A strain of Trichoderma guizhouensis and its application in preventing and controlling anthracnose of oil-tea camellia

Abstract

The invention discloses a strain of Trichoderma guizhouense and its application in preventing and treating anthracnose of oil-tea, belonging to the field of microorganisms. The invention isolates a strain of Trichoderma guizhouense from the rhizosphere of common oil-tea in Changsha, and the preservation number of the fungus is CGMCC No.41486. ​​It is found through experiments that the fungus can produce a large number of conidia, and the conidia can secrete cell wall degrading enzymes to digest the hyphae of pathogenic bacteria; the direct inhibition rate of the growth of the mycelium of the spiny colletotrichum of oil-tea fruit is 84.19%; it has significant prevention and inhibition effects on anthracnose of oil-tea, and the prevention and control effects are as high as 99.7%. Therefore, the invention provides a strain of Trichoderma guizhouense with significant prevention effect on anthracnose of oil-tea, providing new strain resources and environmentally friendly and efficient biocontrol methods for the effective prevention and treatment of anthracnose of oil-tea.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a Guizhou Trichoderma strain and application thereof in preventing and treating anthracnose of oil-tea tree. Background Art

[0002] Camellia oleifera is the most important woody edible oil tree species with the largest cultivation area in my country. Colletotrichum spp. is the pathogen of anthracnose of Camellia oleifera, among which Colletotrichum fructicola is the dominant pathogen, with an isolation rate of 71%.

[0003] At present, the prevention and control of anthracnose of oil-tea camellia mainly relies on chemical fungicides, such as carbendazim, thiophanate-methyl and tebuconazole, etc. However, there is no registered control agent for anthracnose of oil-tea camellia in my country. In addition, the long-term and large-scale application of chemical pesticides is very likely to cause environmental pollution, increase the resistance of pathogens, reduce the quality of oil-tea camellia, and pesticide residues.

[0004] Biological control is to use beneficial microorganisms (such as bacteria, fungi, viruses, etc.) and their metabolites to prevent and treat plant diseases. Compared with chemical control, biological control has the advantages of being safe for the environment, having no residue, and being difficult to develop drug resistance. Trichoderma is a fungus with huge biological control potential. Chinese patents CN115927003A, CN112143654A and CN108587922A disclose that Trichoderma has significant control effects on tea tree anthracnose, litchi anthracnose and winter jujube anthracnose, but there are significant differences in the pathogens of different plant anthracnose, and there is no report that Trichoderma is used to prevent and treat oil tea anthracnose. Therefore, it is of great significance to tap the biological control resources of Trichoderma to the biological control of oil tea anthracnose. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Guizhou Trichoderma and its application in preventing and controlling anthracnose of oil-tea camellia, so as to solve the problems existing in the above-mentioned prior art. The screened Guizhou Trichoderma can effectively prevent and control anthracnose of oil-tea camellia, providing an environmentally friendly and efficient biocontrol approach for the effective prevention and control of anthracnose of oil-tea camellia.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a strain of Trichoderma guizhouense, the preservation number of which is CGMCC No. 41486, the preservation date is August 9, 2024, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides the use of the Guizhou Trichoderma or its spores in inhibiting the pathogenic bacteria of oil-tea anthracnose.

[0009] The present invention also provides the use of the Guizhou Trichoderma or its spores in the preparation of a bacterial agent for inhibiting the pathogenic bacteria of oil-tea anthracnose.

[0010] Preferably, the pathogen of tea oil anthracnose includes Colletotrichum fructicola.

[0011] The present invention also provides the use of the Guizhou Trichoderma or its spores in preventing and / or treating anthracnose of oil tea.

[0012] The present invention also provides the use of the Guizhou Trichoderma or its spores in the preparation of a bacterial agent for preventing and / or treating anthracnose of oil-tea tea.

[0013] The present invention also provides a fungal agent for preventing and / or treating anthracnose of oil-tea tree, comprising the Guizhou Trichoderma or its spores.

[0014] The invention also provides a method for preventing and treating anthracnose of oil tea, comprising the step of inoculating oil tea leaves with the spore suspension of Guizhou Trichoderma.

[0015] Preferably, the method for preparing the spore suspension comprises the following steps: inoculating the Trichoderma kweichowensis onto a PDA plate, and after a large number of spores are produced, washing the spores with sterile water, and then filtering to collect the filtrate to obtain the spore suspension.

[0016] The present invention discloses the following technical effects:

[0017] The present invention separates a Guizhou Trichoderma strain from oil tea leaves, and the direct inhibition rate of the fungus on the growth of the hyphae of the spores of oil tea fruit is 84.19%; the fungus has a significant prevention and inhibition effect on the anthracnose of oil tea, and the prevention and control effect is as high as 99.7%. By observing the hyphae at the junction of the Trichoderma and the pathogen after confrontation culture, it is found that Guizhou Trichoderma produces a large number of spores that are adsorbed and entangled on the pathogen (C. fructicola), and can secrete cell wall degrading enzymes to digest the C. fructicola hyphae into hyphae fragments, thereby inhibiting the growth of the pathogen. It is explained that the present invention obtains a Trichoderma strain that has a preventive effect on oil tea anthracnose, and provides a new strain resource and theoretical basis for the biological control of oil tea anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The morphological characteristics of strain COTG-19; a: Colony morphology of strain COTG-19 after 7 days of culture on PDA plate; b: Conidia; c: Conidiophores;

[0020] Figure 2 The NJ-developmental tree was constructed based on the combined sequence of ITS+fRPB2+TEF-1 using MEGA11;

[0021] Figure 3 The inhibitory effect of strain COTG-19 on the growth of Colletotrichum spp. a: control group; b: inhibitory effect of strain COTG-19 on Colletotrichum spp.

[0022] Figure 4 The effect of strain COTG-19 on Colletotrichum spp. under scanning electron microscope;

[0023] Figure 5 The preventive and therapeutic effects of strain COTG-19 on anthracnose of tea oil; a: pre-inoculate with COTG-19 spore suspension, then inoculate with conidia suspension of Sporangiophora foetida 48 hours later, and observe the disease development on the leaves 5 days after inoculation; b: inoculate with conidia suspension of COTG-19 and Sporangiophora foetida at the same time, and observe the disease development on the leaves 5 days after inoculation. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1 Isolation and identification of Guizhou Trichoderma COTG-19

[0030] 1. Isolation of Trichoderma

[0031] Referring to the method described by Fang Zhongda et al., a strain of Trichoderma was isolated from the rhizosphere of healthy tea oil. The details are as follows:

[0032] Take the rhizosphere of ordinary healthy tea oil plants in Tianxin District, Changsha City, Hunan Province, cut them into 1 cm small pieces with a sterile scalpel, rinse them with sterile water 3 times after disinfection with 2% sodium hypochlorite solution and 75% alcohol, and place them on sterile absorbent paper to absorb the water. Place the leaf tissue on a PDA plate containing streptomycin sulfate (100 μg / mL) and culture it at 28°C. After 24 to 48 hours, use a sterilized toothpick to pick the edge of the colony to a new PDA plate. After the green conidia grow, rinse with suitable sterile water to obtain a spore suspension. After gradient dilution, apply it to a PDA plate containing streptomycin sulfate (100 μg / mL) and culture it at 28°C for 36 to 48 hours. Pick a single colony to obtain a pure isolated strain of a single spore, named COTG-19.

[0033] 2. Morphological observation

[0034] COTG-19 was inoculated on PDA medium to observe the growth rate and colony morphology of the strain. Young mycelium at the edge of the COTG-19 plate after 3 days of culture was taken to observe conidia and conidiophores under a microscope.

[0035] The results are as follows Figure 1As shown, COTG-19 has abundant mycelium on PDA medium, which is cotton-like, yellow-green to green, and produces a large number of conidia, which grow in clusters at the top of the mycelium.

[0036] 3. Molecular identification

[0037] The genomic DNA of strain COTG-19 was extracted by the CTAB method. The primers in Table 1 were used to amplify the ITS, fRPB2 and tef1 sequences of the COTG-19 strain. Each 50 μL PCR reaction system contained 25 μL Green Taq Mix, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 2 μL DNA template and 19 μL ddH2O. The PCR reaction program was: 95°C pre-denaturation for 3 min; 32 amplification cycles (95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min); 72°C extension for 5 min. After the PCR amplification products were sequenced (Sangon Biotechnology Co., Ltd., Shanghai, China), the obtained ITS, fRPB2 and tef1 gene sequences were combined to construct a phylogenetic tree (see Figure 2 ). Combining morphological characteristics and molecular biological identification, COTG-19 was identified as Trichoderma guizhouense.

[0038] Table 1 Primer sequences used

[0039]

[0040] In the above table, Y=C or T; M=A or C; W=A or T; R=A or G.

[0041] The above-mentioned Trichoderma guizhouense COTG-19, classified and named Trichoderma guizhouense, was deposited in the General Microbiology Center of China Microbiological Culture Collection on August 9, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41486.

[0042] Example 2 Inhibitory effect of Guizhou Trichoderma COTG-19 on the pathogenic bacteria of oil-tea anthracnose

[0043] The plate confrontation method was used to determine the inhibitory effect of COTG-19 on C. fructicola, the anthracnose pathogen of tea oil. C. fructicola was inoculated on the left side of a PDA plate with a diameter of 60 mm, and COTG-19 was inoculated 40 mm away from the bacterial cake. The PDA plate inoculated with only C. fructicola was used as a blank control. Each treatment was repeated 3 times and cultured in a 28°C incubator. After 5 days, the radius of the colonies in the treatment group and the control group was recorded, and photos were taken to determine the inhibition rate of COTG-19 on C. fructicola.

[0044] The results are as follows Figure 3 As shown, the average radius of the C. fructicola colony in the blank control group was 35 mm, while in the experimental group, the average diameter of the anthrax pathogen was only 5.5 mm, indicating that Guizhou Trichoderma COTG-19 had a significant inhibitory effect on C. fructicola, with an average inhibition rate of 84.19%.

[0045] Example 3: Hyperparasitism of Guizhou Trichoderma COTG-19 on C. fructicola hyphae

[0046] Guizhou Trichoderma strain COTG-19 was cultured opposite to C. fructicola. After 5 days of culture, the mycelium block at the junction of the two was cut with a sterile blade and fixed in 2.5% glutaraldehyde solution for 4 hours. After washing with double distilled water and dehydrating with ethanol gradient, the mycelium morphology was observed and recorded by scanning electron microscopy (SEM).

[0047] By observing the overlapping hyphae of Guizhou Trichoderma COTG-19 and C. fructicola in confrontation culture under a microscope, it was found that Guizhou Trichoderma COTG-19 produced a large number of spores that were attached to and entangled with C. fructicola, and could secrete cell wall degrading enzymes to decompose C. fructicola hyphae into hyphae fragments, thereby inhibiting the growth of pathogens (see Figure 4 ).

[0048] Example 4 The control effect of Guizhou Trichoderma COTG-19 on anthracnose of Camellia oleifera

[0049] 1. Preparation of inoculated spore suspension

[0050] The strain COTG-19 was inoculated on a PDA plate. After a large number of spores were produced, 2 mL of sterile water was taken with a pipette and washed on the PDA plate to collect the spores. This was repeated 2 to 3 times to obtain a spore-containing aqueous solution. The mycelium was filtered through 2 layers of lens paper to obtain a spore suspension. The COTG-19 spore concentration was adjusted to 1 × 10 5C. fructicola was inoculated into PDB (PDA without agar) liquid culture medium, shaken at 28°C, 180 rpm for 5 days, and then the mycelium was filtered to obtain the fermentation filtrate. The fermentation filtrate was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. The precipitate was added with an appropriate amount of sterile water to obtain the C. fructicola conidia suspension, and then the concentration was adjusted to 1×10 5 pcs / mL for later use.

[0051] 2. Preventive effect

[0052] Take the newly sprouted young leaves of the oil-tea camellia (excellent variety - Asus) plant, place them in a sterilized inoculation box, and poke holes at the inoculation site with a sterile toothpick. Use a pipette to draw 50μL of COTG-19 spore suspension and drop it on the inoculation site. After 48 hours, inoculate 50μL C.fructicola spore suspension at the same location, and use the same amount of sterile water in advance as a control. Place the inoculation box in a 28℃ artificial climate box for moisturizing culture. After 5 days, take photos to record the disease status of the oil-tea camellia leaves, and use ImageJ software to measure the diseased area. The results are as follows Figure 5 As shown in a, the average disease area in the control group was 158.06±3.17mm 2 The average disease area in the treatment group was only 0.41±0.13mm 2 .

[0053] 3. Treatment effect

[0054] The treatment of tea leaves was as described in "2. Preventive effect". 50 μL of COTG-19 and C. fructicola spore suspension were inoculated at the same place, and 50 μL of sterile water and 50 μL of C. fructicola were co-inoculated as a control. The disease incidence was recorded using the above method 5 days after inoculation. The results are shown in Figure 5 As shown in middle b, the average disease area of ​​the control group was 194.98±1.36mm 2 The average disease area in the treatment group was only 0.45±0.14mm 2 .

[0055] Control effect (%) = (the diseased area of ​​the control group - the diseased area of ​​the treatment group) / the diseased area of ​​the control group × 100.

[0056] The results show that the Guizhou Trichoderma COTG-19 provided by the present invention has a significant control effect on tea tree anthracnose, and can almost completely inhibit the pathogenicity of C. fructicola to oil tea leaves. Whether it is a preventive effect or a therapeutic effect, its control effect reaches more than 99%, and it is very likely to be developed into a biocontrol product for the green control of oil tea anthracnose in the future.

[0057] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Trichoderma guizhouensis ( Trichoderma guizhouense ), characterized in that The deposit number of this bacterium is CGMCC No.41486.

2. The Guizhou Trichoderma or its spores as claimed in claim 1 are effective in inhibiting the fruit thorn spore ( Colletotrichum fructicola ) in the application.

3. The Guizhou Trichoderma or its spores as claimed in claim 1 are used in the preparation of a spore inhibiting fruit thorn spore ( Colletotrichum fructicola ) in the application of bacterial agents.

4. Use of Trichoderma guizhouensis or its spores as claimed in claim 1 in preventing and / or treating anthracnose of tea oil.

5. Use of the Guizhou Trichoderma or its spores as claimed in claim 1 in the preparation of a fungal agent for preventing and / or treating anthracnose of oil-tea tea.

6. A fungal agent for preventing and / or treating anthracnose of oil-tea tree, characterized in that: It comprises the Guizhou Trichoderma or its spores as described in claim 1.

7. A method for preventing and treating anthracnose of oil-tea tree, characterized in that: The method comprises the step of inoculating oil tea leaves with the spore suspension of Guizhou Trichoderma according to claim 1.

8. The method according to claim 7, characterized in that The preparation method of the spore suspension comprises the following steps: inoculating the Guizhou Trichoderma on a PDA plate, washing the spores with sterile water after a large number of spores are produced, filtering, and collecting the filtrate to obtain the spore suspension.

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