Plant endophytic fungus JSNL-ZJ63 strain, biocontrol inoculant and application of plant endophytic fungus JSNL-ZJ63 strain

Abstract

The invention belongs to the technical field of plant disease biocontrol, and discloses a plant endophyte penicillium fungus strain, a biocontrol microbial inoculum and application of the biocontrol microbial inoculum, the biocontrol microbial inoculum is named as Penicillium rapai JSNL-ZJ63, the preservation unit is China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.41156. The invention further discloses a preparation method of the biocontrol microbial inoculum. The strain has the capability of remarkably inhibiting various plant pathogenic bacteria, and the pathogenic bacteria comprise one or more of rhizoctonia solani, phomopsis, botrytis cinerea, colletotrichum siamensis and colletotrichum gloeosporioides, are suitable for biological control of plant diseases and can be used for producing biocontrol agents or biopesticides for preventing and treating various plant diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease biocontrol, and relates to a plant endophytic fungus Penicillium raperiJSNL-ZJ63 strain, a biocontrol agent and application thereof. Background Art

[0002] Root rot is a serious soil-borne plant root disease with a high incidence rate, complex pathogens, and compound infections. It is extremely difficult to prevent and control, and is known as "plant cancer". Taking soybean root rot as an example, soybean root rot caused by Rhizoctonia solani can occur from the beginning of seed germination, causing seed rot; after soybean seedlings emerge, the aboveground part shows damping-off symptoms, necrotic spots appear at the base of the seedling stem and the root, and then the spots gradually expand until they surround the stem; the stem and main root pith change color, the growth of the diseased plant is weakened, and sudden wilting occurs in the middle of the growth period, and the diseased plant has fewer pods. When the disease occurs, it can cause a 10%-30% reduction in soybean production, and in severe cases, the loss can reach more than 50%.

[0003] Soybean root rot caused by Phomopsis longicolla is manifested by shrunken and cracked seeds, which are usually chalky and mildewed. They may not germinate or germinate slowly. In the late growth stage of the plant, the disease usually harms the branches and stem nodes, causing dark brown lesions. The epidermis of the affected parts is obviously rotten, ulcerated or necrotic, and is reddish brown or dark brown. The leaves turn yellow or even die, and the flowers and pods develop slowly or even stagnate or die. The disease generally causes a 10%-30% reduction in soybean production. In severe fields, the proportion of diseased plants exceeds 90%, and even leads to a total crop failure.

[0004] Gray mold is widely distributed, spreads rapidly, and causes serious damage, causing huge losses to the yield and quality of fruits and vegetables worldwide, ranking second among the world's top ten plant diseases. Taking strawberry gray mold as an example, the gray mold of strawberry caused by Botrytis cinerea is the most serious fungal disease that harms strawberries after flowering. The disease mainly infects fruits, leaves, fruit stalks, calyx, petals and petioles, causing flower and fruit rot. The diseased fruit rate of susceptible varieties is generally 30%-60%, and in severe cases, even a total crop failure.

[0005] Plant anthracnose has been documented in all regions of the world. In 2009, plant anthracnose was listed as the ninth largest disease in the world. Taking strawberry anthracnose as an example, strawberry anthracnose, caused by the fungus Colletotrichum spp., has been the main disease of strawberries in recent years. It can cause damage to strawberries throughout their entire growth period, mainly during the strawberry seedling period and early planting. When strawberry anthracnose occurs, the leaves, petioles, stipules, runners, petals and fruits of strawberries can all be affected. The disease generally causes a 25%-30% reduction in strawberry yield, up to 80% in severe cases, and even causes the death of the entire greenhouse seedlings.

[0006] The existing technologies for the prevention and control of plant root rot, gray mold and anthracnose mainly include the following methods: 1. Planting disease-resistant varieties. This method is the most economical and effective way to prevent and control plant root rot, gray mold and anthracnose. However, due to the complex field environment and the rapid variation of pathogen virulence, the resistance of varieties is easily lost, making breeding difficult. 2. Chemical control. Although this method can prevent and control plant root rot, gray mold and anthracnose to a certain extent, the large-scale use of chemical agents poses a great threat to the ecological environment and food safety, and will also lead to the continuous enhancement of pathogen resistance.

[0007] Endophytic fungi can help plants obtain nutrients, induce resistance in plants, and improve their ability to survive under biotic or abiotic stress conditions. Since endophytic fungi come from plants, they are safe for plants, environmentally friendly, and not prone to drug resistance. Using these microorganisms to control plant diseases is beneficial to maintaining ecological balance. Therefore, endophytic fungi are a source of new compounds that promote organic agriculture.

[0008] In summary, the prior art still lacks a green and highly effective probiotic for preventing plant root rot, gray mold and anthracnose. Summary of the invention

[0009] In view of the above problems, the present invention provides a biocontrol strain Penicillium raperi JSNL-ZJ63 which can effectively prevent and control plant root rot, gray mold and anthracnose and applications thereof.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] In the first aspect, the present invention provides a plant endophytic fungus JSNL-ZJ63, which is classified and named Penicillium raperi and isolated from the roots of healthy strawberries in Biancheng Town, Jurong City, Zhenjiang City. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC for short) on April 15, 2024, with a deposit number of CGMCC NO.41156, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0012] In a second aspect, the present invention further provides a biocontrol agent for plant root rot, gray mold and anthracnose, wherein the active ingredient of the biocontrol agent includes hyphae or conidia, or extracellular secretions of the plant endophytic fungus JSNL-ZJ63.

[0013] Furthermore, the extracellular metabolites are obtained by culturing the plant endophytic fungus JSNL-ZJ66 in a PDA medium for several days and then removing hyphae and conidia.

[0014] In a third aspect, the present invention also provides a method for preparing the biocontrol agent, comprising inoculating the plant endophytic fungus JSNL-ZJ63 with a preservation number of CGMCC NO.41156 into a sterile culture medium and culturing it in the dark at 22-28°C for several days.

[0015] In a fourth aspect, the present invention further provides a microbial pesticide for preventing and controlling plant root rot and gray mold, wherein the active ingredient of the microbial pesticide comprises the plant endophytic fungus JSNL-ZJ63 described in the first aspect or the biocontrol agent described in the second aspect.

[0016] In a fifth aspect, the present invention further provides the use of the plant endophytic fungus JSNL-ZJ63 described in the first aspect, the biocontrol agent described in the second aspect, or the microbial pesticide described in the fourth aspect in preventing and controlling plant root rot, gray mold and anthracnose.

[0017] Furthermore, the root rot disease includes root rot disease caused by one or both of the pathogens Rhizoctonia solani and Phomopsis solani, and preferably the root rot disease is soybean root rot.

[0018] Furthermore, the gray mold is gray mold caused by Botrytis cinerea.

[0019] Furthermore, the anthrax is caused by one or both of Colletotrichum siamese and Colletotrichum gloeosporioides.

[0020] In a sixth aspect, the present invention further provides a method for preventing and controlling plant root rot and gray mold, the method comprising applying the plant endophytic fungus JSNL-ZJ63 described in the first aspect, the biocontrol agent described in the second aspect, or the microbial pesticide described in the fourth aspect to the plant growth environment.

[0021] In certain embodiments, the plant growth environment is plant roots or leaves.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] 1) The biocontrol bacterium Penicillium raperi JSNL-ZJ63 provided by the present invention can significantly inhibit the mycelial growth of pathogenic bacteria Rhizoctonia solani, Phomopsis longicolla, Botrytis cinerea, Colletotrichum siamense and C. gloeosporioides.

[0024] 2) The biocontrol bacterium Penicillium raperi JSNL-ZJ63 provided by the present invention can significantly reduce the disease severity of soybean root rot and strawberry gray mold, and significantly improve its growth indicators such as germination rate, plant height and fresh weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0026] Figure 1 This is a diagram showing the antagonistic effect of Penicillium raperi JSNL-ZJ63 on different pathogens in Example 1;

[0027] Figure 2 The inhibitory activity of Penicillium raperi JSNL-ZJ63 against different pathogens in Example 1;

[0028] Figure 3 The effect of the extracellular metabolites of Penicillium raperi JSNL-ZJ63 on different pathogens in Example 2;

[0029] Figure 4 The effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by Rhizoctonia solani in Example 3;

[0030] Figure 5 The effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by Rhizoctonia solani in Example 3;

[0031] Figure 6 The effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by Phomopsis longicolla in Example 4;

[0032] Figure 7 The effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by Phomopsis longicolla in Example 4;

[0033] Figure 8 This is the effect of Penicillium raperi JSNL-ZJ63 in Example 5 on strawberry gray mold caused by Botrytis cinerea. DETAILED DESCRIPTION

[0034] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and the protection scope of the present invention cannot be limited by this. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.

[0035] As described in the present invention, the term "biocontrol bacteria" refers to beneficial microorganisms that can prevent and control plant diseases, mainly bacteria, fungi and actinomycetes.

[0036] As described in the present invention, the term "Rhizoctonia solani" belongs to the Imperfecti fungi, Agonomycetales, Agonomycetaceae, Rhizoctoni a, and is a soil-borne pathogenic fungus with a wide host range, strong saprophytic properties, and widespread distribution around the world. It is mainly distributed in the United States, Brazil, Australia, Japan, Canada, Germany, China and other countries.

[0037] As described in the present invention, the term "Phomopsis longicolla" is classified as: Fungi, Ascomycota, Sordariomycete, Diaporthales, Diaporthaceae, Diaporthe. It is widely distributed in the world and causes serious damage in some major soybean producing countries such as the United States, Canada and Argentina.

[0038] As described in the present invention, the term "Botryotinia cinerea" is classified as Fungi, Ascomycota, Leotiomycete, Helotiales, Sclerotiniaceae, Botryotinia. It is mainly distributed in the United States, Sweden, Germany, France, China, Brazil, Japan, South Korea, Argentina, India, Chile and other countries.

[0039] As described in the present invention, the terms "Siamese anthrax" and "Gliosporium anthrax" are classified as Eumycota, Deuteromyeotina, Coelomycetes, Melanconiales, Melanconiaceae, and Colletotrichum. They are mainly distributed in the United States, South Korea, Canada, Australia, Japan, the United Kingdom, China, Belgium, Germany, India, Vietnam, Brazil, Panama, Mexico, Russia, Israel and other countries.

[0040] Example 1 Screening of Penicillium raperi JSNL-ZJ63

[0041] 1. Isolation of potential biocontrol bacteria

[0042] 1.1. Obtaining experimental materials: Select healthy strawberry plants with good growth from the field and take their root tissues.

[0043] 1.2. Isolation and culture of endophytic fungi in strawberry roots: Cut the strawberry root tissue, mix well, weigh 2g, and disinfect the surface. Add 100mL sterile water and 2 drops of Tween 20, shake at 25℃ and 220rpm for 20min, treat with sterile water for 20s, soak in 70% (v / v) ethanol for 30s, soak in 2.5% (v / v) sodium hypochlorite solution for 2min, and finally rinse with sterile water 3-4 times, and dry the surface moisture with sterile absorbent paper. Under sterile conditions, the roots are further cut into smaller segments (about 0.3cm), and 5 segments are randomly selected for each sample and placed on the PDA flat plate culture medium. Seal the culture dish and culture it at 25℃ in the dark for 1-2d. When the mycelium emerges from the root tissue, carefully transfer the small piece of culture medium growing on the edge of the culture medium together with the mycelium to a new PDA plate.

[0044] 2. Screening of antagonistic bacteria by plate confrontation method

[0045] The PDA plate confrontation method was used. The root rot pathogen, gray mold pathogen and anthracnose pathogen cake (5 mm in diameter) and the endophytic fungus cake (5 mm in diameter) cultured for 3-5 days were symmetrically inoculated on a 9 cm diameter plate containing 15 mL of PDA medium 2 cm away from the edge. The plate inoculated with only the root rot pathogen, gray mold pathogen and anthracnose pathogen cake was used as the control. The plate was cultured at 25 ° C in the dark. After 5-10 days, the colony radius of each pathogen was measured and the inhibition rate was calculated. The inhibition rate was calculated according to the formula: inhibition rate = (colony radius of the control group - colony radius of the treatment group) / colony radius of the control group × 100%.

[0046] Finally, a strain with good antagonistic activity against root rot, gray mold and anthracnose pathogens on the plate was screened out, which was specifically manifested by strong inhibition of the growth of pathogenic hyphae. It was numbered JSNL-ZJ63.

[0047] like Figure 1 As shown, compared with the control, the colonies of the pathogenic bacteria in the experimental group were significantly smaller, and the inhibition rates of JSNL-ZJ63 against R. solani, P. longicolla, B. cinerea, C. siamense and C. gloeosporioides were 44.30%, 53.01%, 56.88%, 49.70% and 50.71%, respectively, indicating that the mycelial growth of the above pathogens was inhibited.

[0048] like Figure 2 As shown, the results of optical microscopic observation showed that the mycelium of the pathogenic bacteria in the control group had regular appearance, the contents were evenly distributed in the mycelial cell wall, and the growth was straight; after confrontation culture with JSNL-ZJ63, the internal substances of the mycelium of R.solani, P.longicolla, B.cinerea, C.siamense and C.gloeosporioides condensed, were chaotically distributed, and intertwined, and could not grow normally.

[0049] 3. Molecular biological identification of the strains obtained

[0050] 3.1. The genomic DNA of strain JSNL-ZJ63 was extracted using the kit (DP320-03) from TIANGEN.

[0051] 3.2. Amplify the ITS gene and BenA gene of genomic DNA respectively. DNA amplification uses a 30 μL reaction volume, including 15 μL 2×EasyTaq PCR SuperMix (+dye), 1 μL of primer F / R (10 μM each), 2 μL template DNA and 11 μL ddH 2O, PCR amplification program conditions are as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 54°C annealing for 45 s, 72°C extension for 60 s, for a total of 35 cycles; and finally 72°C extension for 10 min.

[0052] 3.3. The amplified PCR products were subjected to 1% agarose gel electrophoresis and observed under ultraviolet light. The PCR products with target bands were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were as follows: the ITS gene sequence of the strain JSNL-ZJ63 screened in step 2 was SEQ ID NO.1, and the BenA gene sequence was SEQ ID NO.2.

[0053] 3.4. Compare SEQ ID NO.1 and SEQ ID NO.2 on the NCBI database website. The comparison results are shown in Table 1. According to the results in Table 1, it can be inferred that the biocontrol bacterium JSNL-ZJ63 of the present invention is Penicillium raperi, and is named Penicillium raperi JSNL-ZJ63.

[0054] Table 1 Sequence alignment results of SEQ ID NO.1 and SEQ ID NO.2

[0055]

[0056] Example 2 Effects of extracellular metabolites of Penicillium raperi JSNL-ZJ63 on root rot and gray mold pathogens

[0057] 1. Inoculate R.solani, P.longicolla, B.cinerea and Penicillium raperi JSNL-ZJ63 in 9 cm diameter culture dishes containing 15 mL PDA medium, place in a 25°C incubator, and culture in the dark for 3-5 days. Use a cork puncher (5 mm in diameter) to punch holes at the edge of the colony to prepare bacterial plates.

[0058] 2. Acquisition of extracellular metabolites secreted by strain JSNL-ZJ63

[0059] A water filter membrane (pore size 0.22 μm, diameter 5 cm) was covered on a 6 cm diameter PDA medium plate, and the filter membrane was closely attached to the surface of the PDA plate. Then, a Penicillium raperi JSNL-ZJ63 bacterial plate (diameter 5 mm) was inoculated on the filter membrane, and a blank PDA medium was used instead of the JSNL-ZJ63 bacterial plate (diameter 5 mm) for control, and cultured at 25°C in the dark.

[0060] 3. After culturing for 2 days, use tweezers to carefully remove the filter membrane together with the Penicillium raperi JSNL-ZJ63 culture on it. For control, tear off the filter membrane and blank PDA block to prepare plates containing JSNL-ZJ63 extracellular metabolites and blank PDA plates.

[0061] 4. In an ultra-clean workbench, use an inoculation needle to pick up each pathogenic bacteria disc (5 mm in diameter) and inoculate it into the above-prepared flat plate, with the mycelium facing downward. Set up 3 replicates for each.

[0062] 5. Place the inoculated plate in a 25°C incubator, incubate in dark for 2-3 days, and measure the colony size using the cross method. The inhibition rate is calculated according to the formula: inhibition rate = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100%.

[0063] The results are as follows Figure 3 As shown, compared with the control, the colonies of the pathogenic bacteria in the experimental group were significantly smaller, and the inhibition rates of the extracellular metabolites secreted by the strain JSNL-ZJ63 on R.solani, P.longicolla and B.cinerea were 20.94%, 8.80% and 28.76%, respectively. The hyphae at the edge of the colony in the control group were dense, while the hyphae at the edge of the colony in the treatment group were sparse, indicating that the extracellular metabolites secreted by JSNL-ZJ63 had a certain inhibitory effect on the hyphae growth of the above-mentioned root rot and gray mold pathogens.

[0064] Example 3 Effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by R. solani

[0065] 1. Inoculate Penicillium raperi JSNL-ZJ63 and R.solani into 9-cm diameter culture dishes containing 15 mL of PDA culture medium, place them in a 25°C incubator and culture in the dark for 5 days.

[0066] 2. Cut 3 mm × 3 mm mycelium blocks from the edge of the JSNL-ZJ63 colony, transfer 10 mycelium blocks to 400 mL PDB liquid culture medium under sterile conditions, and culture at 25°C with shaking for 6 days. Use a blade to cut the R. solani culture medium into small pieces of 0.5 cm × 0.5 cm.

[0067] 3. Select pots with a diameter of 12 cm and set up two groups for the experiment. The experimental group is: 400 mL of JSNL-ZJ63 bacterial suspension and 2 R. solani flat plates were added to each pot; the control group is: 400 mL of PDB liquid culture medium and 2 R. solani flat plates were added to each pot, and 3 replicates were set for each treatment.

[0068] 3.1. Mix the above treatments with sterilized vermiculite evenly, add to 2 / 3 of the pot, put 10 soybeans (variety: Dongsheng No. 1) in each pot, and then cover them with a thin layer of vermiculite mixture.

[0069] 3.2. Water each group of soybeans with sterile water every other day. Count the germination rate, plant height and fresh weight of soybeans 10 days after sowing.

[0070] The results are as follows Figure 4 , Figure 5 And as shown in Table 2.

[0071] Table 2 Statistical results of soybean emergence rate, plant height and fresh weight

[0072]

[0073] from Figure 4 and Figure 5 It can be seen that the plant height, number of sprouts, root length and luxuriance of the experimental group were significantly better than those of the control group. It can be seen that Penicillium raperi JSNL-ZJ63 has a preventive or therapeutic effect on soybean root rot caused by R.solani.

[0074] Example 4 Effect of Penicillium raperi JSNL-ZJ63 on soybean root rot caused by P. longicolla

[0075] 1. Inoculate Penicillium raperi JSNL-ZJ63 and P. longicolla into 9-cm diameter culture dishes containing 15 mL of PDA medium, place them in a 25°C incubator and culture in the dark for 5 days.

[0076] 2. Cut 3 mm × 3 mm mycelium blocks from the edge of the JSNL-ZJ63 colony, transfer 10 mycelium blocks to 400 mL PDB liquid culture medium under sterile conditions, and culture at 25°C with shaking for 6 days. Use a blade to cut the P. longicolla culture medium into small pieces of 0.5 cm × 0.5 cm.

[0077] 3. Select pots with a diameter of 12 cm and set up two groups for the experiment. The experimental group is: 400 mL of JSNL-ZJ63 bacterial suspension and 2 P. longicolla flat plates were added to each pot; the control group is: 400 mL of PDB liquid culture medium and 2 P. longicolla flat plates were added to each pot, and 3 replicates were set for each treatment.

[0078] 3.1. Mix the above treatments with sterilized vermiculite evenly, add to 2 / 3 of the pot, put 10 soybeans (variety: Dongsheng No. 1) in each pot, and then cover them with a thin layer of vermiculite mixture.

[0079] 3.2. Water each group of soybeans with sterile water every other day. Count the germination rate, plant height and fresh weight of soybeans 22 days after sowing.

[0080] The results are as follows Figure 6 , Figure 7 And as shown in Table 3.

[0081] Table 3 Statistical results of soybean emergence rate, plant height and fresh weight

[0082]

[0083] from Figure 6 and Figure 7 It can be seen that the plant height, number of sprouts, root length and luxuriance of the experimental group were significantly better than those of the control group. It can be seen that Penicillium raperi JSNL-ZJ63 has a preventive or therapeutic effect on soybean root rot caused by P. longicolla.

[0084] Example 5 Penicillium raperi JSNL-ZJ63 induces strawberry resistance to gray mold

[0085] 1. Inoculate Penicillium raperi JSNL-ZJ63 and B. cinerea in 9 cm diameter culture dishes containing 15 mL PDA medium, place in a 25°C incubator, and culture in the dark for 5 days. Use a cork puncher (5 mm in diameter) to punch holes at the edges of the B. cinerea and JSNL-ZJ63 colonies to prepare bacterial plates.

[0086] 1.2. Select healthy strawberry leaves, wash them with tap water, disinfect them with 70% (v / v) ethanol twice, rinse the leaf surface with sterile water three times, prick the left and right sides of the leaf back with a sterilized inoculation needle 3-5 times, place the JSNL-ZJ63 bacterial disc on the wound on the right side of the leaf, with the mycelium facing down, and use a PDA blank medium bacterial disc (5mm in diameter) on the left side as a control, and moisturize with sterile wet absorbent cotton. The inoculated leaves are placed in a sterilized tray, two layers of sterile gauze are laid in the tray and wetted with sterile water, and the tray is covered with plastic wrap to keep it moist. Place it at 25°C in the dark for 24 hours.

[0087] 1.3. Remove the JSNL-ZJ63 and PDA blank culture medium plates on both sides of the leaves, replace them with B. cinerea plates, with the mycelium side facing down, replace with new sterile wet absorbent cotton, cover with plastic wrap and seal the tray to keep moisture. Place at 25℃ in the dark for 3 days.

[0088] Check the disease condition of strawberry leaves, record the diameter of lesions using the cross method, and calculate the average value.

[0089] The results are as follows Figure 8 And as shown in Table 4.

[0090] Table 4 Statistical results of the diameter of lesions on strawberry leaves inoculated with B. cinerea

[0091]

[0092] The numerical expression of colony diameter in the table is: mean ± standard error.

[0093] like Figure 8 As shown, the left side of the front of the leaf is inoculated with strain JSNL-ZJ63 24 hours later and then inoculated with B. cinerea strain, and the right side is inoculated with blank PDA 24 hours later and then inoculated with B. cinerea strain. It can be seen that the lesions in the experimental group are significantly smaller than those in the control group, indicating that the strawberry leaves in the experimental group have developed resistance to B. cinerea.

[0094] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values.

Claims

1. A plant endophytic fungus JSNL-ZJ63, characterized in that: The plant endophytic fungus JSNL-ZJ63 is classified and named Penicillium raperi, the preservation number is CGMCC NO.41156, the preservation time is April 15, 2024, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. A biocontrol agent for plant root rot and gray mold, characterized in that: The active ingredients of the biocontrol agent include hyphae or conidia, or extracellular metabolites of the endophytic fungus JSNL-ZJ63 according to claim 1.

3. The biocontrol agent according to claim 2, characterized in that The extracellular metabolites are obtained by culturing the plant endophytic fungus JSNL-ZJ66 in a PDA medium for several days and then removing hyphae and conidia.

4. A method for preparing the biocontrol agent according to claim 2 or 3, characterized in that: The preparation method comprises inoculating the plant endophytic fungus JSNL-ZJ63 with a preservation number of CGMCC NO.41156 as claimed in claim 1 into a sterile culture medium and culturing it under dark conditions at 22-28°C for several days.

5. A microbial pesticide for preventing and controlling plant root rot, gray mold and anthracnose, characterized in that: The active ingredient of the microbial pesticide comprises the endophytic fungus JSNL-ZJ63 according to claim 1 or the biocontrol agent according to claim 2 or 3.

6. Use of the plant endophytic fungus JSNL-ZJ63 according to claim 1, the biocontrol agent according to claim 2 or 3, or the microbial pesticide according to claim 5 in preventing and controlling plant root rot, gray mold and anthracnose.

7. The use according to claim 5, characterized in that: The root rot disease includes root rot disease caused by one or both of the pathogenic bacteria Rhizoctonia solani and Phomopsis solani. Preferably, the root rot disease is soybean root rot.

8. The use according to claim 5, characterized in that: The gray mold disease is caused by Botrytis cinerea.

9. The use according to claim 5, characterized in that: The anthrax is caused by one or both of Colletotrichum siamese and Colletotrichum gloeosporioides.

10. A method for preventing and treating root rot and gray mold, characterized in that: The method comprises applying the plant endophytic fungus JSNL-ZJ63 according to claim 1, the biocontrol agent according to claim 2 or 3, or the microbial pesticide according to claim 5 to the plant growth environment.

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