing the antibacterial effect of the active fermentation liquid of Trichoderma aspergillus TR41 on mulberry anthracnose fungus Cm-ZJ-1 in one of the examples of the present invention.

[0040] Figure 10 This is the effect of the soluble protein content of Colletotrichum mulberryii Cm-ZJ-1 after treatment with Trichoderma aspergillus TR41 in one of the embodiments of the present invention.

[0041] Figure 11 This is the effect of Trichoderma aspergillus TR41 treatment on the MDA content of Colletotrichum mulberrye Cm-ZJ-1 in one of the examples of the present invention.

[0042] Figure 12 This is the effect of Trichoderma aspergillus TR41 on the root activity of mulberry seedlings in one of the examples of the present invention.

[0043] Figure 13 This is the effect of Trichoderma aspergillus TR41 on the chlorophyll content of mulberry seedlings in one of the examples of the present invention.

[0044] Figure 14 This is one of the examples of the present invention, in which the heavy parasitism of Trichoderma aspergillus TR41 against Colletotrichum mulberrye Cm-ZJ-1 was demonstrated.

[0045] Figure 15 This is the antibacterial effect of the active fermentation liquid of Trichoderma aspergillus TR41 on Colletotrichum truncatum HNRY-9 in one of the examples of the present invention.

[0046] Figure 16This is a curve showing the effect of the active fermentation broth of Trichoderma aspergillus TR41 on the total superoxide dismutase (T-SOD) activity of Colletotrichum truncatula HNRY-9 in one embodiment of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and examples.

[0048] Colletotrichum fructicola Cm-ZJ-1 is a wild-type mulberry anthracnose fungus. (Biological Characteristics and Fungicide Screening of Colletotrichum fructicola Causing Mulberry Anthracnose, Microorganisms 2024, 12, 2386) was provided by Jiangsu University of Science and Technology.

[0049] Colletotrichum spp. HNRY-9 is a wild-type Colletotrichum spp., which was donated by Jiangsu Agricultural and Forestry Vocational Technical College and provided by Jiangsu University of Science and Technology.

[0050] Mulberry seeds: Fengchi mulberry seeds are preserved by the Mulberry Science Research Center of Jiangsu University of Science and Technology / Chinese Academy of Agricultural Sciences.

[0051] Sources of experimental reagents and equipment:

[0052] Experimental reagents: Fresh potatoes, polypeptone, sucrose, agar powder, CTAB, β-mercaptoethanol, EDTA, phenol, chloroform, anhydrous ethanol and isoamyl alcohol, Coomassie brilliant blue, thiobarbituric acid and other reagents were purchased from Sinopharm Group, and RNaseA, Tris-HCl and other reagents were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0053] The peroxidase (POD) enzyme activity kit, plant root activity kit, and plant chlorophyll content kit were provided by Shanghai Yuanye Biotechnology Co., Ltd., POD item number: R30312-50T, SOD item number: A001-1-2.

[0054] The kit for determining the total superoxide dismutase (T-SOD) activity of Colletotrichum anthracis Cm-ZJ-1 and the chitinase kit were provided by Nanjing Jiancheng Biotechnology Co., Ltd.

[0055] Experimental equipment: -80℃ refrigerator (WiseCryo, Korea), high-speed refrigerated centrifuge (Thermo FRESCO17, Thermo Fisher, America), SpectraMaxi3 microplate reader (BUCHI, Switzerland), visible light spectrophotometer, ultra-clean workbench (Zhejiang Fuxia Medical Technology Co., Ltd.), digital display gas bath constant temperature oscillator THZ-82A (Nanjing Lisi Gao Instrument Equipment Co., Ltd.), high pressure sterilizer (Zhiwei Instrument), constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), light incubator (Nanjing Sanshi Biotechnology Co., Ltd.), biological microscope (Nikon ECLIPSE Si RS).

[0056] Solution and test culture medium configuration:

[0057] The composition of PDA liquid culture medium is: 200 g peeled potatoes, 20 g glucose, 20 g agar powder, dilute to 1000 mL, and sterilize at 115°C for 30 min.

[0058] The composition of PDB liquid culture medium is: 200 g peeled potatoes, 18.85 g mannitol, 4.73 g yeast extract, 1000 mL distilled water, sterilized at 115°C for 30 min.

[0059] The composition of PDB solid culture medium is as follows: on the basis of the above-mentioned PDA culture medium, agar powder is not added.

[0060] Example 1

[0061] Soil samples were collected from the base of China Sericulture Research Institute in Zhenjiang, Jiangsu Province. 2 g of sample was added to 98 mL of sterile water to prepare a soil suspension. After vibrating and mixing thoroughly for 24 h, a 10-fold dilution method was used to dilute the suspension into 10 -3 , 10 -4 , 10 -5 Soil suspension. Then take 100 μL of soil dilution and spread it evenly on the PDA culture medium, invert it and culture it in a constant temperature incubator at 28℃, and observe it regularly. After the suspected Trichoderma colony grows on the PDA plate, immediately use a puncher to take the bacterial cake and transfer it to a new PDA plate for purification. Then inoculate the purified strain on the slope of the PDA test tube and culture it at 28℃ until conidia are produced. Store the test tube culture in a 4℃ refrigerator for short-term storage. At the same time, gently scrape the hyphae on the surface of the culture medium with sterile water, take 800 μL of bacterial suspension and add it to the freezing tube, then add 800 μL of 50% glycerol and mix well. After quick freezing with liquid nitrogen, store it in a -80℃ refrigerator for long-term storage.

[0062] Example 2

[0063] Morphological and molecular biological identification of Trichoderma

[0064] (1) Morphological identification

[0065] like Figure 1 As shown in Figure A, the morphological characteristics of the strain screened by the present invention were cultured on a PDA plate for 7 days. The strain grew quickly. After culturing at a constant temperature of 28°C for 3 days, the mycelium could cover a 9 cm PDA plate. The aerial mycelium was well developed and felt-like. The initial mycelium was white, and the later mycelium produced yellow-green concentric circles and expanded to the surrounding area, with a large number of conidia produced. Furthermore, a 5 mm cake of the strain grown on the PDA medium for 5 days was punched out with a hole punch and inoculated into the center of another fresh PDA medium. A coverslip was placed 1 cm away from the cake with sterile tweezers. After culturing at 28°C for 3 days, the coverslip covered with mycelium and spores was taken and turned upside down on a slide with a small amount of sterile water. The morphology of the mycelium and spores was observed under an optical microscope (Nikon ECLIPSE Si RS). Figure 1 B is a spherical or ovoid conidia with smooth outer wall, light yellow-green in color. Conidiophores have septa, grow oppositely, and the main branches are tree-like ( Figure 1 C).

[0066] (2) Molecular biological identification

[0067] The strain was cultured on PDA medium for 5 days, spores and mycelia were collected, and genomic DNA of the strain was extracted using the CTAB method.

[0068] PCR amplification using ITS gene primers ( Figure 2 The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0069] The ITS gene primers are as follows:

[0070] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'

[0071] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0072] The PCR reaction system was 25 μL: 12.5 μL 2×T5 Super PCR Mix (Qingke Biotechnology), 1.5 μL template DNA, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O was added to make up to 25 μL.

[0073] PCR amplification reaction program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, 35 cycles; final extension at 72°C for 2 min.

[0074] The PCR product was verified by 1% agarose gel electrophoresis and then sent to Shanghai Sangon Biotechnology Co., Ltd. for Sanger sequencing. The sequencing result is shown in SEQ ID NO.1. The ITS-rDNA sequence obtained by sequencing was compared and analyzed online on the website of the National Center for Biotechnology Information (NCBI). At the same time, based on the obtained sequence and the sequence information of other strains obtained from Gene Bank, the Neighbor-Joining calculation method of MEGAX software was used to construct a phylogenetic tree to analyze the phylogenetic relationship of the strain and determine the taxonomic attributes of the strain. The results showed that the ITS sequence of the strain was stably clustered in a branch with the strains of the genus Trichoderma ( Figure 3 Based on its morphological, physiological, and biochemical characteristics, the strain was named Trichoderma asperellum TR41, abbreviated as Trichoderma asperellum TR41. The strain is deposited with the Guangdong Provincial Microbial Culture Collection, GDMCC No. 64887, at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou, 510070, China, on July 18, 2024.

[0075] Example 3

[0076] Preparation of Trichoderma aspergillus TR41 bacterial suspension and Colletotrichum mulberryii Cm-ZJ-1 (7-day culture)

[0077] 100 μL TR41 glycerol culture was aspirated and activated in PDA medium. The PDA plate was inverted and incubated in a 28°C constant temperature incubator for 5 days. Mycelia were picked up with a sterile pipette tip and transferred to PDB liquid medium for shaking culture. After the mycelia fully grew, they were scraped with sterile water containing 0.05% Tween-20 to obtain a spore suspension. The concentration was adjusted to 1×10 using a hemocytometer. 7 The culture medium was inoculated into a 250 mL Erlenmeyer flask containing PDB medium at a volume ratio of 5% and shaken at 25°C and 150 rpm for 7 days. The fermentation broth was filtered through two layers of gauze, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the active fermentation broth of TR41.

[0078] 100 μL of anthrax Cm-ZJ-1 glycerol suspension was aspirated and activated in PDA medium. The PDA plate was inverted and incubated in a 28°C constant temperature incubator for 5 days. Mycelia were picked up with a sterile pipette tip and transferred to PDB liquid medium for shaking culture. After the mycelia fully grew, they were scraped with sterile water containing 0.05% Tween-20 to obtain a spore suspension. The concentration was adjusted to 1×10 using a hemocytometer. 7The culture medium was inoculated into a 250 mL Erlenmeyer flask containing PDB culture medium at a volume ratio of 5% and shaken at 25°C and 150 rpm for 7 days. The fermentation broth was filtered through two layers of gauze, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the active fermentation broth of Colletotrichum anthracis Cm-ZJ-1.

[0079] Example 4

[0080] Determination of the antagonistic effect of Trichoderma aspergillus TR41 against mulberry anthracnose fungus Cm-ZJ-1

[0081] The antibacterial test was carried out using the plate confrontation method ( Figure 3 ), in a clean bench, use a 5mm diameter puncher to take the activated mulberry anthracnose fungus Cm-ZJ-1 and Trichoderma aspergillus TR41 cakes (the plate is full of strains), and use sterile tweezers to inoculate the two cakes on the same PDA solid culture medium, with a distance of 5cm between the two, with 3 replicates per group, and use a single inoculation of mulberry anthracnose fungus as a control, and culture in a constant temperature incubator at 28℃ ( Figure 4 The colony radius of mulberry anthracnose fungi towards Trichoderma was measured on the 3rd to 7th day of culture, and the inhibition rate of Trichoderma was calculated ( Figure 4 ).

[0082] Table 1 Antibacterial effect of Trichoderma aspergillus TR41 on mulberry anthracnose fungus Cm-ZJ-1

[0083]

[0084] Example 5

[0085] Determination of the Growth Rates of Trichoderma aspergillus TR41 and Colletotrichum mulberryii Cm-ZJ-1

[0086] The growth rate curves of Trichoderma TR41 and Colletotrichum Cm-ZJ-1 were determined to provide a reference for further understanding of their biological characteristics and mechanisms. In a clean bench, a 5 mm diameter punch was used to take the mulberry anthracnose fungus Cm-ZJ-1 and Trichoderma aspergillus TR41 cakes respectively, and the two cakes were inoculated on PDA solid culture medium, with 3 replicates per group. Culture was carried out in a constant temperature incubator at 28 ° C. After culturing for 1 day, the mycelial diameter was measured every day using the cross-cross method and the growth rate curves of Trichoderma aspergillus TR41 and Colletotrichum Cm-ZJ-1 were drawn ( Figure 5 AB). As can be seen, the growth rate of Trichoderma aspergillus far exceeded that of Colletotrichum on the second day of culture. This helps to screen Trichoderma strains with vigorous growth and strong stress resistance for field biocontrol.

[0087] Example 6

[0088] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the peroxidase (POD) activity of Colletotrichum mulberryii Cm-ZJ-1

[0089] The antibacterial mechanism of the research can be further studied by measuring the effect of Trichoderma fermentation broth on the peroxidase (POD) of mulberry anthrax fungus Cm-ZJ-1. Take 20mL of Trichoderma spinulosa TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a puncher with a diameter of 5mm to take the mulberry anthrax fungus Cm-ZJ-1 cake, put the anthrax fungus Cm-ZJ-1 cake into the TR41 active fermentation broth, and put it into a shaking table at 28°C and 180rpm for cultivation. Each treatment was repeated 3 times, and samples were taken every 2h to measure the OD of each group of mulberry anthrax fungus Cm-ZJ-1. 420 By comparing the absorbance values ​​between different treatment groups, the peroxidase (POD) activity curve of Colletotrichum Cm-ZJ-1 was drawn ( Figure 6 The kit for determining the peroxidase (POD) activity of Cm-ZJ-1 of anthrax fungi (Cat. No. R30312-50T) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The results showed that the peroxidase (POD) activity of Cm-ZJ-1 of anthrax fungi in mulberry trees was significantly reduced after the anthrax fungi were treated with the active fermentation liquid of Trichoderma aspergillus TR41 compared with the anthrax fungi in the control group ( Figure 6 ).

[0090] Example 7

[0091] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the total superoxide dismutase (T-SOD) activity of Colletotrichum mulberryii Cm-ZJ-1

[0092] Determining the effect of Trichoderma fermentation broth on the total superoxide dismutase (T-SOD) of mulberry anthrax fungus Cm-ZJ-1 can further study the antibacterial mechanism of the research. Take 20mL of Trichoderma spinulosa TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a puncher with a diameter of 5mm to take the mulberry anthrax fungus Cm-ZJ-1 cake, put the anthrax fungus Cm-ZJ-1 cake into the TR41 active fermentation broth, and put it into a shaking table at 28°C and 180rpm for cultivation. Each treatment was repeated 3 times, and samples were taken every 2h to measure the OD of each group of mulberry anthrax fungus Cm-ZJ-1. 550 By comparing the absorbance values ​​between different treatment groups, the total superoxide dismutase (T-SOD) activity curve of anthrax fungus Cm-ZJ-1 was drawn ( Figure 7 The kit for determining the total superoxide dismutase (T-SOD) activity of Colletotrichum anthracnose Cm-ZJ-1 (Cat. No. A001-1-2) was provided by Nanjing Jiancheng Biotechnology Co., Ltd. The results showed that the total superoxide dismutase activity of Colletotrichum anthracnose Cm-ZJ-1 was significantly reduced after the active fermentation broth of Trichoderma aspergillus TR41 was treated with the anthracnose fungus compared with the control group ( Figure 7 ).

[0093] Example 8

[0094] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the chitinase activity of Colletotrichum mulberryii Cm-ZJ-1

[0095] The antibacterial mechanism of the research can be further studied by measuring the effect of Trichoderma fermentation broth on the chitinase activity of mulberry anthrax fungus Cm-ZJ-1. Take 20mL of Trichoderma spinulosa TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a puncher with a diameter of 5mm to take the mulberry anthrax fungus Cm-ZJ-1 cake, put the anthrax fungus Cm-ZJ-1 cake into the TR41 active fermentation broth, put it into a shaker at 28°C and 180rpm for cultivation, and culture for 24h. Each treatment was repeated 3 times, and the OD value of each group of mulberry anthrax fungus Cm-ZJ-1 was measured. 540 By comparing the absorbance values ​​between different treatment groups, the chitinase activity curve of Colletotrichum Cm-ZJ-1 was drawn ( Figure 8 The kit for determining the chitinase activity of anthrax fungi Cm-ZJ-1 (Cat. No. A139-1-1) was provided by Nanjing Jiancheng Biotechnology Co., Ltd. The results showed that compared with the control group of anthrax fungi, the chitinase activity of anthrax fungi was reduced after the anthrax fungi were treated with the active fermentation broth of Trichoderma aspergillus TR41, indicating that Trichoderma aspergillus TR41 can reduce the chitinase activity of anthrax fungi ( Figure 8 ).

[0096] Example 9

[0097] Antibacterial Effect of Active Fermentation Broth of Trichoderma aspergillus TR41 on Colletotrichum mulberryii Cm-ZJ-1

[0098] Take 20mL of Trichoderma aspergillus TR41 active fermentation broth (Example 3) and add it to 50mL of PDB liquid culture medium and mix evenly. Use a 5mm diameter punch to take the mulberry anthracnose fungus Cm-ZJ-1 cake, put the anthracnose fungus Cm-ZJ-1 cake into the TR41 active fermentation broth, and place it in a shaker at 28°C and 180rpm for cultivation. Each treatment was repeated 3 times, and samples were taken every 2 hours to measure the OD value of each group of mulberry anthracnose fungus Cm-ZJ-1. 550 ( Figure 9 ). Draw the inhibition rate curve of each time period according to the inhibition rate in different time periods ( Figure 9 ). It was further shown that the active fermentation liquid of Trichoderma aspergillus TR41 had a significant inhibitory effect on the growth of anthrax. After 9 hours of co-culture, the inhibition rate could reach 93.59%, and after 16 hours of co-culture, the inhibition rate could reach 99% ( Figure 9 ).

[0099] Table 2 Antibacterial effect of Trichoderma aspergillus TR41 active fermentation broth on mulberry anthracnose fungus Cm-ZJ-1

[0100]

[0101] Example 10

[0102] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the soluble protein content of Colletotrichum mulberryii Cm-ZJ-1

[0103] The effect of Trichoderma fermentation broth on the soluble protein content of mulberry anthrax Cm-ZJ-1 can be used to further study the antibacterial mechanism of the research. Take 20mL of Trichoderma spinulosa TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a puncher with a diameter of 5mm to take the mulberry anthrax Cm-ZJ-1 bacterial cake, put the anthrax Cm-ZJ-1 bacterial cake into the TR41 active fermentation broth, and put it into a shaker at 28°C and 180rpm for cultivation. Repeat 3 times for each treatment and take samples every 2 hours. Take an appropriate amount of treated sample, use sterile water to prepare a solution, add 5mL of diluted Coomassie Brilliant Blue G-250 solution, mix thoroughly, and react at room temperature for 5-30 minutes. Measure the OD of each group of mulberry anthrax Cm-ZJ-1 at a wavelength of 595nm. 595 By comparing the absorbance values ​​between different treatment groups, the soluble protein content curve of anthrax Cm-ZJ-1 was drawn ( Figure 10 The results showed that compared with the anthrax bacteria in the control group, the soluble protein content of anthrax bacteria treated with the active fermentation broth of Trichoderma aspergillus TR41 was significantly reduced. The content began to decrease after 1.5 hours of co-culture, and decreased by 78% after 6 hours of co-culture. Figure 10 ), further indicating that the active fermentation broth of Trichoderma aspergillus TR41 can reduce the content of soluble protein in osmotic regulating substances.

[0104] Example 11

[0105] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the malondialdehyde content of Colletotrichum mulberryii Cm-ZJ-1

[0106] The effect of Trichoderma fermentation broth on the malondialdehyde (MDA) content of mulberry anthrax fungus Cm-ZJ-1 can be used to further study the antibacterial mechanism of the research. Take 20mL of Trichoderma spinulosa TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a puncher with a diameter of 5mm to take the mulberry anthrax fungus Cm-ZJ-1 cake, put the anthrax fungus Cm-ZJ-1 cake into the TR41 active fermentation broth, and put it into a shaker at 28°C and 180rpm for cultivation. Each treatment is repeated 3 times, and samples are taken every 2 hours. Take an appropriate amount of treated sample, use sterile water to prepare a solution, add an equal amount of thiobarbituric acid solution, and heat in a boiling water bath for 10-15 minutes to allow malondialdehyde and thiobarbituric acid to react with color (the heating time needs to be strictly controlled), and measure the OD of each group of mulberry anthrax fungus Cm-ZJ-1. 600 By comparing the absorbance values ​​between different treatment groups, the MDA content curve of anthrax fungus Cm-ZJ-1 was drawn ( Figure 11 The results showed that compared with the anthrax control group, the malondialdehyde (MDA) content of anthrax bacteria was significantly increased after the anthrax bacteria were treated with the active fermentation liquid of Trichoderma aspergillus TR41. As time went on, the MDA content gradually increased after 4 hours of co-culture, reaching the highest level at 6 hours, which was 3 times that of the control group ( Figure 11 This further indicates that the active fermentation broth of Trichoderma aspergillus TR41 can achieve antibacterial effect by increasing the content of malondialdehyde in cell osmotic regulating substances.

[0107] Example 12

[0108] Effects of Trichoderma aspergillus TR41 fermentation liquid on mulberry root activity

[0109] The growth-promoting effect of Trichoderma can be further studied by measuring the effect of Trichoderma fermentation liquid on the activity of mulberry root system. Take 20mL of Trichoderma aspergillus TR41 active fermentation liquid (Example 3) and add it to 50mL PDB liquid culture medium and mix it evenly. Infiltrate the roots of mulberry seedlings with the mixed liquid, and infiltrate the seedlings of the control group with 70mL PDB liquid culture medium. Each treatment is repeated 3 times, cultured for 7 days, and the OD value of the roots of each group of mulberry seedlings is measured. 595 , calculate its root activity ( Figure 12 The kit for measuring the root activity of mulberry seedlings (Cat. No. R30351-2) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The results showed that compared with the control group, the root activity of mulberry seedlings treated with the active fermentation liquid of Trichoderma aspergillus TR41 was improved ( Figure 12 ), which further illustrates that the active fermentation liquid of Trichoderma aspergillus TR41 has a growth-promoting effect on mulberry seedlings.

[0110] Example 13

[0111] Effects of Trichoderma aspergillus TR41 active fermentation broth on chlorophyll content in mulberry trees

[0112] The growth-promoting effect of Trichoderma can be further studied by measuring the effect of Trichoderma fermentation liquid on the root activity of mulberry trees. Take 20mL of Trichoderma aspergillus TR41 active fermentation liquid (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. The mixed liquid is infiltrated into the roots of mulberry seedlings, and 70mL PDB liquid culture medium is used to infiltrate the seedlings of the control group. Each treatment is repeated 3 times and cultured for 7 days. The OD value of chlorophyll in each group of mulberry seedlings is measured. 665 With OD 649 , calculate the chlorophyll A, chlorophyll B and total chlorophyll content ( Figure 13 The kit for determining the chlorophyll content of mulberry (Cat. No. R30354-50T) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The results showed that compared with the control group, the treatment of the roots of mulberry seedlings with the active fermentation liquid of Trichoderma aspergillus TR41 could significantly increase the chlorophyll A, chlorophyll B and total chlorophyll contents of the mulberry seedlings ( Figure 13 ), which further illustrates that the active fermentation liquid of Trichoderma aspergillus TR41 has a growth-promoting effect on mulberry seedlings.

[0113] Example 14

[0114] Hyperparasitism of Trichoderma aspergillus TR41 against Colletotrichum mulberryii Cm-ZJ-1

[0115] In a clean bench, use a 5mm diameter puncher to take the bacterial cakes of mulberry anthrax fungus Cm-ZJ-1 and Trichoderma TR41, and inoculate the two bacterial cakes on both sides of the same PDA culture medium, 5cm apart, and place a sterile cover glass in the middle of the two bacterial cakes. The inoculated culture medium was placed in a constant temperature incubator at 28℃ for 7 days, and then the cover glass was taken out and the side covered with hyphae was placed upside down on a slide and observed under a microscope. By observing the hyphae effect of Trichoderma TR41 and mulberry anthrax fungus Cm-ZJ-1 after 7 days of confrontation culture, compared with the control ( Figure 14 AB), the study found that after Trichoderma aspergillus TR41 came into contact with Colletotrichum mulberrye Cm-ZJ-1, its hyphae could attach to the hyphae of Colletotrichum mulberrye Cm-ZJ-1 and produce a large number of branches ( Figure 14 CD), some hyphae of Trichoderma aspergillus TR41 can penetrate and embed into the hyphae of Colletotrichum mulberryii Cm-ZJ-1 ( Figure 14 E), the mycelial contents of Colletotrichum mulberryii Cm-ZJ-1 parasitized and attached by Trichoderma aspergillus TR41 decreased and the mycelium became thinner ( Figure 14 FH), cell wall degradation was significant ( Figure 14 ). This further demonstrates that Trichoderma aspergillus TR41 has a strong parasitic effect on mulberry anthracnose fungus Cm-ZJ-1.

[0116] Example 15

[0117] Antibacterial Effect of Active Fermentation Broth of Trichoderma aspergillus TR41 on Colletotrichum truncatum HNRY-9

[0118] Take 20mL of Trichoderma aspergillus TR41 active fermentation broth (Example 3) and add it to 50mL of PDB liquid culture medium and mix evenly. Use a hole punch to take a 5mm diameter strawberry anthrax HNRY-9 bacterial cake, put the strawberry anthrax HNRY-9 bacterial cake into the TR41 active fermentation broth, and place it in a shaker at 28°C and 180rpm for cultivation. Each treatment was repeated 3 times, and samples were taken every 2 hours to measure the OD value of each group of strawberry anthrax HNRY-9. 550 .

[0119] According to the inhibition rate in different time periods, the inhibition rate curve of each time period was drawn ( Figure 15 ). It was further shown that the active fermentation liquid of Trichoderma aspergillus TR41 not only had a strong antibacterial effect on mulberry anthracnose fungus Cm-ZJ-1, but also had a significant inhibitory effect on the growth of strawberry anthracnose fungus HNRY-9. After co-cultivation for 10 hours, the inhibition rate could reach 79.16%, and after co-cultivation for 16 hours, the inhibition rate could reach 83% ( Figure 15 ).

[0120] Table 3 Antibacterial effect of Trichoderma aspergillus TR41 active fermentation broth on Colletotrichum spp. HNRY-9

[0121]

[0122] Example 16

[0123] Effects of the active fermentation broth of Trichoderma aspergillus TR41 on the total superoxide dismutase (T-SOD) activity of Colletotrichum truncatum HNRY-9

[0124] The antibacterial mechanism of the research can be further studied by measuring the effect of Trichoderma fermentation broth on the total superoxide dismutase (T-SOD) of strawberry anthrax fungus HNRY-9. Take 20mL of Trichoderma aspergillus TR41 active fermentation broth (Example 3) and add it to 50mL PDB liquid culture medium and mix evenly. Use a 5mm diameter punch to take the strawberry anthrax fungus HNRY-9 cake, put the strawberry anthrax fungus HNRY-9 cake into the TR41 active fermentation broth, and place it in a shaker at 28°C and 180rpm for cultivation. Each treatment was repeated 3 times, and samples were taken every 2 hours to measure the OD of each group of strawberry anthrax fungus HNRY-9. 550 By comparing the absorbance values ​​between different treatment groups, the total superoxide dismutase (T-SOD) activity curve of anthrax fungus Cm-ZJ-1 was drawn ( Figure 16The total superoxide dismutase (T-SOD) activity kit of Colletotrichum anthracnose fungi Cm-ZJ-1 (Cat. No. A001-1-2) was provided by Nanjing Jiancheng Biotechnology Co., Ltd. The results showed that the total superoxide dismutase activity of Colletotrichum anthracnose fungi HNRY-9 was significantly reduced after treatment with the active fermentation liquid of Trichoderma aspergillus TR41 compared with the control group ( Figure 16 ).

Claims

1. An application of Trichoderma asperellum in inhibiting anthrax pathogens, wherein the Trichoderma asperellum is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum and has been deposited in the Guangdong Provincial Microbial Culture Collection with a deposit number of GDMCC No. 64887 and a deposit date of July 18, 2024.

2. The use according to claim 1, characterized in that The anthracnose pathogen is preferably Colletotrichum fructicola or Colletotrichum siamense.

3. The use according to claim 1, characterized in that The application of the Trichoderma aspergillus TR41 in inhibiting or antagonizing the pathogen of mulberry anthracnose.

4. The use according to claim 1, characterized in that The Trichoderma aspergillus TR41 can not only significantly inhibit the growth of anthrax bacteria, but also significantly inhibit the activities of biological enzymes and biological proteins of anthrax bacteria.

5. The use according to claim 4, characterized in that The Trichoderma aspergillus TR41 inhibits the hyphae growth of anthrax fungi and inhibits the activities of peroxidase (POD), superoxide dismutase (T-SOD) and chitinase in the anthrax fungi.

6. The use according to claim 2, characterized in that The fermentation product or bacterial agent of Trichoderma aspergillus TR41 is used in inhibiting or antagonizing the pathogen of mulberry anthracnose.

7. The use according to claim 5, characterized in that The preparation method of the Trichoderma aspergillus TR41 fermentation product or bacterial agent comprises the following steps: activating the Trichoderma strain TR41 in a PDA culture medium, culturing it upside down in a constant temperature incubator, preparing a conidia suspension, fermenting it in a PDB culture medium, and filtering the fermentation liquid to obtain a filtrate to obtain an active Trichoderma fermentation liquid.

8. An application of Trichoderma asperellum in preventing and controlling mulberry anthracnose, wherein the Trichoderma asperellum is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum and has been deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 64887 and a deposit date of July 18, 2024.

9. An application of Trichoderma asperellum TR41 in promoting the growth of mulberry trees, wherein the Trichoderma asperellum is Trichoderma asperellum TR41, which has been identified as Trichoderma asperellum and has been deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 64887 and a deposit date of July 18, 2024.

10. The use according to claim 9, characterized in that The promoting of mulberry tree growth includes increasing root activity and chlorophyll content.

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