Bacillus velez strain HMQAU20041, biocontrol mixture, preparation method and application thereof

Abstract

The present invention provides a Bacillus velezensis strain HMQAU20041, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee on April 14, 2022, with a deposit registration number of CGMCC No.24676. The present invention also provides an application of the Bacillus velezensis strain HMQAU20041. The present invention also provides a biocontrol mixture, the active ingredients of which include a bacterial suspension, a fermentation liquid or a fermentation filtrate of two bacterial mixtures of Bacillus velezensis strain HMQAU20041 and Bacillus velezensis strain HMQAU20091. The present invention also provides a method for preparing the biocontrol mixture. The present invention also provides an application of the biocontrol mixture in the prevention and control of plant nematode diseases. The present invention also provides a method for preventing and controlling plant diseases. The Bacillus velezensis strain HMQAU20041 provided by the present invention has a broad spectrum of antagonistic activity against plant pathogens and pathogenic nematodes.

Description

Technical Field

[0001] The invention belongs to the field of plant disease biocontrol, and specifically relates to a Velezella bacillus strain HMQAU20041, a biocontrol mixture, and a preparation method and application thereof. Background Art

[0002] There are many stubborn old diseases and some newly emerging diseases in agricultural production, which seriously threaten the pre-production, mid-production and post-production of field crops, vegetables, fruit trees and flowers. In order to explore new means of disease prevention and control in the entire agricultural industry chain, we selected some important local plant diseases such as root-knot nematode disease of cucumber and tomato, stem nematode disease of sweet potato, potato and garlic, gray mold of lily, gray mold of bean, garlic sclerotinia, celery sclerotinia, Chinese cabbage sclerotinia, gray leaf spot of tomato, fusarium wilt and stem rot of wheat, corn anthracnose, grape white rot, blueberry hairy dispora branch blight, spinach Fusarium root rot, blueberry Phytophthora root rot, lettuce leaf spot, spinach Fusarium root rot, blueberry leaf spot, yam Fusarium root rot, soybean and sunflower black spot, quinoa branch blight, carrot soft rot, garlic dry rot, kiwi soft rot, cucumber brown spot, cucumber black spot, cucumber downy mildew and other diseases for relevant research. In production, chemical control is mostly used to prevent and control the above diseases, but chemical pesticides can easily lead to drug resistance in pathogens, pesticide residues, environmental pollution and adverse effects on human health. Therefore, choosing environmentally friendly, efficient and green biological control measures has become a new direction in research on the prevention and control of plant diseases.

[0003] For example, cucumber (Cucumis sativus) has become one of the top ten vegetable crops in the world, with an annual cultivation area of ​​about 146.67 hm2 worldwide. 2 (He Xiaoming et al., 2002). Cucumbers are cultivated in both the south and north of China (Xiong Yan et al., 2016). According to statistics from the Ministry of Agriculture and Rural Affairs, the national cucumber planting area in 2015 was 18.87 million mu, and the output reached 59.38 million tons, accounting for 5.8% and 7.8% of the total vegetables, respectively. The market demand is large, and there are huge economic and social benefits. At present, the production and cultivation area of ​​cucumbers in protected areas has expanded rapidly, and the benefits are significantly higher than those of open field cultivation (Zhao Guoyun et al., 2008). Pseudoperonospora cubensis and Meloidogyne incogita cause downy mildew and root knot nematode disease of cucumbers, respectively. They are important leaf and root diseases in the world's cucumber producing areas (Thomas, 1986), and are also a devastating disease on protected cucumbers, which seriously affect the yield and quality of cucumbers (Fu Shuyun, 1983; Shi Yanxia, ​​2002).

[0004] Ditylenchus destructor Thorne (1945) is an important plant pathogenic nematode in the world and a quarantine pest of agricultural plants in China. It harms a variety of underground fruiting crops such as sweet potatoes, potatoes, and garlic, and can also grow and reproduce on many weeds and fungi. The species of crops and weeds that can be infected by Ditylenchus destructor far exceed 100 species. In agricultural and forestry production, the crops that are most seriously damaged are tubers, bulbs / corms, tuberous roots, and fleshy taproots, including sweet potatoes, potatoes, garlic, angelica, carrots, beets, hops, American ginseng, bulbous irises, dahlias, etc. Ditylenchus destructor prefers coolness and is afraid of heat, and prefers moisture and is afraid of dryness. It is mainly spread and diffused through the asexual propagation materials of host crops.

[0005] However, the prior art lacks biocontrol agents with high efficacy against cucumber downy mildew, root-knot nematode disease, stem nematode rot, etc. How to solve the above problems is a technical problem that needs to be solved in the field of plant disease biocontrol. Summary of the invention

[0006] The present invention aims to provide a Velezella strain HMQAU20041, a biocontrol mixture and a preparation method and application thereof in view of the deficiencies in the prior art.

[0007] The first object of the present invention is to provide a biocontrol bacterium isolated from the leaf surface of cucumber, Bacillus velezensis strain HMQAU20041, which enriches the strain resources of plant disease biocontrol bacteria and lays a foundation for the research and development of biocontrol mixtures. The Bacillus velezensis strain HMQAU20041 is referred to as "strain HMQAU20041" in the present invention. The strain HMQAU20041 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 14, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit registration number is: CGMCC No. 24676.

[0008] The second object of the present invention is to provide an application of the above-mentioned strain HMQAU20041 in inhibiting plant pathogens, preparing products for inhibiting plant pathogens, preventing and controlling diseases caused by plant pathogens, and preparing products for preventing and controlling diseases caused by plant pathogens.

[0009] The third object of the present invention is to provide a biocontrol mixture. The active ingredients of the biocontrol mixture include two bacterial mixtures, Bacillus velezensis strain HMQAU20041 and Bacillus altitudinis strain HMQAU20091. Among them, the Bacillus altitudinis strain HMQAU20091 described in the present invention is an existing known strain, which is recorded in the inventor's earlier authorized patent CN202210621801.4. Its classification name is Bacillus altitudinis, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 14, 2022. The deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit registration number is: CGMCC No. 24677. Bacillus altitudinis strain HMQAU20091 may be referred to as "strain HMQAU20091" in the present invention.

[0010] The fourth object of the present invention is to provide a method for preparing the above-mentioned biocontrol mixture.

[0011] A fifth object of the present invention is to provide an application of a biocontrol mixture.

[0012] The sixth object of the present invention is to provide a method for preventing and controlling plant diseases.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] A Bacillus velezensis strain HMQAU20041, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 14, 2022, with the deposit registration number: CGMCC No.24676.

[0015] The use of the Bacillus velezensis strain HMQAU20041 as described above in the following (a1) or (a2) or (a3) ​​or (a4), (a1) inhibiting plant pathogens; (a2) preparing a product for inhibiting plant pathogens; (a3) ​​preventing and controlling diseases caused by plant pathogens; (a4) preparing a product for preventing and controlling diseases caused by plant pathogens; the plant pathogens are Pseudoperonospora cubensis, Pythium sylvaticum, Sclerotinia sclerotiorum, Botryoshaeria dothidea, Coniella diplodiella, Colletotrichum graminicola, Botrytis cinerea, cinerea), Cladosporium cucumerinum, Lasiodiplodia pseudotheobromae, Pestalotiopsis sp., Phomopsis sp., Pythium irregulare, Rhizoctonia solani, Fusarium oxysporum, Mucor circinelloides, Stemphylium lycopersici, Fusarium asiaticum, Rhizopus sp., Phoma betae, Corynespora cassiicola, Alternaria alternata, Fusariumpseudograminearum, Ulocladium chartarum, Alternaria solani, Fusarium equiseti), Stemphylium botryosum, Fusarium graminearum, Fusarium solani.

[0016] Based on the same inventive concept, the present invention also provides a biocontrol mixture, the active ingredients of which include a bacterial suspension, fermentation broth or fermentation filtrate of two bacterial mixtures, Bacillus velezensis strain HMQAU20041 and Bacillusaltitudinis strain HMQAU20091; the Bacillus velezensis strain HMQAU20041 was deposited in the General Microbiology Center of the China Microorganism Culture Collection on April 14, 2022, with a deposit registration number of CGMCC No. 24676; the Bacillus altitudinis strain HMQAU20091 was deposited in the General Microbiology Center of the China Microorganism Culture Collection on April 14, 2022, with a deposit registration number of CGMCC No. 24677.

[0017] The biocontrol mixture as described above, wherein the active ingredient of the biocontrol mixture is the fermentation filtrate of two bacterial mixtures of Bacillus velez strain HMQAU20041 and Bacillus velez strain HMQAU20091, wherein the total live bacteria concentration of Bacillus velez strain HMQAU20041 and Bacillus velez strain HMQAU20091 in the biocontrol mixture is 1×10 8 ~2×10 8 cfu / mL.

[0018] Based on the same inventive concept, the present invention also provides a method for preparing a biocontrol mixture, the specific steps of which include:

[0019] (1) Cultivation of seed culture solutions of strains HMQAU20041 and HMQAU20091: The strains HMQAU20041 and HMQAU20091 described in claim 3 are inoculated into LB culture solution, respectively, and cultured until the OD values ​​of the respective seed culture solutions are between 0.5 and 0.8, thereby obtaining seed culture solutions of strains HMQAU20041 and HMQAU20091;

[0020] (2) Preparation of fermentation filtrate: The seed culture of strain HMQAU20041 and the seed culture of strain HMQAU20091 are mixed in different volumes, and the mixed seed culture is added to a conical flask containing LB culture medium at a ratio of 1% by volume, shaken and cultured, and then centrifuged and filtered through a 0.22 μm microporous filter membrane to prepare the fermentation filtrate, thereby obtaining the biocontrol mixture. Preferably, the volume ratio of the seed culture of strain HMQAU20041 and the seed culture of strain HMQAU20091 in step (2) is (9:1) to (1:9). Most preferably, the volume ratio of the seed culture of strain HMQAU20041 and the seed culture of strain HMQAU20091 in step (2) is 7:3.

[0021] In the method for preparing the biocontrol mixture as described above, the culture conditions in step (1) refer to shaking culture at 30° C. and 200 rpm for 8 to 10 hours.

[0022] In one embodiment of the present invention, the shaking culture conditions in step (2) are: 30° C., 200 rpm shaking culture for 48 hours.

[0023] In one embodiment of the present invention, the LB culture medium formula in steps (1) and (2) is: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, and the pH value is adjusted to 7.3.

[0024] Preferably, the centrifugation condition in step (2) is 8000 rpm for 15 min.

[0025] In the method for preparing the biocontrol mixture as described above, the formula of the LB culture medium in step (2) is: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L NaCl to adjust the pH value to 7.2-7.4.

[0026] Based on the same inventive concept, the present invention also provides the use of the biocontrol mixture as described above or the biocontrol mixture prepared by the preparation method as described above, in the following (b1) or (b2) or (b3) or (b4), characterized in that (b1) inhibits plant pathogens; (b2) prepares a product for inhibiting plant pathogens; (b3) prevents and treats diseases caused by plant pathogens; (b4) prepares a product for preventing and treating diseases caused by plant pathogens; the plant pathogens are Pseudoperonospora cubensis, Sclerotinia sclerotiorum, Coniella diplodiella, Botryoshaeria dothidea, Rhizoctonia solani, Phytophthora cinnamomi, Pestalotiopsis sp., Colletotrichum siamense, Phomopsis spp. sp.), Stemphylium lycopersici, Cladosporium cucumerinum, Phomabetae, Botrytis cinerea, Mucor circinelloides, Stemphylium botryosum, Rhizopus sp., Corynespora cassiicola, Ulocladium chartarum, Alternaria alternata, Fusarium oxysporum, Fusarium equiseti, Fusarium solani, Lasiodiplodia pseudotheobromae, Pythium sylvaticum, Fusarium graminearum, and Pythium irregulare.

[0027] Based on the same inventive concept, the present invention also provides the use of the biocontrol mixture as described above or the biocontrol mixture prepared by the preparation method as described above in the prevention and control of plant nematode diseases, wherein the plant disease is any one of root-knot nematode disease and stem nematode disease.

[0028] Based on the same inventive concept, the present invention also provides a method for preventing and controlling plant diseases. When preventing and controlling plant diseases, the biocontrol mixture as described above or the biocontrol mixture prepared by the preparation method as described above is directly applied to plants and plant parts using a common treatment method or acts on the surroundings and growth areas thereof, such as by coating, dipping, spraying, atomizing, irrigating, volatilizing, dusting, sowing, foaming, coating, etc., and in the case of propagation materials, especially seeds, it is also applied by one or more layers of coating, etc. as a treatment powder for dry seeds, a solution for treating seeds, and a water-soluble powder for slurry treatment. The microbial agent can also be applied by ultra-low volume, or the microbial agent itself can be injected into the soil. Preferably, when preventing and controlling leaf diseases such as cucumber downy mildew, the biocontrol mixture as described above is sprayed on the diseased part of the plant.

[0029] The beneficial effects of the present invention are:

[0030] 1. The Velez subsp. velutipes strain HMQAU20041 provided by the present invention has a broad-spectrum antagonistic activity against plant pathogens and pathogenic nematodes, and has a control effect on plant diseases caused by pathogens such as Pseudocytin, Pythium, Sclerotinia, Botrytis, White Rot, Graminoides, Botrytis, Cinerea, Pseudocytin ... In particular, the antibacterial rate of forest-dwelling Pythium, Sclerotinia sclerotiorum, Botrytis cinerea, white rot spores, Graminoides fasciata, and Botrytis cinerea reached 100%.

[0031] 2. The present invention provides a biocontrol mixture, wherein the active ingredients of the biocontrol mixture include a bacterial suspension, fermentation liquid or fermentation filtrate of two bacterial mixtures, namely, Bacillus velezensis strain HMQAU20041 and Bacillus subtilis strain HMQAU20091, especially the fermentation filtrate, which has a significant synergistic effect on the prevention and control of cucumber downy mildew.

[0032] 3. The biocontrol mixture provided by the present invention also has a broad-spectrum antagonistic activity against plant pathogens and pathogenic nematodes, and has a control effect on plant diseases caused by pathogens such as Pseudocorhinus cubanius, Sclerotinia sclerotiorum, White Rot Pad Spore, Botrytis cinerea, Rhizoctonia solani, Phytophthora camphora, Pseudodiscus polytrichospore, Siamese anthracnose, Phomopsis spp., Solanum spp., Cucumis spp., Stem Spots of Beet, Mucor volitans, Scolopendra spp., Rhizoctonia spp., Corynosporium spp., Alternaria spp., Fusarium spp., Fusarium equisetifolium, Fusarium solani, Pseudocorhinus spp., Forest-dwelling Pythium, Fusarium graminearum, and Pythium malformis. In particular, the antibacterial rate of Sclerotinia sclerotiorum, White Rot Pad Spore, Botrytis cinerea, and Rhizoctonia solani reaches 100%. In addition, the biocontrol mixture provided by the present invention also has a synergistic effect on the control of root-knot nematode disease and stem nematode disease.

[0033] 4. The biocontrol mixture of the present invention is a biological preparation, and has no series of problems caused by the use of chemical pesticides, so it is conducive to the green production of greenhouse vegetables. Farmers can use or reduce the amount of chemical pesticides, which can not only save expenses for farmers, but also be conducive to the export of vegetables. At the same time, the mixture also has the function of increasing production, which can increase farmers' income. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The colony, Gram staining, and spore staining of strain HMQAU20041 (A, B: colony morphology; C: Gram staining; D: spore staining);

[0035] Figure 2 This is a phylogenetic tree of Bacillus velez constructed by neighbor-joining analysis based on 16S rDNA gene sequences;

[0036] Figure 3 This is a phylogenetic tree of Bacillus velez constructed by neighbor-joining analysis based on the gyrB gene sequence;

[0037] Figure 4 This is the inhibitory effect of Bacillus Velez strain HMQAU20041 on the mycelial growth of different pathogens. DETAILED DESCRIPTION

[0038] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] Example 1 Identification of strain HMQAU20041 and its inhibitory effect on hyphae growth of different pathogens

[0041] 1. Test plant diseases and their pathogens:

[0042] Spinach rot: Pythium sylvaticum HMQAU210092, Pythium irregulare HMQAU210091; Celery sclerotinia: Sclerotinia sclerotiorum HMQAU170216; Quinoa blight: Botryoshaeria dothidea HMQAU190265, Phoma betae HMQAU190266; Grape white rot: Coniella diplodiella HMQAU170082; Maize anthracnose: Colletotrichum graminicola HMQAU 220031; Lily gray mold: Botrytis cinerea HMQAU200037; Cucumber scab: Cladosporium cucumerinum HMQAU180016; Blueberry Lasiodiplodia branch blight: Lasiodiplodia pseudotheobromae HMQAU140073; Blueberry leaf spot: Pestalotiopsis sp., HMQAU180119; Phomopsis sp. HMQAU210069; Garlic sclerotinia: Rhizoctonia solani HMQAU210022; Spinach Fusarium root rot: Fusarium oxysporum HMQAU170173, Fusarium equiseti HMQAU170169; Kiwifruit soft rot: Mucor circinelloides HMQAU150050; Garlic stem rot: Fusarium oxysporum HMQAU210030 and HMQAU210034; Tomato gray leaf spot: Stemphylium solani HMQAU170209; Wheat fusarium: Fusarium asiaticum HMQAU220017, Fusarium graminearum HMQAU170037; Carrot soft rot: Rhizopus sp.HMQAU180037; Chinese cabbage sclerotinia: Rhizoctonia solani HMQAU180110; cucumber brown spot: Corynespora cassiicola HMQAU200087; soybean black spot: Alternaria alternata HMQAU210080; wheat stem rot: Fusariumpseudograminearum HMQAU220027; lettuce leaf spot: Ulocladium chartarum HMQAU210122, Stemphylium botryosum HMQAU210121; sunflower black spot: Alternaria solani HMQAU220028; yam Fusarium root rot: Fusarium solani HMQAU180201. All the above strains were provided by the Mycology Laboratory of Qingdao Agricultural University. .

[0043] 2. Tested biocontrol bacteria:

[0044] The strain HMQAU20041 was isolated from cucumber leaves in Shenxian County, Liaocheng City, Shandong Province using the plate dilution method (Krechel et al., 2002).

[0045] 3. Bacterial identification methods:

[0046] (1) Morphological identification:

[0047] Morphological identification was performed according to the method in the Manual of Identification of Common Bacterial Systems (Dong Xiuzhu and Cai Miaoying, 2001).

[0048] (2) Physiological and biochemical identification:

[0049] According to the morphological characteristics of the target strain, common physiological and biochemical indicators were selected, and physiological and biochemical identification was carried out according to the methods of the Manual of Identification of Common Bacterial Systems (Dong Xiuzhu and Cai Miaoying, 2001).

[0050] (3) Molecular identification and phylogenetic analysis:

[0051] Bacterial genomic DNA was extracted using a bacterial genomic DNA kit and stored in a -20°C refrigerator for later use. Using the extracted bacterial genomic DNA as a template, PCR was performed using universal primers for bacterial 16SrDNA (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') and GyrB gene sequence primers (gyrB-F: 5'-GAAGTCATCATGA CCGTTCTGCAYGCNGGNGGNAARTTYGA-3' and gyrB-FR: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3').

[0052] The total PCR system for 16S rDNA was 25 μL: 2.5 μL 10× PCR reaction buffer, 2 μL dNTPs (10 mmol / L), 1 μL each of primers (10 mmol / L), 0.5 μL Taq DNA polymerase (5 U / μL), 1 μL DNA template, ddH 2 Make up to 25 μL with O. Reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 63℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ extension for 7 min.

[0053] GyrB gene sequence PCR total system (25 μL): 10×PCR Buffer 2.5 μL, dNTP Mixture 0.5 μL (10 mM), gyrB-F / gyrB-R (10 μmol / L) 1 μL each, Pro Taq enzyme (5 U / μL) 0.2 μL, DNA template 1 μL, supplemented with ddH 2 0 to 25 μL. Reaction conditions: 94°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 67°C annealing for 30 s, 72°C extension for 2 min, 30 cycles; 72°C extension for 2 min.

[0054] After PCR, 5 μL of PCR product was mixed with 1 μL of 6× Loading Buffer, and electrophoresed on 1% agarose gel at 120 V for 30 min using DNA marker 2000 as the molecular weight standard. The length and concentration of the amplified product were detected using a gel imaging system.

[0055] The PCR products of the two primers were purified using a kit, and the purified PCR products were recovered and detected by 1% agarose gel electrophoresis. The purification was successful when a single band was displayed. TM18-TVector user manual, product ligation and transformation, single colony using pMD TM 18-T universal primers M13-47: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and RV-M: 5'-GAGCGGATAACAATTTCAC ACACAGG-3' were used for PCR detection. The amplification reaction system and reaction conditions were the same as above. After the PCR was completed, 5 μL of PCR product was electrophoresed on a 1% agarose gel at 120V for 30 minutes, and the length of the amplified product was detected in a gel imaging system. If the length of the target fragment was about 1700 bp, the corresponding colony was a positive clone, and its bacterial solution was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0056] The sequencing results were compared with BLAST on the NCBI database, and the 16S rDNA sequences of the standard strains of the strains in the comparison results were downloaded. The phylogenetic analysis was performed using MEGA5.05 software, and a phylogenetic tree was established and cluster analysis was performed.

[0057] 4. Strain identification results:

[0058] (1) Morphological identification results:

[0059] The strain HMQAU20041 was cultured on LB solid medium at 28°C for 2 days. The colonies were milky white, opaque, round, with irregular edges and wrinkled surfaces. The bacteria were Gram-positive, rod-shaped (0.79±0.09)μm-(2.09±0.11)μm, arranged singly or in pairs, and produced spores. The spores were oval, mesogenic, and the cysts were swollen. Figure 1 shown.

[0060] (2) Physiological and biochemical identification results:

[0061] The results of physiological and biochemical tests showed that strain HMQAU20041 was anaerobic and had strong salt tolerance. It could still grow in 10% NaCl culture medium, but could not grow at 4°C, but could grow at 45°C. It was acid and alkali resistant and could grow at pH 5.5 and 9.0. It could utilize citrate, D-xylose, mannitol, glucose, sorbitol, and D-fructose, but could not oxidize and ferment glucose. Physiological and biochemical tests such as oxidase, indole reaction, denitrification, and methyl red were negative, while physiological and biochemical tests such as catalase, nitrate reduction, VP, and starch hydrolysis were positive, as shown in Table 1. Based on the morphological characteristics and physiological and biochemical characteristics of strain HMQAU20041, the strain can be preliminarily identified as Bacillus.

[0062] Table 1 Physiological and biochemical test results of Bacillus velez strain HMQAU20041

[0063]

[0064]

[0065] (3) Molecular identification and phylogenetic analysis results:

[0066] According to the sequencing results, the lengths of the PCR products of the 16S rRNA gene and gyrB gene sequences of strain HMQAU20041 were 1514 bp and 1259 bp, respectively. The sequenced 16SrDNA gene sequence is shown in SEQ ID No. 1, and the sequenced gyrB gene sequence is shown in SEQ ID No. 2. The BLAST comparison results in the NCBI database showed that the 16S rRNA gene similarity of strain HMQAU20041 and Bacillus velezensis (EF433407) was as high as 99.87%. The phylogenetic tree analysis showed that HMQAU20041 and Bacillus velezensis were on the same branch, see Figure 2 The gyrB gene sequence of HMQAU20041 was further analyzed and a phylogenetic tree was constructed. The results showed that the homology between strain HMQAU20041 and B. velezensis (KY381949) was as high as 99.89%, and it was on the same branch as Bacillus velezensis, see Figure 3 Based on morphological characteristics, sequence alignment analysis, morphological and physiological and biochemical identification results (Li Shengzhang et al., 2019; Yan Zhubing et al., 2021; Wang Qinghua et al., 2019; Huang Huijing, 2022), strain HMQAU20041 was identified as Bacillus velezensis. The identification results are shown in Table 2.

[0067] Table 2 Sequencing alignment results

[0068]

[0069]

[0070] The screened Bacillus velezensis strain HMQAU20041 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 14, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit registration number: CGMCC No. 24676. The requested deposit unit is Qingdao Agricultural University.

[0071] 5. Preparation of fermentation filtrate of strain HMQAU20041:

[0072] (1) Cultivation of seed culture: Inoculate a loop of the strain HMQAU20041 described above into LB culture medium and culture at 30°C and 200 rpm for 8 h until the OD value of the seed culture is between 0.5 and 0.8. Use a hemocytometer to adjust the concentration of the seed culture to 5 × 10 8 CFU / mL, and prepare the seed culture.

[0073] (2) Preparation of fermentation filtrate: The HMQAU20041 seed culture liquid was added to a 150 mL conical flask containing 50 mL LB culture medium at a volume percentage ratio of 1% (in this example, 500 μL of the concentration of 5×10 8 CFU / mL seed bacterial solution was added to 50mL LB culture medium), shaken and cultured at 30°C and 200rpm for 48h, then centrifuged at 8000rpm for 15min, and filtered through a 0.22μm microporous membrane to prepare a sterile filtrate of antagonistic bacteria. The viable bacterial concentration of Bacillus Velezii HMQAU20041 was 1×10 10 CFU / mL.

[0074] The LB culture medium formula is: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and the pH value is adjusted to 7.3.

[0075] 6. Inhibitory effect of fermentation filtrate of strain HMQAU20041 on mycelial growth of different pathogens

[0076] (1) Test method:

[0077] The mycelium growth rate method (Chen Nianchun, 1991) was used to mix the fermentation filtrate stock solution with the melted and cooled PDA medium (45-50°C) (diluted 10 times), and the plate was poured. The PDA medium with an equal volume of LB liquid medium was used as the control, and the pathogenic bacteria cake with a diameter of 5 mm was inoculated in the center of the plate. When the control grew over 3 / 4 of the culture dish, the colony diameters of the control group and the treatment group were measured by the cross method, and the inhibition rate was calculated. Each treatment was repeated 3 times.

[0078] Inhibition rate (%) = (control colony radius (mm) - treated colony radius (mm)) / control colony radius (mm) × 100%.

[0079] (2) Test results

[0080] The test results are shown in Table 3 and Figure 4 shown.

[0081] Table 3 Inhibitory effect of strain HMQAU20041 on hyphae growth of different pathogens

[0082]

[0083]

[0084] Example 2 Screening of the ratio of the biocontrol mixture of strain HMQAU20041 and strain HMQAU20091

[0085] 1. Test method:

[0086] (1) Preparation of the fermentation filtrate of the biocontrol agent

[0087] A. Cultivation of seed culture: strain HMQAU20041 and strain HMQAU20091 were inoculated into LB culture medium at 30°C and 200 rpm for 8 h, and the culture was terminated when the OD value of each seed culture was between 0.5 and 0.8. The concentration of the seed culture was adjusted to 5×10 using a hemocytometer. 8 CFU / mL, and the seed culture solutions of strain HMQAU20041 and strain HMQAU20091 were prepared respectively.

[0088] B. Preparation of fermentation filtrate: The seed culture liquid of strain HMQAU20041 and the seed culture liquid of strain HMQAU20091 were mixed in different volumes, and the mixed seed culture liquid was added into a 150 mL conical flask containing 50 mL LB culture medium at a ratio of 1% by volume, and cultured with shaking at 30°C and 200 rpm for 48 h, and then centrifuged at 8000 rpm for 15 min, and filtered through a 0.22 μm microporous filter membrane to prepare the fermentation filtrate of the biocontrol mixture.

[0089] The LB culture medium formula is: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, and the pH value is adjusted to 7.3.

[0090] The preparation method of the bacterial cell preservation solution is as follows: dilute glycerol with 0.9% physiological saline to a concentration of 30% to obtain the bacterial cell preservation solution.

[0091] (2) Effects of fermentation filtrates of different biocontrol mixtures on the release of sporangia of cucumber downy mildew

[0092] EC treated with different 50 Based on the value, the seed culture liquid of strain HMQAU20041 and the seed culture liquid of strain HMQAU20091 were mixed and fermented with different volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, and the inhibition rate of the fermentation filtrate of the biocontrol mixture against cucumber downy mildew was determined at different volume ratios. If the toxicity ratio (actual inhibition rate / expected inhibition rate) is >1, it is a synergistic effect; if the toxicity ratio is <1, it is an antagonistic effect; if the toxicity ratio is about 1, it is additive. Use sterile tap water to prepare a concentration of 2x105 / mL of sporangium suspension of cucumber downy mildew. Add 20μL of sporangium suspension and fermentation filtrate onto the concave glass slide and mix well. Use sterile tap water and sporangium suspension mixed in equal volumes as a control. Place the concave glass slide in a culture dish covered with wet filter paper, place it in a refrigerator at 4℃, RH>80% in the dark and moisturize for 1h, and then culture it in a 20℃ incubator for 1h. When more than 80% of the sporangia in the control release zoospores, observe the empty spore rate of 100 sporangia under a microscope. Set up 3 replicates for each treatment, and repeat the experiment twice. Using sterile water treatment as a control, calculate the actual inhibition rate of each ratio on cucumber downy mildew.

[0093] The formula for calculating the expected inhibition rate after mixing two single agents A and B is:

[0094] Toxicity ratio = actual inhibition rate / expected inhibition rate

[0095] Actual inhibition rate: the inhibition rate of each ratio tested in the experiment on cucumber downy mildew;

[0096] Expected inhibition rate: A against cucumber downy mildew EC 50 Actual inhibition rate × percentage in the ratio + EC of B against cucumber downy mildew 50 Actual inhibition rate × percentage in the ratio.

[0097] 3. Results of screening of biocontrol mixture ratios

[0098] The test results are shown in Table 4. 50 The strain HMQAU20041 and strain HMQAU20091 were mixed at different doses, and the effects were different in different ratios. Among them, the strain HMQAU20041 and strain HMQAU20091 had the highest toxicity ratio of 1.39, which was greater than 1.2, and the toxicity ratios of other ratios were all lower than 1.2.

[0099] Table 4 Inhibition rate of different ratios of HMQAU20041 and HMQAU20091 on cucumber downy mildew

[0100]

[0101] Example 3 Synergistic Effect Determination of the Effective Ratio of Biocontrol Mixture

[0102] 1. Test method:

[0103] According to Example 2, the mixture with the highest toxicity ratio was screened out by mixing two single agents, and the EC of the effective mixture against cucumber downy mildew was determined by the cotyledon spray method. 50The co-toxicity coefficient was determined by the method introduced by Sun Yunpei (1950). A co-toxicity coefficient of >120 indicates synergistic effect, 80≤co-toxicity coefficient≤120 indicates additive effect, and a co-toxicity coefficient of <80 indicates antagonistic effect. The one with the highest co-toxicity coefficient is the best ratio.

[0104] Actual Toxicity Index (ATI) of the mixture = EC of the standard agent 50 / EC of the test agent 50 ×100

[0105] Theoretical toxicity index (TTI) of mixture = toxicity index of agent A × content of agent A in the mixture (%) + toxicity index of agent B × content of agent B in the mixture (%)

[0106] Co-toxicity coefficient of mixed agent (CTC) = ATI / TTI×100

[0107] 2. Results of the synergistic effect test of the effective ratio of biocontrol mixtures

[0108] The results of the effective ratio synergistic effect test are shown in Table 5.

[0109] Table 5 Effective ratio synergistic effect test results

[0110]

[0111] Example 4 Inhibitory effect of the fermentation filtrate of the biocontrol mixture on different pathogens

[0112] 1. Test plant diseases and their pathogens:

[0113] Celery sclerotinia: Sclerotinia sclerotiorum HMQAU170216; Grape white rot: Coniella diplodiella HMQAU170082; Quinoa branch blight: Botryoshaeriadothidea HMQAU 190265, Colletotrichum siamense HMQAU190267, Phoma betae HMQAU190266; Chinese cabbage sclerotinia: Rhizoctonia solani HMQAU180110; Blueberry Phytophthora root rot: Phytophthora cinnamomi HMQAU180002; Blueberry leaf spot: Pestalotiopsis sp. HMQAU180119, Phomopsis sp.HMQAU210069; tomato gray leaf spot: Stemphylium solani HMQAU170209; cucumber black spot: Cladosporium cucumerinum HMQAU180016; bean gray mold: Botrytis cinereaHMQAU170042; kiwifruit soft rot: Mucor circinelloides HMQAU150050; lettuce leaf spot: Ulocladium chartarum HMQAU210122, Stemphylium botryosumHMQAU210121; garlic sclerotinia: Rhizoctonia solani HMQAU210022; carrot soft rot: Rhizopus sp.HMQAU180037; Gray mold of lily: Botrytis cinerea HMQAU200037; Brown spot of cucumber: Corynespora cassiicola HMQAU 200087; Black spot of soybean: Alternaria alternata HMQAU210080; Fusarium root rot of spinach: Fusarium oxysporum HMQAU170173, Fusarium equiseti HMQAU170169; Dry rot of garlic: Fusarium oxysporum HMQAU210030 and HMQAU210034, Fusarium solani HMQAU210027; Lasiodiplodia pseudotheobromae HMQAU140073; spinach pythium root rot: Pythium sylvaticum HMQAU210092, Pythium irregulare HMQAU210091; wheat fusarium: Fusarium graminearum HMQAU170037; yam fusarium root rot: Fusarium solani HMQAU180201; all of the above were provided by the Mycology Laboratory of Qingdao Agricultural University. .

[0114] 2. Results of the inhibitory effect of the fermentation filtrate of the biocontrol agent on different pathogens

[0115] The LB fermentation filtrate of the mixture of the seed culture liquid of strain HMQAU20041 and strain HMQAU20091 in the optimal volume ratio of 7:3 was diluted 10 times in PDA and the inhibitory effect on different pathogens was determined by the mycelium growth rate method. The results are shown in Table 6. The fermentation filtrate had different antibacterial activities against 30 tested plant pathogenic fungi. Among them, it has a complete inhibitory effect on Sclerotinia sclerotiorum that causes celery sclerotinia, Albugo sclerotiorum that causes grape white rot, Botrytis cinerea that causes quinoa branch blight, and Rhizoctonia solani of Chinese cabbage, with inhibition rates of 100%; followed by Phytophthora camphora that causes blueberry root rot, Pseudomonas aeruginosa that causes blueberry leaf blight, Colletotrichum siamese that causes quinoa branch blight, Phomopsis syringae that causes blueberry leaf blight, Stem Pseudomonas syringae that causes tomato gray leaf spot, Cladosporium cucumerinum that causes cucumber black spot, Stem Pseudomonas aeruginosa that causes quinoa branch blight, and Botrytis cinerea that causes beet gray mold, with inhibition rates all above 93.38%; the inhibition rates on 18 pathogens such as Mucor circinelloides that causes kiwifruit soft rot and Sclerotium stolonifer that causes lettuce leaf spot are also between 46.5% and 89.67%; the inhibition rates on Fusarium graminearum, Pythium tertiorrhiza and Fusarium solani are relatively poor. This indicates that the mixture has a broad antibacterial spectrum, good antibacterial effect, and high biocontrol potential.

[0116] Table 6 Inhibitory effect of the fermentation filtrate of the biocontrol mixture on different pathogens

[0117]

[0118] Note: The letters after the numbers in the same column indicate significant differences at the P < 0.05 level (Duncan's new multiple range method)

[0119] Example 5 The lethal effect of HMQAU20041, HMQAU20091 and the fermentation filtrate of the biocontrol mixture on southern root-knot nematode

[0120] 1. Test methods

[0121] The experiment set up 17 treatments, which were HMQAU20041 and HMQAU20091 single agents and fermentation filtrate stock solutions of the mixture diluted 0 times, 5 times, 10 times, 25 times, and 50 times for standby use. 1 mL of each of the above treatments was added to a 24-well plate, and 200 μl (about 300) root-knot nematodes were added to each well; the drug control was avermectin (2 μg / mL), and the blank control was sterile water; each treatment was repeated 3 times. The 24-well plate was placed in a 25°C incubator for 12 h and 24 h. The morphology of the nematodes was observed under a dissecting microscope. The bending of the nematodes was considered alive, and the stiffness was considered dead. The mortality rate was statistically corrected and the different morphologies of the nematodes were recorded by taking photos. The fermentation filtrate of the strain HMQAU20091 used in the present invention was prepared according to the preparation method of Example 1 of the specification of Chinese patent CN202210621801.4. Among them, the viable bacterial concentration of the strain HMQAU20091 was 1×1010CFU / mL.

[0122] Corrected mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100%

[0123] 2. Lethal effects of each treatment on southern root-knot nematode

[0124] The results are shown in Table 7. When the treatment time was 12h, the corrected mortality of the fermentation filtrate of HMQAU20041 reached 95.26%, the corrected mortality of the fermentation filtrate of HMQAU20091 was 81.82%, and the corrected mortality of the fermentation filtrate of the mixture was 100%, and the difference between the three was significant; when each treatment was diluted 10 times, the corrected mortality was 47.13%, 33.72%, and 54.94%, respectively, and the difference between the three was significant. When the treatment time was 24h, the corrected mortality of the 5-fold dilution of the fermentation filtrate of HMQAU20041, HMQAU20091 and the mixture was 97.58%, 95.27% and 100%, respectively, and the difference was not significant; when each treatment was diluted 50 times, the corrected mortality was 31.97%, 25.58%, and 49.51%, respectively, and the difference between HMQAU20041 and HMQAU20091 and the mixture was significant.

[0125] Table 7 The lethal effects of different treatments on southern root-knot nematodes

[0126]

[0127]

[0128] Note: The letters after the numbers in the same column indicate significant differences at the P < 0.05 level (Duncan's new multiple range method)

[0129] Example 6 The lethal effect of HMQAU20041, HMQAU20091 and the fermentation filtrate of the biocontrol mixture on Ditylenchus destructor

[0130] 1. Test methods

[0131] The experiment set up 13 treatments, which were HMQAU20041 and HMQAU20091 single agent and the fermentation filtrate of the mixture diluted 0 times, 5 times, 10 times, and 25 times. 1 mL of each of the above treatments was added to a 24-well plate, and 200 μl (about 100) of Ditylenchus destructans were added to each well; avermectin was used as the drug control (2 μg / mL), and sterile water was used as the blank control; each treatment was repeated 3 times and placed in a 20°C incubator for 6 hours and 10 hours. The morphology of the nematodes was observed under a dissecting microscope. The bending of the nematodes was considered alive, and the stiffness was considered dead. The mortality rate was statistically corrected and the different morphologies of the nematodes were recorded by taking photos.

[0132] Corrected mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100%

[0133] 2. Lethal effects of each treatment on Ditylenchus destructor

[0134] The results are shown in Table 8. When the treatment time was 6 h, the corrected mortality rates of the fermentation filtrates of HMQAU20041, HMQAU20091 and the mixture were 16.21%, 48.54% and 77.35%, respectively, and there were significant differences among the three. The corrected mortality rates of the 5-fold diluted fermentation filtrates of HMQAU20041, HMQAU20091 and the mixture were 17.41%, 29.55% and 34.25%, respectively, and HMQAU20041 was significantly higher than that of HMQAU20091 and the mixture. The corrected mortality rates of the mixture, HMQAU20041 and HMQAU20091 fermentation filtrates diluted 10 times were 25.72%, 8.74% and 18.47%, respectively, with significant differences among the three; the corrected mortality rates of the mixture, HMQAU20041 and HMQAU20091 fermentation filtrates diluted 25 times were 6.34%, 1.54% and 5.05%, respectively, with significant differences among HMQAU20041, HMQAU20091 and the mixture.

[0135] When the treatment time was 10h, the corrected mortality rates of the fermentation filtrate of HMQAU20041, HMQAU20091 and the mixture were 44.15%, 74.01% and 97.20%, respectively, with significant differences among the three; the corrected mortality rates of the 5-fold diluted fermentation filtrate of HMQAU20041, HMQAU20091 and the mixture were 29.12%, 49.25% and 64.35%, respectively, with significant differences among the three; the corrected mortality rates of the 10-fold diluted fermentation filtrate of HMQAU20041, HMQAU20091 and the mixture were 15.71%, 29.73% and 43.77%, respectively, with HMQAU20041 and HMQAU20091 respectively. There was a significant difference between HMQAU20091 and the mixture; the corrected mortality rates of 25-fold dilutions of the fermentation filtrates of HMQAU20041, HMQAU20091 and the mixture were 5.82%, 10.46% and 15.09%, respectively, and there was a significant difference between HMQAU20041 and the mixture.

[0136] Table 8 The lethal effects of different treatments on Ditylenchus destructans

[0137]

[0138] Note: The letters after the numbers in the same column indicate significant differences at the P < 0.05 level (Duncan's new multiple range method)

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) strain HMQAU20041, characterized in that The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 14, 2022, with the deposit registration number: CGMCC No.24676.

2. The Bacillus Velezii according to claim 1 ( Bacillus velezensis ) Use of strain HMQAU20041 in the following (a1) or (a2) or (a3) ​​or (a4), characterized in that: (a1) inhibiting plant pathogens; (a2) preparing products for inhibiting plant pathogens; (a3) ​​preventing and controlling diseases caused by plant pathogens; (a4) preparing products for preventing and controlling diseases caused by plant pathogens; the plant pathogens are Pseudoperonosporacubensis, Pythium sylvaticum, Pythium sylvaticum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum )、Botrytis cinerea( Botryoshaeria dothidea )、White rot spores( Coniella diplodiella )、Coleosporium graminicola( Colletotrichum graminicola )、Botrytis cinerea( Botrytis cinerea )、Citrus cucurbitae( Cladosporium cucumerinum )、False cocoa hair color two cells ( Lasiodiplodia pseudotheobromae )、Pseudodiscus hirsutus( Pestalotiopsis sp. )、Phomopsis sphaeroides( Phomopsis sp. )、Pythium terrestris( Pythium irregulare )、Rhizoctonia solani( Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum )、Mucor circinelloides( Mucor circinelloides ) Stemphylium lycopersici ), Fusarium asiatica ( Fusarium Asiaticum )、Rhizopus( Rhizopus sp. )、Stoma betae Phoma betae )、Spores of sphaerocephala ( Corynespora cassiicola )、Alternaria alternata( Alternaria alternata ), Pseudofusorium graminearum ( Fusarium pseudograminearum )、Paper-like spores( Ulocladium chartarum ) Alternaria solani ) Fusarium equiseti )、Stolonifera( Stemphylium botryosum ) and Fusarium graminearum ( Fusarium graminearum ) or / and Fusarium solani ( Fusarium solani ).

3. A biocontrol mixture, characterized in that: The active ingredients of the biocontrol mixture include Bacillus velez ( Bacillus velezensis ) strain HMQAU20041 and Bacillus subtilis ( Bacillus altitudinis ) strain HMQAU20091 two bacterial mixture of bacterial suspension, fermentation liquid or fermentation filtrate; the Velez Bacillus ( Bacillus velezensis ) strain HMQAU20041 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 14, 2022, with the deposit registration number: CGMCC No.24676; the highland Bacillus ( Bacillus altitudinis ) strain HMQAU20091 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 14, 2022, with the deposit registration number: CGMCC No.24677.

4. The biocontrol mixture according to claim 3, characterized in that The active ingredient of the biocontrol mixture is the fermentation filtrate of two bacterial mixtures, namely, Bacillus velezensis strain HMQAU20041 and Bacillus altaica strain HMQAU20091.

5. The method for preparing the biocontrol mixture according to claim 4, characterized in that: The specific steps include: (1) Cultivation of seed culture solutions of strain HMQAU20041 and strain HMQAU20091: The strain HMQAU20041 and strain HMQAU20091 described in claim 3 are inoculated into LB culture solution respectively, and the culture is terminated when the OD value of each seed culture solution is between 0.5 and 0.8, thereby obtaining seed culture solutions of strain HMQAU20041 and strain HMQAU20091; (2) Preparation of fermentation filtrate: The seed culture liquid of strain HMQAU20041 and the seed culture liquid of strain HMQAU20091 were mixed in different volumes, and the mixed seed culture liquid was added into a conical flask containing LB culture medium at a ratio of 1% by volume. The mixture was cultured with shaking, and then centrifuged and filtered to prepare the fermentation filtrate to obtain the biocontrol agent.

6. The method for preparing the biocontrol mixture according to claim 5, characterized in that: The culture conditions in step (1) refer to shaking culture at 30° C. and 200 rpm for 8 to 10 hours.

7. The method for preparing the biocontrol mixture according to claim 5, characterized in that: The volume ratio of the seed culture liquid of strain HMQAU20041 to the seed culture liquid of strain HMQAU20091 in step (2) is (1:9) to (9:1).

8. Use of the biocontrol mixture according to claim 3 to 4 or the biocontrol mixture prepared by the preparation method according to any one of claims 5 to 7, characterized in that: The use in the following (b1) or (b2) or (b3) or (b4), characterized in that (b1) inhibits plant pathogens; (b2) prepares products for inhibiting plant pathogens; (b3) prevents and treats diseases caused by plant pathogens; (b4) prepares products for preventing and treating diseases caused by plant pathogens; the plant pathogen is Pseudocerona cubaniensis ( Pseudoperonospora cubensis ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum )、White rot spores( Coniella diplodiella )、Botrytis cinerea( Botryoshaeria dothidea )、Rhizoctonia solani( Rhizoctonia solani )、Cinnamomum camphora( Phytophthora cinnamomi )、Pseudodiscus hirsutus( Pestalotiopsis sp. )、Siamese Anthrax ( Colletotrichum siamense )、Phomopsis sphaeroides( Phomopsis sp. ) Stemphylium lycopersici )、Citrus cucurbitae( Cladosporium cucumerinum )、Stoma betae Phoma betae )、Botrytis cinerea( Botrytis cinerea )、Mucor circinelloides( Mucor circinelloides )、Stolonifera( Stemphylium botryosum )、Rhizopus( Rhizopus sp. )、Spores of sphaerocephala ( Corynespora cassiicola )、Paper-like spores( Ulocladium chartarum )、Alternaria alternata( Alternaria alternata ), Fusarium oxysporum ( Fusarium oxysporum ) Fusarium equiseti )、Fusarium solani( Fusarium solani )、False cocoa hair color two cells ( Lasiodiplodia pseudotheobromae )、Forest-dwelling Pythium Pythium sylvaticum ) and Fusarium graminearum ( Fusarium graminearum ) or / and Pythium terrestris ( Pythium irregulare ).

9. Use of the biocontrol mixture according to claims 3 to 4 or the biocontrol mixture prepared by the preparation method according to claims 5 to 7 in controlling plant nematode diseases, characterized in that: The plant nematode disease is any one of root knot nematode disease and stem nematode disease.

10. A method for preventing and controlling plant diseases, characterized in that: When preventing and controlling plant diseases, the biocontrol mixture according to claims 3 to 4 or the biocontrol mixture prepared by the preparation method according to claims 5 to 7 is used to coat, immerse, spray, atomize, irrigate, dust, broadcast, foam, apply or spray the diseased parts of the plant.

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