Tea tree endophytic fungus myceliophthora verrucosa and biofungicide and application thereof

Abstract

The present application belongs to the technical field of biological control, and particularly relates to a tea tree endophytic fungus Myceliophthora verrucosa, a biological fungicide and application thereof. The preservation number of the tea tree endophytic fungus Myceliophthora verrucosa is CCTCC NO: M20232089. The tea tree endophytic fungus strain Myceliophthora verrucosa JH101 provided in the present application has good antagonistic activity on several common tea tree pathogenic bacteria, can be used as a plant pathogenic bacteria biocontrol fungus, provides an excellent starting strain for development and preparation of a microbial source biological fungicide, can be used for safe, efficient and pollution-free plant disease control, is conducive to reducing pollution to an ecological environment, improving quality and edible safety of crops, and has wide application potential and important economic, ecological and environmental protection values.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to the endophytic fungus Myceliophthoraverrucosa of tea trees, biological fungicides, and their applications. Background Technology

[0002] Plant endophytes are a group of microorganisms that can live in the intercellular spaces and cells of various plant organs and tissues during part or all of their life cycle, establishing a harmonious symbiotic relationship with the plant without causing obvious pathological symptoms in the host. Endophytes, including bacteria, fungi, and actinomycetes, have been isolated and identified from various plants such as wheat, cotton, rice, peanut, potato, tomato, lemon, citrus, and tea. As an integral part of the plant body, endophytes play a wide range of biological roles in plant growth and development, biomass accumulation, and resistance to pests and diseases. Endophytes colonize within the host plant, promoting plant growth and enhancing the host plant's resistance to biotic stresses (such as plant pathogen infection and pest invasion) and abiotic stresses. The biocontrol mechanisms of plant endophytes include antagonistic effects (producing enzymes, antibiotics, bacteriocins, and volatile substances) and the induction of systemic resistance (inducing the production of polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase, and superoxide dismutase).

[0003] In agricultural and forestry production, crops are attacked by various pathogens. Chemical fungicides are the main means of controlling plant diseases, but their drawbacks are becoming increasingly prominent, such as inducing pathogens to develop resistance, polluting the ecological environment, disrupting the ecological balance, and threatening human health. Therefore, research on the biological control of plant diseases is receiving increasing attention. Utilizing antimicrobial active substances produced by plant endophytes for the biological control of plant pathogenic diseases has advantages such as short cycle time, high safety, ease of production, and low toxicity. It is currently one of the important options to replace chemical control methods, opening up a bright future for the research and application of biofungicides. Isolating biocontrol endophytic bacterial strains with antagonistic effects against pathogens is the prerequisite and foundation for developing and utilizing plant endophytes for the control of plant diseases.

[0004] The tea plant (Camellia sinensis) is a perennial evergreen shrub primarily grown for its fresh leaves and is an important economic crop in my country. However, pests and diseases continuously occur during tea cultivation, severely damaging tea plant growth and tea production. Traditional chemical control methods have had serious negative impacts on tea quality and drinking safety. Screening antagonistic strains from the endophytic fungi of tea plants that inhibit plant pathogens is of great significance for developing biological fungicides, ensuring the green, healthy, and sustainable development of the tea industry, and protecting food safety.

[0005] Currently, research on endophytic fungi in tea trees started relatively late and is limited, with few reports on the preventive effects of utilizing endophytic fungal resources on tea tree diseases. Therefore, the discovery and exploration of endophytic fungi in tea trees, and the screening of new antagonistic strains with antibacterial activity, are of great significance and have broad prospects for the green, efficient, and pollution-free control of tea tree pathogens. Summary of the Invention

[0006] To address the shortcomings of existing technologies, such as the unsafety of chemical pesticides and the limited variety of microbial biological pesticides, this invention provides a tea tree endophytic fungus, Myceliophthora verrucosa, and a biological fungicide containing Myceliophthora verrucosa. It also provides the application of the aforementioned Myceliophthora verrucosa strain and biological fungicide in the prevention and control of tea tree diseases and pests, aiming to solve some of the problems in the existing technologies or at least alleviate some of them.

[0007] This invention is specifically achieved through the following technical solutions:

[0008] The first aspect of this invention provides an endophytic fungus of tea tree, Myceliophthora verrucosa, with accession number CCTCC NO: M 20232089, deposited on October 31, 2023 at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University), and the culture name is Myceliophthora verrucosa JH101.

[0009] This application describes the isolation of a novel endophytic fungus from tea plants, identified morphologically and molecularly as *Myceliophthora verrucosa*, and named *Myceliophthora verrucosa* JH101. Pathogen control tests were conducted on *Myceliophthora verrucosa* JH101 of this invention. Plate confrontation bioactivity evaluation experiments showed that *Myceliophthora verrucosa* JH101 exhibited good antagonistic and antibacterial activity against several common pathogens on tea plants. On PDA plates, after 5 days of confrontation, the mycelial growth inhibition rate against three pathogens—*Fusarium sp.*, *Colletotrichum gloeosporioides*, and *Phoma sp.*—reached over 53%. On 7 days, the inhibition rate against *Fusarium sp.*, *Colletotrichum gloeosporioides*, *Phoma sp.*, and the tea leaf spot pathogen *Pseudopestalotiopsis* was significantly increased. The inhibition rates of the four pathogenic fungi (Camelliae sinensis) were all above 58%, with a particularly significant antagonistic effect against *Colletotrichum gloeosporioides*, exhibiting a mycelial growth inhibition rate as high as 70% after 7 days. This indicates that the endophytic fungus *Myceliophthora verrucosa* JH101 provided by this invention has a good control effect on tea tree diseases and provides an excellent starting strain for the further research and development of microbial biofungicides. It can be applied to the green, safe, and efficient control of plant diseases, possessing broad application potential and significant economic, ecological, and environmental value. Furthermore, there are currently no reports on the application of *Myceliophthora* fungi as biocontrol agents or for the control of plant pathogens; the implementation of this invention expands the germplasm resources of microorganisms.

[0010] A second aspect of the invention provides a biological fungicide comprising the tea plant endophytic fungus Myceliophthora verrucosa as described above.

[0011] The advantages of the aforementioned biological fungicide over existing technologies are the same as those of the tea tree endophytic fungus Myceliophthora verrucosa over existing technologies, as described above, and will not be repeated here.

[0012] Furthermore, it also includes acceptable functional additives.

[0013] Acceptable functional adjuvants are components that do not interfere with the efficacy of the active ingredient, namely the biological activity of the endophytic fungus *Myceliophthoraverrucosa*, and do not have significant toxicity or pathological effects on organisms (including humans, animals, or plants) at the applied concentration. These include any one or a combination of at least two of solvents, dispersants, stabilizers, wetting agents, emulsifiers, synergists, thickeners, defoamers, suspending agents, antioxidants, lyophilizers, preservatives, and pH adjusters. The use of these components in microbial preparations is well known in the art. Solvents may include water and / or organic reagents, as well as mixtures of these solvents; antioxidants may include one or more of benzoic acid, ascorbic acid, 2,6-di-tert-butyl-4-methylphenol, sodium sulfite, or sodium bisulfite; pH adjusters may include one or more of sodium carbonate, sodium bicarbonate, phosphoric acid, dipotassium hydrogen phosphate, sodium hydroxide, and ammonia. The biocide of the present invention can be prepared into wettable powder, oil, aqueous solution, suspension or lyophilized agent by means of the aforementioned acceptable functional additives.

[0014] A third aspect of the invention provides the application of the tea tree endophytic fungus Myceliophthora verrucosa as described above or the biological fungicide as described above in the prevention and control of tea tree diseases.

[0015] The advantages of the application of the endophytic fungus Myceliophthora verrucosa or biological fungicide in the prevention and control of tea tree diseases compared with the prior art are the same as the advantages of the endophytic fungus Myceliophthora verrucosa compared with the prior art as described above, and will not be repeated here.

[0016] Furthermore, the pathogens causing the tea tree diseases are selected from at least one of Fusarium sp., Colletotrichum gloeosporioides (C. gloeosporioides), Phona sp., and Pseudopestalotiopsis camelliae-sinensis (P. camelliae). Fusarium sp. causes tea wilt, Colletotrichum gloeosporioides causes tea anthracnose and leaf blight, Phona sp. causes tea leaf blight, and Pseudopestalotiopsis camelliae-sinensis causes tea ring spot.

[0017] The advantages and positive effects of this invention are as follows:

[0018] The endophytic fungal strain *Myceliophthora verrucosa* JH101 provided in this application exhibits good antagonistic activity against several common tea tree pathogens (Fusarium, *Colletotrichum gloeosporioides*, *Pseudomonas aeruginosa*, and *Pseudomonas aeruginosa*), and can serve as a biocontrol agent for plant pathogens, providing an excellent starting strain for the research and development of microbial biological fungicides. Compared to traditional chemical control, the biocontrol agent *Myceliophthora verrucosa* JH101 provided by this invention can safely, efficiently, and pollution-free control plant diseases, which helps reduce pollution to the ecological environment, improve crop quality and food safety, and has broad application potential and significant economic, ecological, and environmental value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a PDA plate culture morphology diagram of Myceliophthora verrucosa JH101, an endophytic fungus of tea plant, in Example 1 of the present invention.

[0021] Figure 2 This is a phylogenetic tree diagram of Myceliophthora verrucosa JH101, an endophytic fungus of tea plant in Embodiment 1 of the present invention.

[0022] Figure 3 This invention demonstrates the antifungal effect of the endophytic fungus Myceliophthora verrucosa JH101 plate on the mycelial growth of Fusarium oxysporum in tea plantation in Example 2 of the present invention. The upper part represents the treatment group, and the lower part represents the control group.

[0023] Figure 4 This is a diagram showing the antibacterial effect of the endophytic fungus Myceliophthora verrucosa JH101 on the growth of mycelium of Colletotrichum gloeosporioides in tea trees on a plate according to Example 2 of the present invention. The upper part is the treatment group and the lower part is the control group.

[0024] Figure 5 This is a diagram showing the antibacterial effect of the endophytic fungus Myceliophthora verrucosa JH101 on the growth of hyphae of Pseudomonas aeruginosa in tea plantation according to Example 2 of the present invention. The upper part is the treatment group and the lower part is the control group.

[0025] Figure 6 This is a diagram showing the antibacterial effect of the endophytic fungus Myceliophthora verrucosa JH101 plate against the mycelial growth of tea stem buds in Example 2 of the present invention. The upper part is the treatment group and the lower part is the control group.

[0026] Figure 7 The bar chart shows the change over time in the inhibition rate of the endophytic fungus Myceliophthora verrucosa JH101 on four pathogenic fungi of tea plants in Example 2 of this invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0029] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, etc., should in all cases be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range. Furthermore, it should be noted that the terms "comprising," "including," "containing," "having," etc., are non-limiting in meaning, allowing for the addition of other steps and other components that do not affect the result.

[0030] Unless otherwise specified, all embodiments of the present invention are performed under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the normal temperature is controlled between 10-30°C, preferably between 15-25°C.

[0031] In the following examples, four pathogenic fungi of tea were provided by the State Key Laboratory of Tea Biology and Resource Utilization of Anhui Agricultural University: Fusarium sp. (causative agent of tea wilt), C. gloeosporioides (causative agent of tea anthracnose / cloud leaf blight), P. camelliae (causative agent of tea ring spot disease), and Phona sp. (causative agent of tea blight).

[0032] It should be noted that experimental methods without specific conditions in the following embodiments are generally performed according to the conditions recommended by the manufacturer or conventional experimental methods in the art.

[0033] Example 1: Isolation and Identification of the Endophytic Fungus Myceliophthora verrucosa from Tea Plants

[0034] Take root, stem and leaf tissues from healthy tea trees, wash off the surface soil with tap water, soak them in 70% alcohol for 1 minute, then soak them in 3% sodium hypochlorite solution for 4 minutes, then soak them in 70% alcohol for 1 second, and finally rinse them 5 times with sterile water. Take 100 μL of the sterile water from the last rinse and spread it on PDA medium as the medium control group.

[0035] Isolating endophytic fungi from tea plant root, stem, and leaf tissues includes the following steps: After surface sterilization, tea plant tissue is cut into 5mm × 5mm pieces, and the cut surfaces are placed on PDA medium. The pieces are incubated at 25℃ for 7 days, with daily observation. Newly grown mycelia are then isolated and cultured separately using a transfer loop. Figure 1 The image shows the colony morphology of the endophytic strain isolated in this invention after 7 days of culture on a PDA plate. The image shows that the colonies are milky white and flocculent, with a round shape and relatively regular edges. The hyphae are septate, transparent or translucent, which is consistent with the colony morphology characteristics of the genus *Hymenopterus*.

[0036] DNA was extracted from the endophytic strain, and the ITS and 18S genes of the isolated endophytic bacteria were sequenced. The sequenced sequences were then aligned using the NCBI BLAST online tool, yielding a similar species named *Myceliophthora verrucosa*. Furthermore, ITS sequences of existing species in the *Myceliophthora* genus were downloaded from GenBank, and multiple sequence alignments were performed on the isolated endophytic strain and these known species using MEGA-X software. A phylogenetic tree of the isolated endophytic strain and known *Myceliophthora* species was constructed using the Neighbor-Joining method with MEGA-X software. The isolated endophytic strain clustered with *Myceliophthora verrucosa* (GenBank gene code KM527251.1:25-571) in the same branch, and the molecular phylogenetic results supported the morphological identification results.

[0037] The endophytic strain isolated in this invention was identified as Myceliophthora verrucosa by morphological and molecular biological methods, and named as: Myceliophthora verrucosa JH101.

[0038] The sequence of the ITS gene obtained from sequencing is shown below:

[0039] (See SEQ ID NO: 1).

[0040] The aforementioned Myceliophthora verrucosa JH101 was deposited at the China Center for Type Culture Collection (CCTCC) on October 31, 2023, with accession number CCTCC NO: M 20232089. The deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University). The culture is classified as Myceliophthora verrucosa.

[0041] Example 2: Biocontrol Antagonism Experiment of Myceliophthora verrucosa JH101

[0042] In this embodiment, the endophytic fungus *Myceliophthora verrucosa* JH101 was subjected to a plate confrontation experiment with several pathogenic fungi of tea plants. The diameter of the colonies was counted at different incubation times, and the inhibition rate was calculated. The pathogenic fungi used were preserved and provided by the State Key Laboratory of Tea Tree Biology and Resource Utilization of Anhui Agricultural University. The four pathogenic fungi of tea plants were: *Fusarium* sp. (causative agent of tea wilt), *C. gloeosporioides* (causative agent of tea anthracnose / cloud leaf blight), *P. camelliae* (causative agent of tea leaf spot), and *Phoma* sp. (causative agent of tea blight).

[0043] The specific steps are as follows:

[0044] Plate confrontation method: A 5 mm radius fungal disc was cut from the edge of the pathogen colony and inoculated onto the center of a PDA medium plate with the hyphae facing down. Four endophytic fungal discs of Myceliophthora verrucosa JH101 were cut in the same way and inoculated at four directions 2.5 cm away from the pathogen discs. A plate without Myceliophthora verrucosa discs was used as a control. The plates were incubated at 25°C for 7 days. The diameter of the pathogen colony was measured daily, and the inhibition rate was calculated.

[0045] Method for calculating the antibacterial rate: Antibacterial rate of antagonistic bacteria (%) = ((D) 对照组 -5)-(D 处理组 -5)) / (D 对照组 -5); where D 对照组 D represents the colony diameter in the control group. 处理组 Indicates the diameter of the colonies in the treatment group.

[0046] Figure 3-6 The antifungal effect of the endophytic fungus Myceliophthora verrucosa JH101 plate on the mycelial growth of four tea tree pathogens is shown in the figure. The upper part of the figure is the treatment group and the lower part is the control group. Figure 7The study shows the time-dependent inhibition rate of the endophytic fungus *Myceliophthora verrucosa* JH101 against four pathogenic fungi of tea. The results indicate that *Myceliophthora verrucosa* JH101 inhibited the mycelial growth of three pathogenic fungi of tea—*Fusarium*, *Pseudomonas aeruginosa*, and *Colletotrichum gloeosporioides*—by more than 53% after 5 days of culture. Furthermore, the antagonistic effect became more pronounced with prolonged culture time. By day 7 of culture, *Myceliophthora verrucosa* JH101 achieved inhibition rates of over 58% against all four selected pathogenic fungi, particularly against *Colletotrichum gloeosporioides*, where the mycelial growth inhibition rate reached as high as 70% after 7 days.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An endophytic fungus of tea trees, characterized in that, The accession number is CCTCC NO: M 20232089, and the taxonomic name is Myceliophthora verrucosa.

2. A biological bactericide, characterized in that, Including the endophytic fungi of tea trees as described in claim 1.

3. The biocidal agent according to claim 2, characterized in that, It also includes acceptable functional additives, which include one or more of solvents, dispersants, stabilizers, wetting agents, emulsifiers, synergists, thickeners, defoamers, suspending agents, antioxidants, lyophilizers, preservatives, and pH adjusters.

4. The application of the endophytic fungi of tea trees as described in claim 1 or the biological fungicide as described in any one of claims 2-3 in the prevention and control of tea tree diseases, wherein the pathogenic fungi of the tea tree diseases are selected from at least one of Fusarium, Colletotrichum gloeosporioides, Pseudomonas spp. and Pseudomonas chapensis.

5. The application of the endophytic fungi or biological fungicide of tea trees according to claim 4 in the prevention and control of tea tree diseases, characterized in that, The tea tree diseases mentioned are at least one of the following: tea wilt, tea anthracnose, tea leaf blight, tea ring spot, and tea leaf blight.

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