Plant endophytic fungus Penicillium adamantium JSNL-ZJ87 strain, biocontrol agent and application thereof

Abstract

The present invention belongs to the technical field of plant disease biocontrol, and discloses a plant endophyte Penicillium adametzii strain, a biocontrol agent and its application. The Penicillium adametzii strain is named Penicillium adametzii JSNL-ZJ87, and the deposit unit is the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC NO.40489. The strain has the ability to significantly inhibit a variety of plant pathogens and promote plant growth. The pathogens include one or more of Botrytis cinerea, Pythium ultimum, Pythium rotundum, Pythium spinulosum, Pythium malformis, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani, and is suitable for biological control of plant diseases, and can be used to produce biological fungicide products with the ability to prevent and control a variety of 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 adamantium JSNL-ZJ87 strain, a biocontrol agent and application thereof. Background Art

[0002] Gray mold caused by Botrytis cinerea is an important plant disease. The pathogen can infect more than 1,200 plants such as strawberries, grapes, cucumbers, tomatoes, leeks, peppers, blueberries, and loofahs. 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 top ten plant diseases in the world. Taking strawberry gray mold as an example, gray mold is the most serious fungal disease that causes damage to strawberries after flowering. The disease mainly infects fruits, and also infects leaves, fruit stalks, calyxes, 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 occurs, causing huge losses to strawberry production.

[0003] Root rot is a serious soil-borne plant root disease worldwide. There are many types of pathogens. The pathogens reported at home and abroad include: Pythium spp., Fusarium spp., Rhizoctonia sp., Colletotrichum sp., Pestalotiopsis sp., Phytophthora sp. and Macrophoma sp., etc. The incidence of plant root rot is high, the pathogens are active, and it is easy to spread between plants. It is difficult to prevent and control, and it is easy to cause large-scale plant death. It is known as "plant cancer". At present, it can only be prevented in production, and there is no effective cure. Therefore, plant root rot has become a more difficult disease faced by all parts of the world.

[0004] The prevention and control of plant gray mold and root rot in the prior art mainly include the following methods: 1. Breeding disease-resistant varieties. This method is the most economical and effective way to prevent and control plant gray mold and root rot. However, due to the wide host range, large spore production, fast reproduction speed and complex pathogenic population of the pathogenic bacteria causing the disease, the resistance of the varieties is easily lost, making breeding difficult. 2. Chemical control. Although this method can prevent and control plant gray mold and root rot 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, resulting in the phenomenon of ineffective pesticide control.

[0005] Endophytes are defined as microorganisms that live in plant tissues but do not cause disease symptoms. They are one of the key factors affecting plant growth, nutrition and health. They can help plants obtain nutrients, inhibit plant pathogens, and resist biotic and abiotic stresses. Since endophytic fungi originate 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.

[0006] In summary, the prior art still lacks a green and efficient probiotic for preventing plant gray mold and root rot. Summary of the invention

[0007] In view of the above problems, the present invention provides a biocontrol bacterium Penicillium adametzii JSNL-ZJ87 and its application which can effectively prevent and control plant gray mold and root rot.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a plant endophytic fungus JSNL-ZJ87, which is classified and named Penicillium adametzii 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 the China Microbiological Culture Collection Administration (CGMCC for short) on January 16, 2023, with a deposit number of CGMCC NO.40489, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] In a second aspect, the present invention further provides a biocontrol agent for plant gray mold and root rot, wherein the active ingredient of the biocontrol agent includes hyphae or conidia, or extracellular secretions of the plant endophytic fungus JSNL-ZJ87.

[0011] In a third aspect, the present invention also provides a method for preparing the biocontrol agent, comprising inoculating the plant endophytic fungus JSNL-ZJ87 with a preservation number of CGMCC NO.40489 into a sterile culture medium and culturing it under dark conditions at 22-28°C for several days.

[0012] In a fourth aspect, the present invention further provides a microbial pesticide for preventing and controlling plant gray mold and root rot, wherein the active ingredient of the microbial pesticide comprises the plant endophytic fungus JSNL-ZJ87 described in the first aspect or the biocontrol agent described in the second aspect.

[0013] In the fifth aspect, the present invention also provides the use of the plant endophytic fungus JSNL-ZJ87 described in the first aspect, the biocontrol agent described in the second aspect, or the microbial pesticide described in the fourth aspect in inhibiting the growth of pathogenic bacteria, characterized in that the pathogenic bacteria are one or more of Botrytis cinerea, Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium tertiorrhiza, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

[0014] In a sixth aspect, the present invention further provides the use of the plant endophytic fungus JSNL-ZJ87 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 gray mold and root rot.

[0015] In certain embodiments, the gray mold disease is strawberry gray mold disease, and the root rot disease is strawberry root rot disease or soybean root rot disease.

[0016] In certain embodiments, the gray mold is gray mold caused by Botrytis cinerea.

[0017] In some embodiments, the root rot is a root rot of various plants such as legumes, strawberries, melons, solanaceae, bananas, cotton, Chinese cabbage and flowers. Preferably, the root rot is strawberry root rot or soybean root rot.

[0018] In certain embodiments, the root rot comprises root rot caused by one or more of the pathogens Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium maltose, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae, and Rhizoctonia solani.

[0019] In a seventh aspect, the present invention further provides a method for preventing and controlling gray mold and root rot, the method comprising applying the endophytic fungus JSNL-ZJ87 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.

[0020] In certain embodiments, the plant growth environment is the hypocotyl growth area, root or leaf of a plant seedling.

[0021] In certain embodiments, the plant is preferably strawberry or soybean.

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

[0023] 1) The biocontrol bacteria Penicillium adametzii JSNL-ZJ87 provided by the present invention can significantly inhibit the mycelial growth of pathogenic bacteria Botrytis cinerea, Pythium ultimum, P. helicoides, P. spinosum, P. irregulare, P. aphanidermatum, Fusarium solani, F. oxysporum, Colletotrichum siamense, C. gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

[0024] 2) The biocontrol bacterium Penicillium adametzii JSNL-ZJ87 provided by the present invention can significantly reduce the disease severity of strawberry gray mold and soybean root rot, 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 adametzii JSNL-ZJ87 on different pathogens in Example 1;

[0027] Figure 2 The inhibitory activity of Penicillium adametzii JSNL-ZJ87 against different pathogens in Example 1;

[0028] Figure 3 The effect of the extracellular metabolites of Penicillium adametzii JSNL-ZJ87 on different pathogens in Example 2;

[0029] Figure 4 The effect of Penicillium adametzii JSNL-ZJ87 on soybean plant growth in Example 3;

[0030] Figure 5The effect of Penicillium adametzii JSNL-ZJ87 on strawberry gray mold caused by Botrytis cinerea in Example 4;

[0031] Figure 6 The effect of Penicillium adametzii JSNL-ZJ87 on soybean root rot caused by Pythiumultimum in Example 4;

[0032] Figure 7 This is the effect of Penicillium adametzii JSNL-ZJ87 on plant height and sprout number of soybean root rot caused by Pythiumultimum in Example 4.

[0033] Figure 8 This is the effect of Penicillium adametzii JSNL-ZJ87 in Example 4 on soybean root rot caused by Pythium ultimum. 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 "Botryotinia cinerea" is classified as: Fungi, Ascomycota, Leotiomycete, Helotiales, Sclerotiniaceae, Botryotinia. It has a wide range of hosts and can infect plants such as Leguminosae, Rosaceae, Asteraceae, Cucurbitaceae, Solanaceae, Amaryllisaceae and Liliaceae, forming a gray-black mold layer at the site of disease, causing huge losses to the yield and quality of fruits and vegetables worldwide.

[0037] As described in the present invention, the terms "ultimate Pythium", "rotten Pythium", "thorny Pythium", "teratogenic Pythium", and "aphantom Pythium" are classified as: Chromista, Oomycota, Peronosporales, Pythiacea, and Pythium. They are mainly spread through soil and have a wide host range, infecting plants of the Leguminosae, Asteraceae, Cucurbitaceae, Solanaceae, and Rosaceae families. They usually infect the roots and seeds of seedlings and cause symptoms such as damping-off, seed rot, root rot, and fruit rot, causing serious agricultural economic losses.

[0038] As described in the present invention, the terms "Siamese Anthrax" and "Gliosporium Anthrax" are classified as Eumycota, Deuteromyeotina, Coelomycetes, Melanconiales, Melanconiaceae, and Colletotrichum. It is widely distributed in the world and has many species. It can infect flowers, fruit trees, vegetables, field crops, etc. After infecting strawberries, root crown rot will occur. As the disease spreads, the disease will also appear on the plant runners, leaves, petioles, flowers and fruits.

[0039] As described in the present invention, the terms "Fusarium oxysporum" and "Fusarium solani" belong to the Imperfectifungi, Moniliales, Tuberculariaceae, and Fusarium genus. It is a soil-borne pathogenic fungus distributed worldwide with a wide host range. It can cause root rot of many plants such as legumes, strawberries, melons, Solanaceae, bananas, cotton, cabbage and flowers.

[0040] As described in the present invention, the term "Phytophthora sojae" is classified as follows: Chromista, Oomycota, Peronosporales, Melanconiaceae, Phytophthora. It is mainly distributed in the United States, Canada, Brazil, Argentina, Japan, Australia, the United Kingdom, Hungary, Nigeria, India, China, Egypt, Israel, South Africa, Germany, Switzerland, New Zealand and other countries. Soybean Phytophthora root rot caused by Phytophthora sojae occurs widely in soybean planting areas around the world, causing a large amount of economic losses.

[0041] As described in the present invention, the term "Rhizoctonia solani" belongs to the class Imperfecti fungi, the order Agonomycetales, the family Agonomycetaceae, and the genus Rhizoctonia. It is a plant soil-borne pathogenic fungus with a wide host range, strong saprophytic properties, and widespread distribution throughout the world.

[0042] Example 1 Screening of Penicillium adametzii JSNL-ZJ87

[0043] 1. Isolation of potential biocontrol bacteria

[0044] 1.1. Obtaining experimental materials: Select healthy strawberry plants with good growth from the field (Jiangsu Agricultural Expo Park) and take their root tissues.

[0045] 1.2. Isolation and culture of endophytic fungi in strawberry roots: Cut the strawberry root tissue, mix well, weigh 2g, and disinfect the surface. To ensure that all epiphytic microorganisms are removed, add 100mL of 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 were further cut into smaller segments (about 0.3cm), and 5 segments were randomly selected from each sample and placed on PDA flat culture medium containing ampicillin (50mg / L) and rifampicin (50mg / L). The culture dish was sealed and cultured at 25℃ in the dark for 1-2d. When the mycelium appeared from the leaf tissue, the small piece of culture medium growing on the edge of the culture medium was carefully transferred to a new PDA plate together with the mycelium.

[0046] 2. Screening of antagonistic bacteria by plate confrontation method

[0047] The PDA plate confrontation method was used. The gray mold pathogen, root rot pathogen strain 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 gray mold pathogen and root rot pathogen strain 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 root rot 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%.

[0048] Finally, a strain with good antagonistic activity against gray mold and root rot pathogens on the plate was screened out, which was specifically manifested by strong inhibition of pathogen hyphae growth. It was numbered JSNL-ZJ87.

[0049] like Figure 1 As shown in the figure, compared with the control, the colonies of the pathogens in the experimental group were significantly smaller. JSN L-ZJ87 was very effective against Botrytis cinerea, Pythium ultimum, P. helicoides, P. spinosum, P. irregulare, P. aphanidermatum, Fusarium solani, F. oxysporum, Colletotrichum siamense, C. gloeosporioides, Phytophthora sojae and Rhizoctonia solani. solani) were 61.88%, 75.81%, 70.81%, 75.64%, 74.19%, 52.21%, 66.89%, 57.84%, 50.38%, 56.41%, 58.67% and 54.82%, respectively, indicating that the mycelial growth of the above-mentioned gray mold and root rot pathogens were inhibited.

[0050] 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-ZJ87, the internal substances of the mycelium of B. cinerea, F. solani, F. oxysporum, C. gloeosporioides and R. solani were condensed and distributed in a chaotic manner, the mycelium was uneven in thickness and deformed, and could not grow normally.

[0051] 3. Molecular biological identification of the strains obtained

[0052] 3.1. The genomic DNA of strain JSNL-ZJ87 was extracted using the kit (DP320-03) from TIANGEN.

[0053] 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), 2 μL template DNA and 11 μL ddH2O. The PCR amplification program conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 60 s, a total of 35 cycles; and finally 72℃ extension for 10 min.

[0054] 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-ZJ87 screened in step 2 was SEQ ID NO.1, and the Ben A gene sequence was SEQ ID NO.2.

[0055] 3.4. Compare the two sequences of 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 bacteria JSNL-ZJ87 of the present invention is Penicillium adametzii, and is named Penicillium adametzii JSNL-ZJ87.

[0056] Table 1 Sequence alignment results of SEQ ID NO.1 and SEQ ID NO.2

[0057]

[0058] Example 2 Effect of extracellular metabolites of Penicillium adametzii JSNL-ZJ87 on gray mold and root rot pathogens

[0059] 1. Inoculate B. cinerea, P. ultimum, P. helicoides, P. spinosum, P. irregulare, F. solani, F. oxysporum, C. siamense, C. gloeosporioides and Penicillium adametzii JSNL-ZJ87 in a 9 cm diameter culture dish containing 15 mL of 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.

[0060] 2. Acquisition of extracellular metabolites secreted by strain JSNL-ZJ87

[0061] 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 adametzii JSNL-ZJ87 bacterial plate (diameter 5 mm) was inoculated on the filter membrane, and a blank PDA medium was used instead of the JSNL-ZJ87 bacterial plate (diameter 5 mm) for control, and cultured at 25°C in the dark.

[0062] 3. After culturing for 2 days, use tweezers to carefully remove the filter membrane together with the Penicillium adametzii JSNL-ZJ87 culture on it. For control, tear off the filter membrane and blank PDA block to prepare plates containing JSNL-ZJ87 extracellular metabolites and blank PDA plates.

[0063] 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.

[0064] 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%.

[0065] The results are as follows Figure 3 As shown in the data, compared with the control, the colonies of each pathogenic strain in the experimental group were significantly smaller, and the inhibition rates of the extracellular metabolites secreted by the strain JSNL-ZJ87 on Pythium ultimum, P. spinosum, P. irregulare and P. helicoides reached 100%, and the inhibition rates on F. solani, Botrytis cinerea, C. gloeosporioides, F. oxysporum and C. siamense were 57.78%, 47.37%, 32.35%, 36.72% and 30.69%, respectively, indicating that the extracellular metabolites secreted by JSNL-ZJ87 had a certain inhibitory effect on the hyphae growth of the above pathogens.

[0066] Example 3 Effect of Penicillium adametzii JSNL-ZJ87 on Plant Growth

[0067] 1. Inoculate the strain JSNL-ZJ87 into a 9-cm diameter culture dish (containing 15 ml of PDA medium) and culture at 25°C in the dark for 7 days.

[0068] 2. Select a pot with a diameter of 12 cm, and cut the culture medium of Penicillium adametzii JSNL-ZJ87, which has been cultured for 7 days, into 0.5 cm × 0.5 cm flat blocks. The experiment set up two groups, the control group: add 2 0.5 cm × 0.5 cm PDA flat blocks to each pot; the experimental group: add 2 0.5 cm × 0.5 cm Penicillium adametzii JSNL-ZJ87 flat blocks to each pot. Mix various flat blocks evenly with sterile water and sterilized vermiculite, add to the pot to 1 / 3, evenly spread 10 soybean seeds (Zhonghuang 37) in each pot, and then cover with a thin layer of mycelium block and vermiculite mixture. Set up 3 replicates for each treatment.

[0069] 3. Water each group of soybeans with sterile water every other day. After 10 days, calculate the germination rate, plant height and fresh weight.

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

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

[0072]

[0073] Among them, the numerical expressions of germination rate, plant height and fresh weight in the table are: mean ± standard error.

[0074] from Figure 4 As shown in the data in Table 2, the plant height and fresh weight of the experimental group were significantly better than those of the control group, indicating that Penicillium adametzii JSNL-ZJ87 has a growth-promoting effect on soybean plants.

[0075] Example 4 Effects of Penicillium adametzii JSNL-ZJ87 on strawberry gray mold caused by B. cinerea, strawberry root rot caused by P. irregulare, and soybean root rot caused by P. ultimum

[0076] 1. Strain JSNL-ZJ87 induces strawberry resistance to gray mold

[0077] 1.1. Penicillium adametzii JSNL-ZJ87 and B. cinerea were inoculated into 9 cm diameter culture dishes containing 15 mL PDA medium, respectively, and placed in a 25°C incubator 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-ZJ87 colonies to prepare bacterial plates.

[0078] 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-ZJ87 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.

[0079] 1.3. Remove the JSNL-ZJ87 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 it moist. Place at 25℃ in the dark for 3 days.

[0080] Check the disease condition of strawberry leaves, record the diameter of lesions using the cross method, and calculate the average value.

[0081] The results are as follows Figure 5 And as shown in Table 3.

[0082] Table 3 Statistical results of the diameter of lesions on strawberry leaves inoculated with B. cinerea

[0083]

[0084] The numerical expression of colony diameter in the table is: mean ± standard error.

[0085] like Figure 5 As shown, the left side of the front of the leaf was inoculated with strain JSNL-ZJ87 24 hours later and then with B. cinerea strain, and the right side was inoculated with blank PDA 24 hours later and then 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.

[0086] 2. Effect of strain JSNL-ZJ87 on strawberry root rot caused by P. irregulare

[0087] 2.1. Penicillium adametzii JSNL-ZJ87 and P. irregulare were inoculated into 9-cm diameter culture dishes containing 15 mL PDA culture medium, respectively, and placed in a 25°C incubator in the dark for 5 and 3 days.

[0088] 2.2. Prepare two sterile trays, cover each with two layers of sterile gauze and moisten with sterile water. Select three healthy strawberry seedlings (variety Hongyan) with uniform growth, cut their roots neatly with scissors, and use a hole puncher (diameter 5mm) to punch the edge of the culture medium full of JSNL-ZJ87 and P.irregulare to take bacterial cakes. The experiment set up two groups. The control group was: 3 pieces of P.irregulare and 3 blank PDA bacterial cakes were attached to both sides of the strawberry roots, wrapped with sterile moist absorbent paper, and then wrapped tightly with sterile tin foil on the outside, and placed in a tray; the experimental group was: 3 pieces of P.irregulare and 3 pieces of JSNL-ZJ87 bacterial cakes were attached to both sides of the strawberry roots, wrapped with sterile moist absorbent paper, and then wrapped tightly with sterile tin foil on the outside, and placed in a tray. Three replicates were set for each group.

[0089] 2.3. Seal with plastic wrap, culture at 25°C, and record the disease occurrence 5 days after inoculation.

[0090] The results are as follows Figure 6 shown.

[0091] from Figure 6 It can be seen that Penicillium adametzii JSNL-ZJ87 can significantly reduce the disease severity of strawberry root rot, indicating that JSNL-ZJ87 has a preventive or control effect on strawberry root rot caused by P. irregulare.

[0092] 3. Effect of strain JSNL-ZJ87 on soybean root rot caused by P. ultimum

[0093] 3.1. Penicillium adametzii JSNL-ZJ87 and P. ultimum were inoculated into 9-cm diameter culture dishes containing 15 mL of PDA culture medium, respectively, and placed in a 25°C incubator in the dark for 5 days.

[0094] 3.2. Cut 3 mm × 3 mm mycelium blocks at the edge of the JSNL-ZJ87 colony, transfer 10 mycelium blocks into 400 mL PDB liquid culture medium under sterile conditions, and culture at 25°C with shaking or static conditions for 21 days.

[0095] 3.3. Pots with a diameter of 12 cm were selected and two groups were set up for the experiment. The experimental group was: 400 mL of JSNL-ZJ87 bacterial suspension and 2 0.5 cm × 0.5 cm flat plates of P. ultimum were added to each pot; the control group was: 400 mL of PDB liquid culture medium and 2 0.5 cm × 0.5 cm flat plates of P. ultimum were added to each pot. Each treatment was set up with 3 replicates.

[0096] 3.4. 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.

[0097] 3.5. Water each group of soybeans with sterile water every other day. Count the germination rate, plant height and fresh weight of soybeans 9 days after sowing.

[0098] The results are as follows Figure 7 , Figure 8 And as shown in Table 4.

[0099] Table 4 Statistical results of soybean emergence rate, plant height and fresh weight

[0100]

[0101] from Figure 7 and Figure 8 It can be seen that the plant height, number of buds, root length and luxuriance of the experimental group were significantly better than those of the control group. It can be seen that Penicillium adametzii JSNL-ZJ87 has a preventive or therapeutic effect on soybean root rot caused by P. ultimum.

[0102] 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-ZJ87, characterized in that: The plant endophytic fungus The classification of JSNL-ZJ87 is named Penicillium adamantium ( Penicillium adametzii ), deposited by: China General Microbiology Culture Collection Center, with the deposit number being CGMCC NO. 40489.

2. A biocontrol agent for plant gray mold and root rot, characterized in that: The active ingredients of the biocontrol agent include hyphae or conidia of the endophytic fungus JSNL-ZJ87 according to claim 1, the gray mold is strawberry gray mold, the root rot is strawberry root rot and soybean root rot, the gray mold is gray mold caused by Botrytis cinerea, and the root rot includes root rot caused by one or more pathogens including Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium tertiorrhiza, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

3. A method for preparing the biocontrol agent according to claim 2, characterized in that: The preparation method comprises inoculating the plant endophytic fungus JSNL-ZJ87 with a preservation number of CGMCC NO. 40489 as described in claim 1 into a sterile culture medium and culturing it under dark conditions at 22-28° C. for several days.

4. A microbial pesticide for preventing and controlling plant gray mold and root rot, characterized in that: The active ingredient of the microbial pesticide comprises the plant endophytic fungus JSNL-ZJ87 according to claim 1 or the biocontrol agent according to claim 2, the gray mold is strawberry gray mold, the root rot is strawberry root rot and soybean root rot, the gray mold is gray mold caused by Botrytis cinerea, and the root rot includes root rot caused by one or more of the pathogens Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium tertiorrhiza, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

5. Use of the plant endophytic fungus JSNL-ZJ87 according to claim 1, the biocontrol agent according to claim 2 or the microbial pesticide according to claim 4 in inhibiting the growth of pathogenic bacteria, characterized in that: The pathogenic bacteria are one or more of Botrytis cinerea, Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium tertiorrhiza, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

6. Use of the plant endophytic fungus JSNL-ZJ87 according to claim 1, the biocontrol agent according to claim 2 or the microbial pesticide according to claim 4 in preventing and controlling gray mold and root rot, characterized in that: The gray mold disease is strawberry gray mold disease, the root rot disease is strawberry root rot disease and soybean root rot disease, the gray mold disease is gray mold disease caused by Botrytis cinerea, and the root rot diseases include root rot diseases caused by one or more of the pathogenic bacteria Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium tertiorrhiza, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

7. A method for preventing and treating gray mold and root rot, characterized in that: The method comprises applying the endophytic fungus JSNL-ZJ87 of claim 1, the biocontrol agent of claim 2 or the microbial pesticide of claim 4 into a plant growth environment, the gray mold is strawberry gray mold, the root rot is strawberry root rot or soybean root rot, the gray mold is gray mold caused by Botrytis cinerea, and the root rot comprises root rot caused by one or more of the pathogenic fungi Pythium ultimum, Pythium involutum, Pythium spinulosum, Pythium malformis, Pythium aphanidermatum, Fusarium solani, Fusarium oxysporum, Colletotrichum siamese, Colletotrichum gloeosporioides, Phytophthora sojae and Rhizoctonia solani.

Images