A strawberry biocontrol endophytic fungus JSNL-ZJ66 and its application

Abstract

The invention discloses a strawberry gray mold, anthracnose and wilt biocontrol bacteria and applications thereof, wherein the biocontrol bacteria is named Aspergillus qilianyuensis JSNL-ZJ66, and the deposit unit is the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.41157. The strain has the ability to significantly inhibit a variety of plant pathogens, including one or more of Botrytis cinerea, Siamese anthracnose, Gloeosporium anthracnose and Fusarium oxysporum, and is suitable for biological control of plant diseases, and can be widely used in the prevention and control of a variety of plant fungal diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease biocontrol and relates to a strawberry biocontrol endophytic fungus JSNL-ZJ66 and an application thereof. Background Art

[0002] Strawberry gray mold is caused by Botrytis spp., which can infect more than 1,400 important economic crops such as tomatoes, cucumbers, grapes, blueberries, leeks, eggplants, peppers, and loofahs in addition to strawberries. Gray mold is widely distributed, spreads rapidly, and causes serious damage, causing huge losses to the yield and quality of fruits and vegetables around the world. Strawberry gray mold is the most serious fungal disease that causes damage to strawberries after flowering. The disease mainly infects fruits, as well as leaves, fruit stalks, calyxes, petals, and petioles, causing flower and fruit rot. The incidence rate of fruit damage is as high as 13.2%-78%, and in severe cases, even the harvest is completely lost, causing huge losses to strawberry production.

[0003] Strawberry anthracnose is caused by the fungus Colletotrichum spp., which 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 raising period and the early stage of planting. Anthracnose has a wide host range. In addition to infecting strawberries, it can also infect plants such as grapes, bananas, apples, peppers, mangoes, citrus, peaches, pears and Dendrobium officinale. When strawberry anthracnose occurs, strawberry leaves, petioles, stipules, runners, petals and fruits 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 entire greenhouse seedlings, bringing devastating damage to strawberry seedling raising and production.

[0004] Strawberry wilt is a soil-borne disease caused by Fusarium spp., a fungus that harms not only strawberries, but also tomatoes, watermelons, cucumbers, melons, eggplants, potatoes, cotton, tobacco and many other plants. When strawberry plants are infected with wilt bacteria, the heart leaves turn yellow or yellow-green in the early stages of the disease, some curl and become deformed, the leaves lose their luster, and the growth of the plants stops. As the disease worsens, the vascular bundles of the petioles and roots turn brown or dark brown, the underground roots of the plants turn dark brown, and new roots grow little or not at all, causing the entire plant to wilt and die. In severe cases, it can cause large-scale seedling death and poor fruit quality, affecting the yield and quality of strawberries.

[0005] In the prior art, the prevention and control of strawberry gray mold, anthracnose and root rot mainly include the following methods: 1. Cultivating high-quality disease-resistant seedlings. This method is the most economical and effective way to prevent and control diseases in strawberry production. However, since this method is currently difficult to put into production on a large scale and the relevant mechanism is relatively unclear, it cannot be widely promoted and applied for the time being. 2. Chemical control. Although this method can prevent and control strawberry gray mold, anthracnose 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.

[0006] As the country vigorously develops ecological agriculture, biological control has become a key research direction for disease control. Endophytic fungi can help plants obtain nutrients, induce resistance in plants, and improve the survival ability of plants under biotic or abiotic stress conditions. Since endophytic fungi come from plants, they are safe for plants, environmentally friendly, and not easy to develop drug resistance. Using these microorganisms to control plant diseases is conducive to maintaining ecological balance. Therefore, endophytic fungi are a new type of biological control method to promote organic agriculture.

[0007] In summary, the prior art still lacks a green and highly effective probiotic for preventing strawberry gray mold, anthracnose and root rot. Summary of the invention

[0008] In view of the above-mentioned problems, the present invention provides a biocontrol strain Aspergillus qilianyuensis JSNL-ZJ66 capable of effectively preventing and controlling strawberry gray mold, anthracnose and root rot and applications thereof.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a plant endophytic fungus JSNL-ZJ66, which is classified and named Aspergillus qilianyuensis 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 CGMCCNO.41157, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0011] In a second aspect, the present invention further provides a biocontrol agent for strawberry gray mold, anthracnose and root rot, wherein the active ingredient of the biocontrol agent includes hyphae or conidia, or extracellular metabolites of the endophytic fungus JSNL-ZJ66.

[0012] 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.

[0013] In a third aspect, the present invention also provides a method for preparing the biocontrol agent, comprising inoculating the plant endophytic fungus JSNL-ZJ66 with a preservation number of CGMCC NO.41157 into a sterile culture medium and culturing it under dark conditions at 22-28°C for several days.

[0014] In a fourth aspect, the present invention further provides a microbial pesticide for preventing and controlling gray mold, anthracnose and root rot of strawberry, wherein the active ingredient of the microbial pesticide comprises the endophytic fungus JSNL-ZJ66 described in the first aspect or the biocontrol agent described in the second aspect.

[0015] In a fifth aspect, the present invention further provides the use of the plant endophytic fungus JSNL-ZJ66 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 strawberry gray mold, anthracnose and root rot.

[0016] Furthermore, the gray mold, anthracnose and wilt are respectively strawberry gray mold caused by Botrytis cinerea, strawberry anthracnose caused by Colletotrichum siamese and Colletotrichum gloeosporioides, and strawberry wilt caused by Fusarium oxysporum.

[0017] In a sixth aspect, the present invention further provides a method for preventing and controlling gray mold, anthracnose and root rot of strawberry, the method comprising applying the endophytic fungus JSNL-ZJ66 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.

[0018] Furthermore, the plant growth environment is plant leaves.

[0019] Furthermore, the plant is preferably strawberry.

[0020] Furthermore, the gray mold, anthracnose and wilt are respectively strawberry gray mold caused by Botrytis cinerea, strawberry anthracnose caused by Colletotrichum siamese and Colletotrichum gloeosporioides, and strawberry wilt caused by Fusarium oxysporum.

[0021] Compared with the prior art, the present invention has the following technical effects:

[0022] Compared with the control, the biocontrol bacteria Aspergillus qilianyuensis JSNL-ZJ66 provided by the present invention has inhibition rates of 52.73%, 49.11%, 42.14% and 53.64% on Botrytis cinerea, C. siamense, C. gloeosporioides and F. oxysporum, respectively. Meanwhile, the extracellular metabolites secreted by the strain Aspergillus qilianyuensis JSNL-ZJ66 also have high inhibition effects on B. cinerea, C. siamense, C. gloeosporioides and F. oxysporum. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 The figure is a diagram showing the antagonistic effect of Aspergillus qilianyuensis JSNL-ZJ66 on different pathogens in Example 1;

[0025] Figure 2 The inhibitory activity of Aspergillus qilianyuensis JSNL-ZJ66 against different pathogens in Example 1;

[0026] Figure 3 This is the effect of the extracellular metabolites of Aspergillus qilianyuensis JSNL-ZJ66 in Example 2 on different pathogens. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] As described in the present invention, the term "Fusarium oxysporum" belongs to the class Imperfecti fungi, the order Moniliales, the family Tuberculariaceae, and the genus Fusarium, and is mainly distributed in Australia, Japan, South Korea, China, the United States, Mexico, Spain and other countries.

[0032] Example 1 Screening of Aspergillus qilianyuensis JSNL-ZJ66

[0033] 1. Isolation of potential biocontrol bacteria

[0034] 1.1. Obtaining experimental materials: Select healthy strawberry plants with good growth from the field and take their root tissues.

[0035] 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.

[0036] 2. Screening of antagonistic bacteria by plate confrontation method

[0037] The PDA plate confrontation method was used. Strawberry gray mold pathogen, anthracnose pathogen and wilt pathogen strain cake (5 mm in diameter) and the endophytic fungus cake (5 mm in diameter) were symmetrically inoculated 2 cm from the edge of a 9 cm diameter plate containing 15 mL of PDA culture medium for 3-5 days. The plate inoculated with only the gray mold pathogen, anthracnose pathogen and wilt pathogen strain cake was used as a control. The plate was cultured at 25 ° C in the dark. After 7-10 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 = (control group colony radius - treatment group colony radius) / control group colony radius × 100%.

[0038] Finally, a strain with good antagonistic activity against gray mold, anthracnose and wilt pathogens on the plate was screened out, which was specifically manifested by strong inhibition of the mycelial growth of the pathogens. It was numbered JSNL-ZJ66.

[0039] like Figure 1 As shown, compared with the control, the colonies of the pathogenic bacteria strains in the experimental group were significantly smaller, and the inhibition rates of JSNL-ZJ66 against B. cinerea, C. siamense, C. gloeosporioides and F. oxysporum were 52.73%, 49.11%, 42.14% and 53.64%, respectively, indicating that the mycelial growth of the above-mentioned gray mold, anthracnose and wilt diseases was inhibited.

[0040] like Figure 2As shown, the results of optical microscopic observation showed that the hyphae of the pathogen in the control group had regular shape and grew straight; after being cultured against JSNL-ZJ66, the hyphae of B. cinerea, C. siamense, C. gloeospori oides and F. oxysporum were chaotically distributed, uneven in thickness and deformed, and could not grow normally.

[0041] 3. Molecular biological identification of the strains obtained

[0042] 3.1. The genomic DNA of strain JSNL-ZJ66 was extracted using the kit (DP320-03) from TIANGEN.

[0043] 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.

[0044] 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-ZJ66 screened in step 2 was SEQ ID NO.1, and the BenA gene sequence was SEQ ID NO.2.

[0045] 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-ZJ 66 of the present invention is Aspergillus qilianyuensis, and is named Aspergillus qilianyuensis JSNL-ZJ66.

[0046] Table 1 Sequence alignment results of SEQ ID NO.1 and SEQ ID NO.2

[0047]

[0048] Example 2 Effects of extracellular metabolites of Aspergillus qilianyuensis JSNL-ZJ66 on pathogens of gray mold, anthracnose and wilt

[0049] 1. Inoculate B. cinerea, C. siamense, C. gloeosporioides, F. oxysporum and Aspergillus qilianyuensis JSNL-ZJ66 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.

[0050] 2. Acquisition of extracellular metabolites secreted by strain JSNL-ZJ66

[0051] 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 Aspergillus qilianyuensis JSNL-ZJ66 bacterial plate (diameter 5 mm) was inoculated on the filter membrane, and a blank PDA medium was used instead of the JSNL-ZJ66 bacterial plate (diameter 5 mm) for control, and cultured at 25°C in the dark.

[0052] 3. After culturing for 2-3 days, use tweezers to carefully remove the filter membrane together with the Aspergillus qilianyuensis JSNL-ZJ66 culture on it. For control, tear off the filter membrane and blank PDA block to prepare a plate containing JSNL-ZJ66 extracellular metabolites and a blank PDA plate.

[0053] 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.

[0054] 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%.

[0055] The results are as follows Figure 3 As shown, 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 strain JSNL-ZJ66 on B. cinerea, C. siamense, C. gloeosporioides and F. oxysporum were 90.56%, 90.26%, 42.57%, and 64.84%, respectively, indicating that the extracellular metabolites secreted by JSNL-ZJ66 had a certain inhibitory effect on the hyphae growth of the above pathogens.

[0056] 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. Application of plant endophytic fungus JSNL-ZJ66 in preventing and controlling gray mold, anthracnose and wilt, characterized in that: The plant endophytic fungus JSNL-ZJ66 is classified and named Aspergillus qilianyuensis, with a preservation number of CGMCCNO.41157, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. The use according to claim 1, characterized in that: The gray mold, anthracnose and wilt are respectively gray mold of strawberry caused by Botrytis cinerea, strawberry anthracnose caused by Colletotrichum siamese and Colletotrichum gloeosporioides, and strawberry wilt caused by Fusarium oxysporum.

3. The use of a biocontrol agent for plant gray mold, anthracnose and wilt in preventing and controlling gray mold, anthracnose and wilt, characterized in that: The active ingredients of the biocontrol agent include hyphae or conidia, or extracellular metabolites of the endophytic fungus JSNL-ZJ66 described in claim 1.

4. The use according to claim 3, 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.

5. The use according to claim 3, characterized in that: The preparation method of the biocontrol agent comprises the following steps: inoculating the plant endophytic fungus JSNL-ZJ66 with the accession number of CGMCC NO.41157 as claimed in claim 1 into a sterile culture medium and culturing it under dark conditions at 22-28° C. for several days.

6. The use according to claim 3, characterized in that: The gray mold, anthracnose and wilt are respectively gray mold of strawberry caused by Botrytis cinerea, strawberry anthracnose caused by Colletotrichum siamese and Colletotrichum gloeosporioides, and strawberry wilt caused by Fusarium oxysporum.

7. The use of a microbial pesticide for preventing and controlling gray mold, anthracnose and wilt of strawberry in preventing and controlling gray mold, anthracnose and wilt, characterized in that: The active ingredient of the microbial pesticide comprises the endophytic fungus JSNL-ZJ66 described in claim 1 or the biocontrol agent described in any one of claims 3-6.

8. The use according to claim 7, characterized in that: The gray mold, anthracnose and wilt are respectively gray mold of strawberry caused by Botrytis cinerea, strawberry anthracnose caused by Colletotrichum siamese and Colletotrichum gloeosporioides, and strawberry wilt caused by Fusarium oxysporum.

9. A method for preventing and treating gray mold, anthracnose and wilt of strawberry, characterized in that: The method comprises applying the endophytic fungus JSNL-ZJ66 described in claim 1, the biocontrol agent described in any one of claims 3 to 6, or the microbial pesticide described in any one of claims 7 to 8 to the strawberry growth environment.

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