A biocontrol bacterium for preventing and controlling Phomopsis sojae and its application

Abstract

The present invention discloses a biocontrol bacterium for preventing and controlling Phomopsis seed decay of soybeans caused by Phomopsis longicolla and its application. The biocontrol bacterium is named Aspergillus qilianyuensis JSNL-ZJ66, and the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO. 41157. This strain is mainly applied in preventing and controlling Phomopsis seed decay of soybeans caused by P. longicolla, can significantly reduce the disease severity of Phomopsis seed decay of soybeans, and significantly improve the growth indexes such as germination rate, plant height and fresh weight.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control of Phomopsis sojae, and relates to a biocontrol bacterium for preventing and controlling Phomopsis sojae and its application. Background Art

[0002] Phomopsis sojae Phomopsis / Diaporthe longicolla is an important plant pathogen that can infect various plants such as soybean, mung bean, pea, lima bean, clover, peanut, garlic, onion, tomato, pepper, Xanthium sibiricum, Rumex crispus, etc. The infection of this pathogen on soybean can cause seed rot and stem / pod blight, and it has been reported in the United States, Canada, Argentina, Italy, South Korea and other places. In 2009, China first reported stem blight caused by Phomopsis sojae in Jiamusi City, Heilongjiang Province, and then the occurrence of this disease was successively found in Guangdong, Jiangxi, Hubei and other places.

[0003] The main damage period of Phomopsis sojae is the seed storage period and the seedling stage. The infected parts of soybean seeds do not show symptoms, and some seeds are shriveled, cracked, chalky, and the seed coat is covered with hyphae. Infected seeds usually germinate slowly or do not germinate, the plants are short, the growth is retarded or even rotten. Infected fields generally cause a 10%-30% reduction in soybean yield, and in severe cases, even a complete crop failure. This pathogen can also co-infect with pathogenic fungi of the same genus, causing diseases such as soybean stem canker and leaf spot disease, resulting in huge losses to soybean production.

[0004] The prevention and control of Phomopsis sojae in the prior art mainly include the following methods: 1. Planting disease-resistant varieties, which is the most economical and effective way to control diseases in soybean production. However, due to the complex field environment and the rapid variation of pathogen virulence, the resistance of soybean varieties is easily lost. 2. Chemical control, although this method can control Phomopsis sojae to a certain extent, the large use of chemical agents poses a great threat to the ecological environment and food safety, and also leads to the continuous increase of pathogen drug resistance.

[0005] Plant endophytic fungi can help plants obtain nutrients, induce plants to produce resistance, and improve the survival ability of plants under biotic stress or abiotic stress conditions. Since plant endophytic fungi are derived from plants, they are safe for plants, friendly to the environment and not easy to produce drug resistance. Using these microorganisms to control plant diseases is beneficial to maintaining ecological balance. Therefore, plant endophytic fungi are a source of new compounds for promoting organic agriculture.

[0006] In summary, the prior art still lacks a green and efficient probiotic for preventing Phomopsis sojae. Summary of the Invention

[0007] In view of the above problems, the present invention provides a biocontrol strain that can effectively prevent and control Phomopsis sojae Aspergillus qilianyuensis JSNL-ZJ66 and its applications.

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

[0009] In a first aspect, the present invention provides a plant endophytic fungus JSNL-ZJ66, and the taxonomic nomenclature of the plant endophytic fungus JSNL-ZJ66 is Aspergillus qilianyuensis , which is isolated from the roots of healthy strawberries in Biancheng Town, Jurong City, Zhenjiang City. This strain was deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC) on April 15, 2024, with the deposit number CGMCC NO. 41157 and the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] In a second aspect, the present invention further provides a biocontrol agent for Phomopsis sojae, and the active ingredient of the biocontrol agent includes the mycelium or conidia of the plant endophytic fungus JSNL-ZJ66, or extracellular metabolites.

[0011] Further, the extracellular metabolites are the culture obtained by culturing the plant endophytic fungus JSNL-ZJ66 in a PDA medium for several days and then removing the mycelium and conidia.

[0012] In a third aspect, the present invention further provides a preparation method of the biocontrol agent, and the preparation method includes inoculating the plant endophytic fungus JSNL-ZJ66 with the deposit number CGMCC NO.41157 in a sterile medium and culturing it for several days under dark conditions at 22-28°C.

[0013] In a fourth aspect, the present invention further provides a microbial pesticide for preventing and controlling Phomopsis sojae, and the active ingredient of the microbial pesticide includes the plant endophytic fungus JSNL-ZJ66 described in the first aspect or the biocontrol agent described in the second aspect.

[0014] In a fifth aspect, the present invention further provides the application 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 Phomopsis sojae.

[0015] In a sixth aspect, the present invention further provides a method for preventing and controlling Phomopsis sojae, and the method includes applying 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 to the plant growth environment.

[0016] In some embodiments, the plant growth environment is the roots of plants.

[0017] In some embodiments, the plant is soybean.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] The biocontrol bacterium provided by the present invention Aspergillus qilianyuensis JSNL-ZJ66 has an inhibition rate of 61.20% against Phomopsis longicolla Hobbs P. longicolla ), and can significantly inhibit the mycelial growth of the pathogen Phomopsis longicolla Hobbs Phomopsis longicolla ). At the same time, the biocontrol bacterium Aspergillus qilianyuensis JSNL-ZJ66 provided by the present invention can significantly reduce the disease severity of Phomopsis longicolla Hobbs, and significantly improve the growth indexes such as the germination rate, plant height and fresh weight of soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments.

[0021] Figure 1 For Example 1 Aspergillus qilianyuensis Effect diagram of JSNL-ZJ66 inhibiting Phomopsis longicolla Hobbs on the plate;

[0022] Figure 2 For Example 1 Aspergillus qilianyuensis Inhibitory activity of JSNL-ZJ66 against Phomopsis longicolla Hobbs;

[0023] Figure 3 For Example 2 Aspergillus qilianyuensis Effect of extracellular metabolites of JSNL-ZJ66 on Phomopsis longicolla Hobbs;

[0024] Figure 4 For Example 3 Aspergillus qilianyuensis Effect of JSNL-ZJ66 on the growth of soybean plants;

[0025] Figure 5 For Example 4 Aspergillus qilianyuensis Effect of JSNL-ZJ66 on Phomopsis longicolla Phomopsis longicolla Hobbs-induced soybean seed rot;

[0026] Figure 6 For Example 4 Aspergillus qilianyuensis Effect of JSNL-ZJ66 on Phomopsis longicolla Phomopsis longicolla Hobbs-induced soybean seed rot. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The embodiments of the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, so they are only used as examples and cannot be used to limit the protection scope of the present invention. It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meaning 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 including bacteria, fungi and actinomycetes.

[0029] As described in the present invention, the term "Phomopsis longicolla Hobbs Phomopsis / Diaporthe longicolla )" Taxonomic status: Fungi, Ascomycota, Sordariomycete, Diaporthales, Diaporthaceae, Diaporthe Diaporthe ). It has a wide host range and can infect plants of Leguminosae, Solanaceae, Compositae, Liliaceae, Polygonaceae, etc. It is widely distributed worldwide and causes serious damage in some major soybean-producing countries such as the United States, Canada and Argentina.

[0030] Example 1 Aspergillus qilianyuensis Screening of JSNL-ZJ66

[0031] 1. Isolation of potential biocontrol bacteria

[0032] 1.1. Obtaining test materials: Select healthy strawberry plants with good growth from the field and take their root tissues.

[0033] 1.2. Isolation and culture of endophytic fungi in strawberry roots: Cut the strawberry root tissues, mix them evenly, weigh 2 g, and perform surface disinfection. Add 100 mL of sterile water and 2 drops of Tween 20, shake at 25 °C and 220 rpm for 20 min, treat with sterile water for 20 s, soak in 70% (v / v) ethanol for 30 s, soak in 2.5% (v / v) sodium hypochlorite solution for 2 min, 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 0.3 cm), and 5 segments are randomly selected from each sample and placed on a PDA plate medium. Seal the culture dish and culture it at 25 °C in the dark for 1-2 d. When mycelia appear from the root tissues, carefully transfer the small piece of medium together with the mycelia growing on the edge of the medium to a new PDA plate.

[0034] 2. Screening of antagonistic bacteria by the plate confrontation method

[0035] Using the PDA plate confrontation method, inoculate the mycelial discs (5 mm in diameter) of Phomopsis longicolla Hobbs and the tested endophytic fungi (5 mm in diameter) cultured for 3 - 5 days symmetrically at a distance of 2 cm from the edge of a PDA medium plate with a diameter of 9 cm containing 15 mL. Use the plate inoculated only with the mycelial disc of Phomopsis longicolla Hobbs as the control. Incubate under the conditions of 25°C and darkness. After 7 days, measure the colony radius of Phomopsis longicolla Hobbs and calculate the inhibition rate. The inhibition rate is 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%.

[0036] Finally, one strain with good antagonistic activity against Phomopsis longicolla Hobbs on the plate was screened out, which was specifically manifested as strong inhibition of the mycelial growth of the pathogenic bacterium, and its number was JSNL-ZJ66.

[0037] As Figure 1 shown, compared with the control, the colonies of the pathogenic bacterium strains in the experimental group were significantly smaller. The inhibition rate of JSNL-ZJ66 against Phomopsis longicolla Hobbs ( P. longicolla ) was 61.20%, indicating that the mycelial growth of the above-mentioned pathogenic bacterium of Phomopsis longicolla Hobbs was inhibited.

[0038] As Figure 2 shown, the results of optical microscopy observation showed that: the mycelia of the pathogenic bacterium in the control group had regular shapes and grew straight; after confrontation culture with JSNL-ZJ66, P. longicolla the mycelia were distributed chaotically and intertwined, and could not grow normally.

[0039] 3. Molecular biological identification of the screened strain

[0040] 3.1 Extract the genomic DNA of strain JSNL-ZJ66 using the kit (DP320-03) from TIANGEN Company.

[0041] 3.2 Amplify the ITS gene and BenA gene of the genomic DNA respectively. The DNA amplification uses a reaction volume of 30 μL, which includes 15 µL 2×EasyTaq PCR SuperMix (+dye), 1 μL of each primer F / R (10 μM), 2 μL of template DNA and 11μL ddH 2 O. The PCR amplification program conditions are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 45 s, extension at 72°C for 60 s, for a total of 35 cycles; finally, extension at 72°C for 10 min.

[0042] 3.3 After the amplified PCR products are electrophoresed on a 1% agarose gel and observed under an ultraviolet lamp, the PCR products with target bands are sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results show that the ITS gene sequence of the strain JSNL-ZJ66 screened in step 2 is SEQ ID NO.1, and the BenA gene sequence is SEQ ID NO.2.

[0043] 3.4 Align the two sequences of SEQ ID NO.1 and SEQ ID NO.2 on the NCBI database website. The alignment results are shown in Table 1. According to the results in Table 1, it can be deduced that the biocontrol bacterium JSNL-ZJ66 of the present invention is Aspergillus qilianyuensis , named Aspergillus qilianyuensis JSNL-ZJ66.

[0044] Table 1 Sequence alignment results of SEQ ID NO.1 and SEQ ID NO.2

[0045]

[0046] Example 2 Aspergillus qilianyuensis Effect of extracellular metabolites of JSNL-ZJ66 on the pathogen of Phomopsis sojae

[0047] 1. Inoculate the pathogen of Phomopsis sojae ( P. longicolla ) and Aspergillus qilianyuensis JSNL-ZJ66 into petri dishes with a diameter of 9 cm containing 15 mL of PDA medium respectively, place them in an incubator at 25°C, and culture them in the dark for 3 - 5 d. Use a puncher (with a diameter of 5 mm) to punch holes at the edge of the colonies to prepare bacterial discs.

[0048] 2. Obtaining the extracellular metabolites secreted by the strain JSNL-ZJ66

[0049] Cover a layer of aqueous filter membrane (with a pore size of 0.22 µm and a diameter of 5 cm) on a PDA medium plate with a diameter of 6 cm, and make the filter membrane closely adhere to the surface of the PDA plate. Then inoculate Aspergillus qilianyuensis JSNL-ZJ66 bacterial discs (with a diameter of 5 mm) on the filter membrane. Use a blank PDA medium to replace the JSNL-ZJ66 bacterial discs (with a diameter of 5 mm) as a control, and culture them at 25°C in the dark.

[0050] 3. After culturing for 2 d, carefully remove the filter membrane together with the Aspergillus qilianyuensis JSNL-ZJ66 culture on it with forceps. For the control, tear off the filter membrane and the blank PDA block to obtain a plate containing the extracellular metabolites of JSNL-ZJ66 and a blank PDA plate.

[0051] 4. Use an inoculation needle to pick up each pathogen disc (5 mm in diameter) in a laminar flow hood and inoculate it onto the prepared plate, with the mycelial side facing downwards, and set up 3 replicates for each.

[0052] 5. Place the inoculated plate in an incubator at 25 °C and incubate it in the dark for 2 - 3 d. 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%.

[0053] The results are as Figure 3 shown. Compared with the control, the colonies of the pathogen strains in the experimental group were significantly smaller, and the extracellular metabolites secreted by the strain JSNL-ZJ66 P. longicolla had an inhibition rate of 76.25% against

[0054] Example 3 Aspergillus qilianyuensis Effect of JSNL-ZJ66 on the growth of plants themselves

[0055] 1. Inoculate the strain JSNL-ZJ66 into a petri dish with a diameter of 9 cm (containing 15 ml of PDA medium), and incubate it at 25 °C in the dark for 5 d. Cut a 3 mm × 3 mm mycelial block at the edge of the JSNL-ZJ66 colony, and transfer 10 mycelial blocks to 400 mL of PDB liquid medium under sterile conditions, and incubate it at 25 °C with shaking or statically for 4 d.

[0056] 2. Select pots with a diameter of 12 cm, set up 2 groups in the experiment. The experimental group is: add 400 mL of the bacterial suspension of JSNL-ZJ66 to each pot; the control group is: add 400 mL of PDB liquid medium to each pot. Mix each treatment evenly with sterilized vermiculite, add it to the pot to 2 / 3, evenly sprinkle 10 soybean seeds (Dongsheng No. 1) into each pot, and then cover it with a thin mixture of mycelial blocks and vermiculite. Set up 3 replicates for each treatment.

[0057] 3. Water each group of soybeans every other day with sterile water, and count the germination rate, plant height, and fresh weight after 14 d.

[0058] The results are as Figure 4 and Table 2 show.

[0059] Table 2 Statistical results of soybean emergence rate, plant height, and fresh weight

[0060]

[0061] Among them, the numerical expressions of emergence rate, plant height, and fresh weight in the table are: mean ± standard error.

[0062] From Figure 4 and the data in Table 2, it can be seen that the plant height and fresh weight of the experimental group are better than those of the control group, but the difference is not significant. It can be seen that Aspergillus qilianyuensis JSNL-ZJ66 has a growth-promoting effect on soybean plants, but it is not obvious.

[0063] Example 4 Aspergillus qilianyuensis The effect of JSNL-ZJ66 on P. longicolla Phomopsis sojae

[0064] 1. Inoculate Aspergillus qilianyuensis JSNL-ZJ66 and P. longicolla into petri dishes with a diameter of 9 cm containing 15 mL of PDA medium respectively, and place them in an incubator at 25 °C for dark culture for 5 d.

[0065] 2. Cut a 3 mm × 3 mm mycelium block from the edge of the JSNL-ZJ66 colony. Under sterile conditions, transfer 10 mycelium blocks to 400 mL of PDB liquid medium and culture them with shaking at 25 °C for 6 d. Use a blade to cut P. longicolla the medium into small pieces of 0.5 cm × 0.5 cm.

[0066] 3. Select flowerpots with a diameter of 12 cm. Set up 2 groups in the experiment. The experimental group is: add 400 mL of the bacterial suspension of JSNL-ZJ66 and 2 P. longicolla platelets to each flowerpot; the control group is: add 400 mL of PDB liquid medium and 2 P. longicolla platelets to each flowerpot. Set 3 replicates for each treatment.

[0067] 3.4. Mix the above treatments evenly with sterilized vermiculite, add them to the flowerpots to 2 / 3, add 10 soybeans (variety: Dongsheng No. 1) to each flowerpot, and then cover each with a thin layer of vermiculite mixture.

[0068] 3.5. Water the soybeans in each group with sterile water every other day, and count the germination rate, plant height and fresh weight of the soybeans 14 d after sowing.

[0069] The results are as shown in Figure 5 、 Figure 6 and Table 3.

[0070] Table 4 Statistical results of soybean emergence rate, plant height and fresh weight

[0071]

[0072] From Figure 5 and Figure 6 it can be seen that the plant height, number of germinated buds, root length and lushness of the experimental group are significantly better than those of the control group. It can be seen that Aspergillus qilianyuensis JSNL-ZJ66 on P. longicollaIt has a preventive or control effect on the resulting Phomopsis sojae.

[0073] Unless otherwise specifically stated, the numerical values set forth in these examples do not limit the scope of the present invention. In all examples shown and described herein, any specific value should be construed as merely exemplary and not as a limitation, and thus other examples of the exemplary embodiments may have different values.

Claims

1. A method for controlling soybean phomopsis seed rot fungus ( Phomopsis longicolla ) caused by soybean phomopsis seed rot, characterized in that: The biocontrol bacteria are Aspergillus qilianyuensis JSNL-ZJ66, the deposit number is CGMCC NO. 41157, and the depository is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. A biocontrol agent for soybean phomopsis seed rot, characterized in that: The active ingredient of the biocontrol agent includes hyphae or conidia of the biocontrol bacterium JSNL-ZJ66 according to claim 1.

3. A method for preparing the biocontrol agent according to claim 2, characterized in that: The preparation method comprises inoculating the biocontrol bacteria JSNL-ZJ66 with the deposit 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.

4. A microbial pesticide for preventing and controlling soybean phomopsis seed rot, characterized in that: The active ingredient of the microbial pesticide comprises the biocontrol bacterium JSNL-ZJ66 according to claim 1 or the biocontrol agent according to claim 2.

5. Use of the biocontrol bacterium JSNL-ZJ66 according to claim 1, the biocontrol agent according to claim 2 or the microbial pesticide according to claim 4 in preventing and controlling soybean phomopsid seed rot.

6. The use according to claim 5, characterized in that: The soybean phomopsis seed rot is P. longicolla Soybean stem rot caused by soybean stem rot.

7. A method for preventing and treating soybean phomopsis seed rot, characterized in that: The method comprises applying the biocontrol bacteria JSNL-ZJ66 of claim 1, the biocontrol agent of claim 2 or the microbial pesticide of claim 4 to the plant growth environment.

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