Plant endophyte Microcera larvarum ML-5 and application thereof in prevention and treatment of carya illinoensis diseases

Abstract

The invention discloses a plant endophyte Microcera larvarum ML-5 and application of the plant endophyte Microcera larvarum ML-5 in prevention and treatment of carya illinoensis diseases, and belongs to the technical field of microorganism application. The preservation number of the plant endophyte is CCTCC (China Center for Type Culture Collection) NO: M 2025636, and the preservation number of the plant endophyte is CCTCC NO: M 2025636. The compound can effectively inhibit carya illinoensis black spot colletotrichum gloeosporioides, carya illinoensis leaf spot alternaria, fusarium oxysporum, white rot, fusarium oxysporum and watermelon gummy stem blight. The plant diseases caused by the pathogenic bacteria can be prevented and treated. The plants can be colonized in the plants to form a symbiotic relationship with the plants, so that the immunity of the plants is enhanced, and invasion of pathogenic bacteria is effectively resisted. The endophytic fungi can promote plant growth, improve yield and quality, and bring sustainable development to agricultural production. Therefore, prevention and treatment of diseases by using the plant endophytic fungi is an important direction of agricultural green prevention and control in the future.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a plant endophyte Microcera larvarum ML-5 and application thereof in preventing and controlling diseases of pecan trees. Background Art

[0002] Pecan, commonly known as pecan, belongs to the Juglandaceae family and is a large tree that can reach 50 meters in height. Pecan has large fruits with thin shells, a high kernel yield, and rich nutrition, making it a top-quality health food. Its kernels have a high oil content, with a large proportion of unsaturated fatty acids, making it a top-quality edible oil. At the same time, it has a tall tree shape and excellent wood, making it an ideal material for construction and furniture, and an excellent greening tree species. So far, research on pecan has been limited to its edible and economic value, and there are few reports on the endophytic fungi associated with it.

[0003] During their growth, thin-shelled pecan trees face threats from a variety of diseases, such as black spot, anthracnose, powdery mildew, and a variety of pests. These diseases not only affect the healthy growth of the tree, but also lead to a decrease in fruit yield and quality. Therefore, improving the disease resistance of thin-shelled pecan trees is an important measure to ensure their stable production. Among them, anthracnose is the most serious disease of thin-shelled pecans. Anthracnose initially causes brown circular lesions on the peel, sometimes arranged in concentric rings. In severe cases, the entire fruit rots, shrinks, and falls off. At the same time, the leaves have irregular shapes or strip-shaped yellow spots on both sides of the veins. In severe cases, the entire leaf dries up and falls off. Anthracnose fungi overwinter in tissues such as susceptible branches and tips, old ulcer lesions, diseased buds and scales, and residual diseased fruits. In the spring and summer of the following year, the pathogen produces conidia, which are spread by wind, rain, insects, etc., and invade from wounds, insect holes, lenticels, etc. The pathogen lurks in the site of invasion and causes harm when the temperature and humidity are suitable and the host's disease resistance is poor.

[0004] Traditional agricultural control measures are regional, not necessarily effective for specific diseases, and cannot be used as emergency measures to deal with large outbreaks of diseases. Chemical control may lead to soil and water pollution, destroy ecological balance, enhance pest resistance, affect human health, and increase economic costs. Traditional chemical control, if used improperly, will cause plant damage and pathogens to develop resistance. In addition, it may kill beneficial microorganisms in the plant microenvironment and cause poisoning in humans and livestock. Use biological agents for control, such as Beauveria bassiana. These biological agents not only prevent pathogens, but are also environmentally friendly and meet the requirements of green production.

[0005] The Chinese patent application document with the publication number CN111349568A discloses plant endophytic fungal strains, strains and their isolation and application methods. The plant endophytic flora consists of at least two plant endophytic strains, namely (Parengyodontium SP.) MD313901 and (Purpureocillium SP.) MD313902. The plant endophytic flora is derived from natural plant medicinal materials, and the two strains may come from the same plant medicinal material or different plant medicinal materials. The preparation method of the plant endophytic flora includes plant tissue pretreatment, extraction of plant tissue endophytes, isolation and purification of plant tissue endophytes. However, this endophytic fungus does not have the effect of resisting walnut pathogens, so further research and improvement are needed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to provide a plant endophyte and its application in preventing and controlling the diseases of Carya illinoinensis trees.

[0007] The present invention solves the above technical problems by the following technical means:

[0008] In the first aspect of the present invention, a plant endophyte Microcera larvarum ML-5 is provided, and the preservation number is CCTCC NO: M 2025636.

[0009] The plant endophyte is classified and named as Microcera larvarum ML-5, preserved in the China Center for Type Culture Collection, the preservation date is March 31, 2025, and the preservation number is CCTCC NO: M 2025636.

[0010] In the second aspect of the present invention, an inactivated cell body, extract or fermentation product of the above plant endophyte Microcera larvarum ML-5 is provided.

[0011] Preferably, the extract of the plant endophyte Microcera larvarum ML-5 includes the lysate extracted from the plant endophyte.

[0012] Preferably, the fermentation product of the plant endophyte Microcera larvarum ML-5 is obtained by inoculating the Microceralarvarum ML-5 strain cake into a PDB medium and culturing it at a constant temperature of 25-27 °C with shaking.

[0013] In the third aspect of the present invention, a composition is provided, and its main active ingredient includes the plant endophyte Microceralarvarum ML-5, or its inactivated cell body, extract or fermentation product.

[0014] Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipient.

[0015] "Pharmaceutically acceptable" means a non-toxic material that does not reduce the activity of the active ingredient. Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington’s Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd edition, edited by A. Kibbe, Pharmaceutical Press (2000)).

[0016] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, and disintegrants.

[0017] The fourth aspect of the present invention provides the use of the above-mentioned endophytic fungus Microcera larvarum ML-5, or its inactivated cells, extracts or fermentation products, or the above-mentioned composition in inhibiting pathogenic bacteria or controlling plant diseases.

[0018] Preferably, the pathogenic bacteria include one or more of Colletotrichum fructicola causing pecan black spot, Alternaria alternata causing pecan leaf spot, Ceriporiopsis subvermispora, Gloeophyllum trabeum, Fusarium decemcellulare, and Stagonosporopsis cucurbitacearum causing watermelon gummy stem blight.

[0019] Preferably, the control of plant diseases includes the control of pecan black spot, white rot, brown rot, gummy stem blight, etc.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The Microcera genus fungus Microcera larvarum ML-5 isolated in the present invention can effectively inhibit Colletotrichum fructicola causing pecan black spot, Alternaria alternata causing pecan leaf spot, Gloeophyllum trabeum, Ceriporiopsis subvermispora, Fusarium decemcellulare, and Stagonosporopsis cucurbitacearum causing watermelon gummy stem blight. It can control plant diseases caused by the above-mentioned pathogenic bacteria.

[0022] 2. The fermentation broth products of the Microcera larvarum ML-5 strain of the present invention all have a certain growth inhibitory effect on 6 plant pathogenic bacteria. The inhibitory effects of the fermentation broth products of the endophytic fungus Microcera larvarum ML-5 strain on the 6 pathogenic bacteria are, from low to high, Alternaria alternata of pecan leaf spot (81%), Gloeophyllum trabeum (80%), Colletotrichum fructicola of pecan anthracnose (79%), Didymella bryoniae of watermelon (76%), Fusarium decemcellulare (59%), and Coniella diplodiella (36%). Among them, the inhibitory effects on 4 pathogenic bacteria reach more than 70%.

[0023] 3. The method of the present invention is used to control pecan diseases. As a precious resource in nature, plant endophytic fungi have significant advantages in preventing plant diseases. They can colonize in plants, form a symbiotic relationship with plants, enhance the immunity of plants, and effectively resist the invasion of pathogenic bacteria. This biological control method is environmentally friendly and safe, reduces the use of chemical pesticides, and is conducive to protecting the ecological environment and biodiversity. At the same time, endophytic fungi can also promote plant growth, improve yield and quality, and bring sustainable development to agricultural production. Therefore, using plant endophytic fungi to control diseases is an important direction for future green prevention and control in agriculture. Description of the Drawings

[0024] Figure 1 It is the morphological appearance diagram and microscopic diagram of the endophytic fungus Microcera larvarum ML-5 strain in Example 1 of the present invention; among them, A and B are the appearance diagrams of the endophytic fungus Microcera larvarum ML-5 strain on the 10th day of growth, A is the front view of the strain, B is the back view of the strain, and C is the microscopic diagram of the endophytic fungus Microcera larvarum ML-5 strain on the 10th day of growth;

[0025] Figure 2 It is the gene phylogenetic tree of the endophytic fungus Microcera larvarum ML-5 strain in Example 1 of the present invention.

[0026] Figure 3Antagonistic effects of the endophytic fungus Microcera larvarum ML-5 strain against 6 common plant pathogens in Example 2 of the present invention; among them, A is the confrontation plate of Microcera larvarum ML-5 strain and pecan anthracnose; B is the confrontation plate of Microcera larvarum ML-5 strain and pecan leaf spot; C is the confrontation plate of Microcera larvarum ML-5 strain and white rot fungus; D is the confrontation plate of Microcera larvarum ML-5 strain and Gloeophyllum trabeum; E is the confrontation plate of Microcera larvarum ML-5 strain and Fusarium decemcellulare; F is the confrontation plate of Microcera larvarum ML-5 strain and watermelon gummy stem blight;

[0027] Figure 4 Inhibitory rate chart of the endophytic fungus Microcera larvarum ML-5 strain against 6 common plant pathogens in Example 2 of the present invention;

[0028] Figure 5 Fermentation broth diagram of the endophytic fungus Microcera larvarum ML-5 and two pathogens in Example 2 of the present invention, where A is the fermentation broth of the endophytic fungus Microcera larvarum ML-5, B is the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola, and C is the fermentation broth of Colletotrichum fructicola; a is the fermentation broth of the endophytic fungus Microcera larvarum ML-5, b is the mixed fermentation broth of Microcera larvarum ML-5 and Alternaria alternata, and c is the fermentation broth of Alternaria alternata;

[0029] Figure 6This is a graph showing the proportion of each strain in the mixed fermentation broth of endophytic fungus Microcera larvarum ML-5 and two pathogenic bacteria in Example 2 of the present invention. Among them, A is the proportion of Microcera larvarum ML-5 in the mixed fermentation broth of Microcera larvarum ML-5 and two pathogenic bacteria, B is the proportion of Colletotrichum fructicola in the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola, and C is the proportion of Alternaria alternata in the mixed fermentation broth of Microcera larvarum ML-5 and Alternaria alternata;

[0030] Figure 7 This is the KEGG enrichment graph of the mixed fermentation broth of endophytic fungus Microcera larvarum ML-5 and two pathogenic bacteria in Example 2 of the present invention; among them, A is Microcera larvarum ML-5 and Colletotrichum fructicola, and B is Microcera larvarum ML-5 and Alternaria alternata;

[0031] Figure 8 This is the total component analysis graph of the mixed fermentation broth of endophytic fungus Microcera larvarum ML-5 and two pathogenic bacteria in Example 2 of the present invention; among them, A is Microcera larvarum ML-5 and Colletotrichum fructicola, and B is Microcera larvarum ML-5 and Alternaria alternata;

[0032] Figure 9 This is the heat map of metabolic pathways of the mixed fermentation broth of endophytic fungus Microcera larvarum ML-5 and two pathogenic bacteria in Example 2 of the present invention; among them, A is Microcera larvarum ML-5 and Colletotrichum fructicola, and B is Microcera larvarum ML-5 and Alternaria alternata;

[0033] Figure 10This is a diagram showing the preventive effect of the endophytic fungus Microcera larvarum ML-5 on detached thin-shelled pecan leaves in Example 2 of the present invention; wherein A is Colletotrichum fructicola + Microcera larvarum ML-5, and B is pure water + Microcera larvarum ML-5;

[0034] Figure 11 This is a diagram showing the preventive effect of the endophytic fungus Microcera larvarum ML-5 on in vitro thin-shelled pecan fruit in Example 2 of the present invention; wherein A is Colletotrichum fructicola + Microcera larvarum ML-5, and B is pure water + Microcera larvarum ML-5. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0037] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0038] The bacterial strain Microcera larvarum ML-5 is isolated from a walnut tree.

[0039] Example 1: Isolation and screening of endophytic fungi in walnuts

[0040] 1. Experimental Materials and Methods

[0041] 1. Experimental materials: The tissue of thin-shelled pecan was collected from the teaching demonstration base of Anhui Agricultural University

[0042] 2. Isolation and purification of endophytic fungi

[0043] The collected healthy plant samples without pests and diseases were rinsed with running water to remove surface impurities, and the surface moisture of the plant samples was blotted dry with filter paper. Inside a laminar flow hood, the samples were soaked in 75% ethanol solution for 1 min, then soaked in 2.5% sodium hypochlorite solution for 5 min, and finally rinsed 3 - 5 times with sterile water. 100 μL of the sterile water used to wash the experimental materials for the last time was aspirated and smeared on fresh PDA medium as a sterile control to check whether the disinfection was complete. The endophytic fungi of Carya illinoinensis were isolated by tissue isolation method. Inside a laminar flow hood, the disinfected experimental materials were cut into 5×5 mm tissue blocks with a sterile scalpel, and then placed in PDA medium containing streptomycin for culture with sterilized forceps. Four tissue blocks were placed in each petri dish and incubated at a constant temperature of 25°C in an incubator. When hyphae grew out from the edges of the plant tissue blocks in the petri dish, the tip hyphae picking method was used to pick different morphological hyphae on the plate and transfer them to fresh PDA medium without antibiotics for culture. The growth status of the hyphae was observed every day and purified multiple times until colonies with consistent morphology and neat edges, namely purified endophytic fungi, were obtained. The strains isolated in this experiment were inoculated into 20% glycerol solution and stored frozen in an ultra-low temperature freezer (-80°C). And it was named Microcera larvarum ML-5.

[0044] 3. Morphological identification of the endophytic fungus Microcera larvarum ML-5 strain

[0045] The Microcera larvarum ML-5 strain was inoculated on PDA medium and incubated at a constant temperature of 25°C. The colony morphology and size of the strain on PDA medium were observed daily. On the 10th day, the morphological characteristics of the colony were observed and recorded (as Figure 1 shown), the growth diameter of the colony was measured by the cross method, with 3 replicates. Then, a small amount of hyphae was picked and placed on a glass slide to observe its hyphal morphology and spore-bearing structure under a microscope. Based on these characteristics and relevant materials such as "Fungal Identification Handbook", the fungus was preliminarily identified.

[0046] 4. Molecular biological identification of the endophytic fungus Microcera larvarum ML-5 strain

[0047] The Microcera larvarum ML-5 strain was inoculated on PDA medium and cultured at 25°C for 7 days. The hyphae were scraped off, and DNA was extracted using a fungal DNA extraction kit.

[0048] Using the universal primer ITS4 (5′-TCCTCCGCTTATTG ATATGC-3′ SEQ ID NO:1)

[0049] and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′ SEQ ID NO:2),

[0050] LR5 (5′-ATCCTGAGGGAAACTTC-3′ SEQ ID NO:3)

[0051] and LROR (5′-GTACCCGCTGAACTTAAGC-3′ SEQ ID NO:4),

[0052] bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′ SEQ ID NO:5)

[0053] and bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′ SEQ ID NO:6) were used for PCR amplification to isolate the ITS rDNA, LSU rDNA and TUB rDNA sequences of endophytic fungi. Using Tilachlidium brachiatum as an outgroup, a phylogenetic tree was constructed by the Neighbor-Joining method (NJ) in the software IQ-TREE for species identification. The sequenced results were analyzed by BLAST sequence alignment in the GenBank database to find the strain with the highest similarity and determine the taxonomic status of the strain.

[0054] II. Experimental Results

[0055] 1. Morphological identification of the endophytic fungus Microcera larvarum ML-5 strain

[0056] The endophytic fungus Microcera larvarum ML-5 strain was cultured at a constant temperature of 25°C on PDA medium. The mycelium was milky white with concentric rings. The colony was regular or irregularly round. The colony was initially white. After 1-2 days of culture growth, new mycelia could be seen and yellow pigment was secreted. On the 10th day of growth, the back of the colony was light yellow with white mycelia at the edge. The endophytic fungus Microcera larvarum ML-5 strain was preliminarily identified as the genus Microcera.

[0057] 2. Molecular biological identification of the endophytic fungus Microcera larvarum ML-5 strain

[0058] After DNA sequencing, the obtained sequence was subjected to homology alignment on GenBank using BLAST. Closely related species within the same genus with homology ≥ 98% were selected to construct an ITS-LSU-TUB three-gene phylogenetic tree. Through sequence alignment, it can be seen that the endophytic fungus Microcera larvarum ML-5 strain belongs to the Ascomycetes class, the Microcera genus, and is most closely related to Microcera larvarum CBS169.30 and Microcera larvarum CBS133964. Therefore, the endophytic strain was named Microcera larvarum ML-5 (as Figure 2 shown).

[0059] Example 2: Application of the obtained pecan endophytic fungus Microcera larvarum ML-5

[0060] I. Experimental materials and methods

[0061] 1. Experimental strains

[0062] The pecan endophytic fungus Microcera larvarum ML-5 strain isolated in Example 1;

[0063] Plant pathogens: Colletotrichum fructicola causing pecan black spot, Alternaria alternata causing pecan leaf spot, Ceriporiopsis subvermispora, Gloeophyllum trabeum, Fusarium decemcellulare, Stagonosporopsis cucurbitacearum causing watermelon gummy stem blight, etc. These pathogens were all provided by the RCEF strain bank of the Anhui Key Laboratory of Microbial Control (http: / / mc.ahau.edu.cn / index.htm).

[0064] 2. Culture media

[0065] Potato Dextrose Agar Medium (PDA): 200 g of potatoes, 20 g of glucose, 15 g of agar powder, 1000 mL of distilled water. Potato Dextrose Medium (PDB): 200 g of potatoes, 20 g of glucose, 1000 mL of distilled water.

[0066] 3. Antagonistic effect of the endophytic fungus Microcera larvarum ML-5 strain against 6 common plant pathogens

[0067] Determined by the plate confrontation method. Six plant pathogens, namely *Xanthomonas campestris* pv. *caryae*, *Alternaria alternata*, *Phanerochaete velutina*, *Gloeophyllum trabeum*, *Fusarium decemcellulare*, and *Didymella bryoniae* (5 mm), were respectively inoculated in the center of PDA plates. The mycelial plugs (5 mm) of *Microcera larvarum* ML-5 strain were inoculated at four points on the ≥20 mm cross line centered on the mycelial plug by the cross method. Each treatment had 3 replicates, and the treatment with only plant pathogens inoculated was used as the control, also with 3 replicates. After culturing at 25 °C for 7 d, the colony diameter was measured, and the inhibition rate (I / %) was calculated according to the following formula. (As Figure 3 and Figure 4 shown)

[0068] I / % = (C - T) / (C - 5)×100

[0069] Where: C is the colony diameter of the control, and T is the colony diameter of the treatment group.

[0070] 4. Antibacterial activity of the fermentation product of endophytic fungus *Microcera larvarum* ML-5 strain

[0071] Five mycelial plugs of *Microcera larvarum* ML-5 strain and five mycelial plugs of *Xanthomonas campestris* pv. *caryae* were punched out with a puncher (5 mm), and then inoculated into a conical flask containing 100 ml of sterilized PDB medium. The control group repeated the above operation, and each treatment had 6 replicates. Fermentation was carried out on a shaker at 25 °C and 120 r / min. After fermentation, the fermentation broth was taken out and aliquoted into centrifuge tubes, centrifuged at 2800 r / min for 15 min, and part of the supernatant was poured out. The remaining fermentation product was stored in liquid nitrogen.

[0072] 5. Biocontrol effect of the fermentation product of endophytic fungus *Microcera larvarum* ML-5 strain on detached leaves and fruits of *Carya illinoinensis* against *Xanthomonas campestris* pv. *caryae*

[0073] Prevention experiment: Fresh, healthy leaves and fruits without pests and diseases were collected. First, they were disinfected with 75% ethanol solution, then rinsed 3 - 4 times with sterile water and dried naturally. Then, a quantitative and fixed-concentration fermentation product was sprayed on the leaves and fruits of *Carya illinoinensis* using a sprayer. The control group was sprayed with an equal amount of sterile water. After the leaves and fruits were dry, artificial inoculation was carried out for in vitro inoculation. The leaves and fruits were pricked with a sterile needle, and a mycelial block of *Xanthomonas campestris* pv. *caryae* was placed at the pricked site, and the wound was wrapped with moist sterile cotton. The treated leaves and fruits were placed in a petri dish; the control group repeated the above operation, and each treatment had 3 replicates. After marking respectively, they were placed in an incubator at 25 °C, and the environment in the dish was kept moist during the period.

[0074] II. Experimental results

[0075] 1. Antagonistic effect of the endophytic fungus Microcera larvarum ML-5 strain against 6 common plant pathogens

[0076] The antagonistic activity of Microcera larvarum ML-5 strain against 6 plant pathogens was determined by the plate confrontation method. The inhibition rate of the endophytic fungus was calculated based on the colony diameter of the pathogen on the 7th day. The inhibition rates against 7 pathogens from high to low were 79% for Gnomonia leptostyla, 81% for Cercospora illinoinensis, 36% for Trichaptum abietinum, 80% for Gloeophyllum trabeum, 59% for Fusarium decemcellulare, and 76% for Didymella bryoniae. Compared with the control group, the inhibitory effects on Gnomonia leptostyla and Cercospora illinoinensis were better, while the inhibitory effects on Trichaptum abietinum, Gloeophyllum trabeum, Fusarium decemcellulare, and Didymella bryoniae were relatively weaker.

[0077] 2. Bacteriostatic activity of the fermentation broth products of the endophytic fungus Microcera larvarum ML-5 strain

[0078] As Figures 5 - 6 shown, Microcera larvarum ML-5 was mixed and fermented with two pathogen strains respectively. When Microcera larvarum ML-5 was mixed and fermented with Colletotrichum fructicola, the content of Microcera larvarum ML-5 decreased significantly, while when Microcera larvarum ML-5 was mixed and fermented with Alternaria alternata, the content of Alternaria alternata decreased to 0. This shows that Microcera larvarum ML-5 has a more obvious inhibitory effect on Alternaria alternata, the pathogen of Cercospora illinoinensis of pecan.

[0079] As Figure 7As shown in the figure, we used the phper function in R software to perform KEGG (Kyoto Encyclopedia of Gene and Genomes) functional enrichment analysis. The results showed that the differential genes of Microcera larvarum ML-5 + Colletotrichum fructicola were mainly enriched in ribosome, oxidative phosphorylation, and fatty acid elongation. The differential genes of Microcera larvarum ML-5 + Alternaria alternata were mainly enriched in oxidative phosphorylation, phagosome, ribosome, and RNA degradation.

[0080] As Figure 8 shown in the figure, in the total component analysis of the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola, the proportion of Microcera larvarum ML-5 was greater than that of Colletotrichum fructicola, indicating that Microcera larvarum ML-5 had an obvious inhibitory effect on Colletotrichum fructicola, the pathogen of pecan anthracnose. In the total component analysis of the mixed fermentation broth of Microcera larvarum ML-5 and Alternaria alternata, there was an obvious intersection in the spatial distribution maps of the three components, which might be the reason for the obvious inhibitory effect of Microcera larvarum ML-5 on Alternaria alternata, the pathogen of pecan leaf spot.

[0081] As Figure 9As shown, in the fermentation broth of Microcera larvarum ML-5 and the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola, the expression levels of myrcene and dihydroactinidiolide in the fermentation broth of Microcera larvarum ML-5 are higher than those in the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola. This may be the reason for the antagonistic effect of Microcera larvarum ML-5 against Colletotrichum fructicola. In the fermentation broth of Microcera larvarum ML-5 and the mixed fermentation broth of Microcera larvarum ML-5 and Alternaria alternata, the expression levels of allothreonine and arginylisoleucine in the fermentation broth of Microcera larvarum ML-5 are significantly higher than those in the mixed fermentation broth of Microcera larvarum ML-5 and Alternaria alternata. This may be the reason for the antagonistic effect of Microcera larvarum ML-5 against Alternaria alternata. From the two figures, it can be seen that the gene expression level of the fermentation broth of Microcera larvarum ML-5 is lower than that of the mixed fermentation broth of Microcera larvarum ML-5 and Colletotrichum fructicola. Therefore, the inhibitory effect of the strain Microcera larvarum ML-5 on Colletotrichum fructicola is weaker than that on the strain Alternaria alternata.

[0082] As Figure 10 , 11 shown, the fermentation products of this strain have a certain preventive effect on pecan anthracnose on detached leaves and fruits.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant endophyte Microcera larvarum ML-5, characterized in that, The preservation number is CCTCC NO: M2025636.

2. The inactivated cells, extracts or fermentation products of the plant endophyte Microcera larvarum ML-5 as claimed in claim 1.

3. The inactivated cells, extracts or fermentation products of the endophytic bacterium Microcera larvarum ML-5 according to claim 2, characterized in that, The extract of the plant endophyte Microcera larvarum ML-5 includes the lysate extracted from the plant endophyte.

4. The inactivated cells, extracts or fermentation products of the endophytic bacterium Microcera larvarum ML-5 according to claim 2, characterized in that, The fermentation product of the plant endophyte Microcera larvarum ML-5 is obtained by inoculating the mycelial cake of Microcera larvarum ML-5 strain into PDB medium and culturing it at a constant temperature of 25-27 °C with shaking.

5. A composition, characterized in that, Its main active ingredient includes the plant endophyte Microcera larvarum ML-5 as claimed in claim 1, or the inactivated cells, extracts or fermentation products of the plant endophyte Microcera larvarum ML-5 as claimed in any one of claims 2-4.

6. The composition according to claim 5, wherein The composition is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier and / or excipient.

7. The composition according to claim 6, wherein The pharmaceutically acceptable carrier includes diluents, binders, surfactants, humectants, adsorption carriers, lubricants, fillers, disintegrants.

8. The application of the plant endophyte Microcera larvarum ML-5 as claimed in claim 1, or the inactivated cells, extracts or fermentation products of the plant endophyte Microcera larvarum ML-5 as claimed in any one of claims 2-4, or the composition as claimed in claim 5 in inhibiting pathogenic bacteria or preventing and controlling plant diseases.

9. The application according to claim 8, characterized in that, The pathogenic bacteria include one or more of Colletotrichum fructicola causing pecan anthracnose, Alternaria alternata causing pecan leaf spot, Sclerotium cepivorum, Gloeophyllum trabeum, Fusarium decemcellulare and Didymella bryoniae.

10. The application according to claim 8, wherein The prevention and control of plant diseases include preventing and controlling pecan anthracnose, white rot, brown rot, gummy stem blight.

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