CN116676213A - Bacillus bailii strain HMQAU20041, biocontrol agent and preparation method and application thereof - Google Patents
Bacillus bailii strain HMQAU20041, biocontrol agent and preparation method and application thereof Download PDFInfo
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- CN116676213A CN116676213A CN202310475267.5A CN202310475267A CN116676213A CN 116676213 A CN116676213 A CN 116676213A CN 202310475267 A CN202310475267 A CN 202310475267A CN 116676213 A CN116676213 A CN 116676213A
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- 230000009466 transformation Effects 0.000 description 1
- 239000004552 water soluble powder Substances 0.000 description 1
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Abstract
The invention provides a bacillus beleiensis (Bacillus velezensis) strain HMQAU20041, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 th month and 14 th year of 2022, with the preservation registration number: CGMCC No.24676. The invention also provides application of the bacillus belicus strain HMQAU 20041. The invention also provides a biocontrol agent, and the active ingredients of the biocontrol agent comprise bacterial suspension, fermentation liquor or fermentation filtrate of two bacterial agents, namely bacillus belicus strain HMQAU20041 and bacillus highland strain HMQAU 20091. The invention also provides a preparation method of the biocontrol agent. The invention also provides application of the biocontrol agent in preventing and controlling plant nematode diseases. The invention also provides a method for preventing and controlling plant diseases. The bacillus belgium strain HMQAU20041 provided by the invention has broad-spectrum antagonistic activity on plant pathogenic bacteria and pathogenic nematodes.
Description
Technical Field
The invention belongs to the field of plant disease biocontrol, and in particular relates to bacillus beijerinckii strain HMQAU20041, biocontrol agents, a preparation method and application thereof.
Background
There are many intractable old diseases and some new diseases in agricultural production, which seriously threaten the prenatal, prenatal and postnatal production of field crops, vegetables, fruit trees and flowers. In order to explore new means for disease control in the agricultural full industry chain, related researches are carried out by selecting some important plant diseases in the local area, such as root knot nematode diseases of cucumbers, tomatoes and the like, rot stem nematode diseases of sweet potatoes, potatoes and garlic, lily gray mold, bean gray mold, garlic sclerotinia, celery sclerotinia, cabbage sclerotinia, tomato gray leaf spot, scab and stem rot of wheat, corn anthracnose, grape white rot, blueberry dichotoma, spinach Pythium root rot, blueberry phytophthora root rot, lettuce leaf spot, spinach fusarium root rot, blueberry leaf spot, chinese yam fusarium root rot, black spot of soybeans and sunflowers, quinoa branch rot, carrot soft rot, garlic dry rot, kiwi soft rot, cucumber brown spot, cucumber scab, cucumber downy mildew and the like. In production, chemical control is mostly adopted for controlling the diseases, but chemical pesticides are easy to cause pathogenic bacteria to generate drug resistance, pesticide residues and pollute the environment and cause adverse effects on human health, so that the selection of environment-friendly, efficient and green biological control measures becomes a new direction for researching and controlling plant diseases.
For example, cucumber (cucumber sativus) has become one of ten vegetable cultivation crops worldwide, and the annual cultivation area worldwide is about 146.67hm 2 (He Xiaoming et al, 2002). Cucumber is cultivated (Xiong Yan, 2016) in both the south and the north of China, the planting area of the national cucumber in 2015 is 1887 mu according to statistics of agricultural rural areas, the yield is 5938 mu ton, the cucumber accounts for 5.8% and 7.8% of the total vegetable, and the cucumber has large market demand and huge economic and social benefits. The current cucumber protected field production and cultivation area is rapidly enlarged, and the benefit is obviously higher than that of open field cultivation (Zhao Guoyun and the like, 2008). The downy mildew (Pseudoperonospora cubensis) and the root knot nematode (Meloidogyne incogita) of cucumbers cause downy mildew and root knot nematode disease respectively, are important leaf and root diseases in cucumber producing areas in the world (Thomas, 1986), are also destructive diseases on cucumbers in protected areas, and seriously affect the yield and quality of the cucumbers (Fu Shuyun, 1983; dan Yanxia, 2002).
The rotting stem nematodes (Ditylenchus destructor Thorne, 1945) are important plant pathogenic nematodes in the world and Chinese agricultural plant quarantine pests, endanger various underground maturing crops such as sweet potatoes, garlic and the like, and can grow and reproduce on a plurality of weeds and fungi. The species of crops and weeds which can be infected by the rotten stem nematodes are far more than 100, and the crops with heavy attack on agriculture and forestry production are mainly tubers, bulbs/corms, tubers and fleshy straight root crops, including sweet potatoes, garlic, angelica sinensis, carrots, beetles, hops, american ginseng, bulb iris, dahlia and the like. The rotten stem nematodes are cool and hot and wet and dry, and are mainly transmitted and spread through asexual propagation materials of host crops.
However, the prior art lacks a biocontrol agent with higher control effect on various plant diseases such as cucumber downy mildew, root knot nematode disease, rotting stem nematode and the like. How to solve the problems is a technical problem which needs to be solved in the field of plant disease biocontrol at present.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the invention provides a bacillus beijerinckii strain HMQAU20041, a biocontrol agent, a preparation method and application thereof.
The first aim of the invention is to provide a biocontrol bacterium, namely bacillus beliensis (Bacillus velezensis) strain HMQAU20041, which is separated from cucumber leaf surfaces, enriches strain resources of biocontrol bacteria of plant diseases and lays a foundation for researching and developing an anti-mixture. The bacillus belicus (Bacillus velezensis) strain HMQAU20041 is called as "strain HMQAU20041" in the invention, wherein the strain HMQAU20041 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year 2022, month 4 and 14, and the preservation address is North Chen West Lu No. 1, 3 of the Korean region of Beijing city, and the preservation registration number is the institute of microorganisms of the national academy of sciences of China: CGMCC No.24676.
A second object of the present invention is to provide an application of the strain HMQAU20041 in inhibiting plant pathogenic bacteria, preparing a product for inhibiting plant pathogenic bacteria, preventing and treating diseases caused by plant pathogenic bacteria, and preparing a product for preventing and treating diseases caused by plant pathogenic bacteria.
The third object of the invention is to provide a biocontrol agent. The active ingredients of the biocontrol mixture comprise two bacterial mixtures, namely bacillus beijerinus (Bacillus velezensis) strain HMQAU20041 and bacillus altitudinariacus (Bacillus altitudinis) strain HMQAU 20091. The strain HMQAU20091 of the bacillus highland is a known strain, and is recorded in the patent CN202210621801.4 of the inventor, which is named as bacillus highland (Bacillus altitudinis), and is deposited in the general microbiological center of the China general microbiological culture Collection center for 4 months and 14 days in 2022, with a deposit address of the national institute of microbiological culture Collection, national institute of sciences, accession No. 3 of north chen west way 1, the region of korea, beijing city: CGMCC No.24677. The strain of geobacillus highland HMQAU20091 is herein or simply referred to as "strain HMQAU20091".
The fourth object of the present invention is to provide a method for preparing the biocontrol agent.
The fifth object of the invention is to provide an application of the biocontrol agent.
The sixth object of the present invention is to provide a method for controlling plant diseases.
The above object of the present invention is achieved by the following technical scheme:
Bacillus bailii (Bacillus velezensis) strain HMQAU20041 deposited at China general microbiological culture Collection center, accession number: CGMCC No.24676.
The use of bacillus belgium (Bacillus velezensis) strain HMQAU20041 as described above in (a 1) or (a 2) or (a 3) or (a 4) as follows, (a 1) to inhibit plant pathogenic bacteria; (a 2) preparing a product for inhibiting plant pathogenic bacteria; (a 3) controlling diseases caused by plant pathogenic bacteria; (a4) Preparing a product for controlling diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are Pythium gulum (Pseudoperonospora cubensis), pythium Lin Qi (Pythium sylvaticum), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), botrytis cinerea (Botryoshaeria dothidea), pachyrhizus gracilis (Coniella diplodiella), pachyrhizus gracilis (Colletotrichum graminicola), botrytis cinerea (Botrytis cinerea), cladosporium cucumericum (Cladosporium cucumerinum), pachyrhizus pseudocurcas (Lasiodiplodia pseudotheobromae), pachyrhizus moellendon (Pestalotiopsis sp.), phomopsis pseudophoma (Pythium irregulare), pythium irregulare (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), mucor circinelloides (Mucor circinelloides), pythium solani (Stemphylium lycopersici), fusarium asiaticum (Fusarium asiaticum), rhizopus sp), fusarium betanae (Phoma betatae), fusarium roseum (Corynespora cassiicola), alternaria (Alternaria alternata), fusarium graminearum (Fusarium pseudograminearum), pachytrium gracilii (Ulocladium chartarum), alternaria solani (Alternaria solani), fusarium roseum (676, fusarium roseum (633) and Fusarium roseum (633.
Based on the same inventive concept, the invention also provides a biocontrol agent, and the active ingredients of the biocontrol agent comprise bacterial suspension, fermentation liquor or fermentation filtrate of two bacterial agents, namely bacillus belicus (Bacillus velezensis) strain HMQAU20041 and bacillus highland (Bacillus altitudinis) strain HMQAU 20091; the bacillus belicus (Bacillus velezensis) strain HMQAU20041 was deposited in the China general microbiological culture Collection center, accession number: CGMCC No.24676; the strain HMQAU20091 of Geobacillus (Bacillus altitudinis) was deposited in China general microbiological culture Collection center, accession number: CGMCC No.24677.
The active ingredients of the biocontrol agent are fermentation filtrate of two bacterial agents of bacillus beliensis bacterial strain HMQAU20041 and bacillus highland bacterial strain HMQAU20091, wherein the total viable bacteria concentration of bacillus beliensis bacterial strain HMQAU20041 and bacillus highland bacterial strain HMQAU20091 in the biocontrol agent is 1 multiplied by 10 8 ～2×10 8 cfu/mL。
Based on the same inventive concept, the invention also provides a preparation method of the biocontrol mixture, which comprises the following specific steps:
(1) Culturing bacterial strain HMQAU20041 and bacterial strain HMQAU20091 seed bacterial liquid: respectively inoculating the strain HMQAU20041 and the strain HMQAU20091 of claim 3 into LB culture solution for culture until the OD value of each seed bacterial solution is between 0.5 and 0.8, and obtaining the seed bacterial solutions of the strain HMQAU20041 and the strain HMQAU 20091;
(2) And (3) preparing fermentation filtrate: mixing the seed bacterial liquid of the strain HMQAU20041 and the seed bacterial liquid of the strain HMQAU20091 according to different volumes, adding the mixed seed bacterial liquid into an conical flask filled with LB culture liquid according to the volume percentage content of 1%, carrying out shaking culture, centrifuging, and filtering with a microporous filter membrane of 0.22 mu m to prepare fermentation filtrate, thus obtaining the biocontrol agent. Preferably, in the step (2), the volume ratio of the bacterial strain HMQAU20041 seed bacterial solution to the bacterial strain HMQAU20091 seed bacterial solution is (9:1) - (1:9). Most preferably, the volume ratio of the bacterial strain HMQAU20041 seed bacterial solution to the bacterial strain HMQAU20091 seed bacterial solution in the step (2) is 7:3.
The above-mentioned method for producing a biocontrol agent, wherein the culture conditions in the step (1) are shaking culture at 30℃and 200rpm for 8 to 10 hours.
In one embodiment of the present invention, the shaking culture conditions in the step (2) are: the culture was performed at 30℃and 200rpm for 48 hours.
In one embodiment of the present invention, the formula of the LB culture solution in the steps (1) and (2) is: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl, and adjusting the pH value to 7.3.
Preferably, the centrifugation conditions in step (2) are 8000rpm for 15min.
The preparation method of the biocontrol agent comprises the following steps: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl are used for regulating the pH value to 7.2-7.4.
Based on the same inventive concept, the invention also provides the application of the biocontrol agent or the biocontrol agent prepared by the preparation method, as described above, in the following (b 1) or (b 2) or (b 3) or (b 4), which is characterized in that (b 1) inhibits plant pathogenic bacteria; (b 2) preparing a product for inhibiting plant pathogenic bacteria; (b 3) controlling diseases caused by phytopathogens; (b4) Preparing a product for controlling diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are Pythium gulum (Pseudoperonospora cubensis), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), pachyrhizus gracilis (Coniella diplodiella), portugal cavity bacteria (Botryoshaeria dothidea), rhizoctonia solani (Rhizoctonia solani), phytophthora camphorata (Phytophthora cinnamomi), mucor pseudodisc (Pestalotiopsis sp.), rhizopus nii (Colletotrichum siamense), phytophthora pseudoginseng sp.), pythium solani (Stemphylium lycopersici), cladosporium cucumerium, beta beta (Phoma betae), botrytis cinerea (Botrytis cinerea), mucor circinelloides (Mucor circinelloides), rhizopus (Stemphylium botryosum), rhizopus Rhizopus sp., torulaspis (Corynespora cassiicola), alternaria papyriferus (Ulocladium chartarum), fusarium oxysporum (Alternaria alternata), fusarium oxysporum (Fusarium oxysporum), fusarium equi (lanum) Fusarium, fusarium pseudoginseng (Lasiodiplodia pseudotheobromae), fusarium roseum (Pythium, pythium venenatum (Pythium sp.) and Pythium gracilium.
Based on the same inventive concept, the invention also provides application of the biocontrol agent or the biocontrol agent prepared by the preparation method in preventing and controlling plant nematode diseases, wherein the plant diseases are any one of root knot nematode diseases and rotting stem nematode diseases.
Based on the same inventive concept, the present invention also provides a method for controlling plant diseases, in which the biocontrol agent as described above or the biocontrol agent prepared by the preparation method as described above is directly applied or acted on plants and plant parts and organs using a usual treatment method, for example, by coating, dipping, spraying, atomizing, irrigating, volatilizing, dusting, scattering, foaming, coating, etc., and in the case of propagation material, especially seeds, as a treatment powder for drying seeds, a solution for treating seeds, a water-soluble powder for slurry treatment, etc., by one or more layers of coating, etc. The microbial inoculum may also be applied by ultra low volume means or may be poured into the soil itself. Preferably, the biocontrol agent as described above is sprayed onto the affected parts of plants in the control of leaf diseases such as cucumber downy mildew.
The beneficial effects of the invention are as follows:
1. the bacillus belgium strain HMQAU20041 provided by the invention has broad-spectrum antagonistic activity on plant pathogenic bacteria and pathogenic nematodes, and has control effect on plant diseases caused by pathogenic bacteria such as Pythium gulum, pythium Lin Qi, sclerotinia sclerotiorum, botrytis cinerea, white rot fungus, cephalosporium graminearum, botrytis cinerea, pseudococoa hair two cells, mucor pulmonale, phomopsis, pythium irregulare, rhizoctonia solani, fusarium oxysporum, mucor solani, fusarium asiaticum, rhizopus, rhizopus, alternaria lablab, alternaria, fusarium pseudograminearum, alternaria papyriferum, fusarium equiseti, fusarium graminearum, fusarium putida and the like, and nematodes such as meloidogyne and rotten nematodes. In particular to the antibacterial rate of Pythium praeparatum, sclerotinia sclerotiorum, botrytis cinerea, white rot aschersonia, celastracellum graminearum and Botrytis cinerea reaching 100 percent.
2. The active ingredients of the biocontrol mixture provided by the invention comprise bacterial suspension, fermentation liquor or fermentation filtrate of two bacterial mixtures of bacillus beijerinckii strain HMQAU20041 and bacillus highland strain HMQAU20091, and particularly the fermentation filtrate has remarkable control and synergy effects on cucumber downy mildew.
3. The biocontrol agent provided by the invention has broad-spectrum antagonistic activity on plant pathogenic bacteria and pathogenic nematodes, and has control effect on plant diseases caused by pathogenic bacteria such as pseudomonas gulfweed, sclerotinia sclerotiorum, aschersonia aleyrodis, botrytis cinerea, rhizoctonia solani, phytophthora camphorata, trichoderma pseudodisc, colletotrichum glomeratum, phomopsis, trichoderma reesei, phomopsis, rhizopus, lablab album, alternaria papyriferus, alternaria alternifolia, fusarium oxysporum, fusarium equisetosum, fusarium beatification, fusarium pseudococoa hair color two cells, lin Qi Pythium gracile, fusarium graminearum, pythium irregulare and the like. In particular to sclerotinia sclerotiorum, aschersonia aleyrodis, botrytis cinerea and rhizoctonia solani, and the antibacterial rate reaches 100 percent. In addition, the biocontrol agent provided by the invention has synergistic effect on preventing and treating root-knot nematode disease and rot stem nematode disease.
4. The biocontrol mixture is a biological agent, and a series of problems caused by the use of chemical pesticides are completely avoided, so that the biocontrol mixture is beneficial to green production of greenhouse vegetables, farmers can not use or reduce the dosage of the chemical pesticides, the expense can be saved for the farmers, and the export of the vegetables is facilitated. Meanwhile, the mixture has the function of increasing yield, and can increase the income of peasants.
Drawings
FIG. 1 shows bacterial strain HMQAU20041 colony, gram stain, spore stain (A, B: colony morphology; C: gram stain; D: spore stain);
FIG. 2 is a phylogenetic tree of the adjacency analysis of Bacillus belicus constructed based on the 16S rDNA gene sequence;
FIG. 3 is a phylogenetic tree of the adjacency analysis of Bacillus bailii constructed based on the gyrB gene sequence;
FIG. 4 shows the inhibition of hyphal growth of different pathogenic bacteria by Bacillus belicus strain HMQAU 20041.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1 identification of Strain HMQAU20041 and its inhibition effect on the growth of mycelium of different pathogenic bacteria
1. Plant disease and pathogenic bacteria for the test:
pythium ultimum root rot of spinach: lin Qi Pythium ultimum Pythium sylvaticum HMQAU210092 and Pythium irregulare Pythium irregulare HMQAU210091; apicomplexation disease: sclerotinia sclerotiorum Sclerotinia sclerotiorum HMQAU170216; quinoa branch blight: phycomyces viticola Botryoshaeria dothidea HMQAU190265, phoma betanae Phoma betae HMQAU190266; white rot of grape: white rot aschersonia Coniella diplodiella HMQAU170082; corn anthracnose: disc grass 5. Spinosa Colletotrichum graminicola HMQAU 220031; lily gray mold: botrytis cinerea Botrytis cinerea HMQAU200037; cucumber scab: guacladosporium cucumerinum Cladosporium cucumerinum HMQAU180016; branch blight of blue berry hair color two spore: pseudo cocoa hair color two cells Lasiodiplodia pseudotheobromae HMQAU140073; blueberry leaf spot: pestalotiopsis sp., HMQAU180119; phomopsis sp.hmqau210069; garlic sclerotinia disease: rhizoctonia solani Rhizoctonia solani HMQAU210022; fusarium root rot of spinach: fusarium oxysporum Fusarium oxysporum HMQAU170173, fusarium equisetum Fusarium equiseti HMQAU 170169; soft rot of kiwi fruit: mucor circinelloides Mucor circinelloides HMQAU150050; dry rot of garlic: fusarium oxysporum Fusarium oxysporum HMQAU210030 and HMQAU210034; tomato gray leaf spot: the process comprises the steps of (1) carrying out the process of preparing the phoma solanacearum Stemphylium solani HMQAU170209; wheat scab: fusarium asiaticum Fusarium asiaticum HMQAU220017, fusarium graminearum Fusarium graminearum HMQAU170037; soft rot of carrot: rhizopus sp.HMQAU180037; sclerotinia rot of Chinese cabbage: rhizoctonia solani Rhizoctonia solani HMQAU180110; brown spot of cucumber: the lentil is dried by a process of Corynespora cassiicola HMQAU200087; black spot of soybean: alternaria alternata Alternaria alternata HMQAU210080; wheat stem basal rot: fusarium pseudograminearum Fusarium pseudograminearum HMQAU220027; lettuce leaf spot disease: acidocella papyrifera Ulocladiumchartarum HMQAU210122, acidocella stolonifera Stemphylium botryosum HMQAU210121; black spot of sunflower: alternaria solani Alternaria solani HMQAU220028; fusarium root rot of Chinese yam: fusarium solani Fusarium solani HMQAU180201. All of the above strains were supplied by the Qingdao university of agriculture mycological laboratory.
2. Test biocontrol bacteria:
the strain HMQAU20041 was isolated from cucumber leaf surfaces in the chat city of Shandong province by plate dilution (Krechel et al, 2002).
3. Bacterial identification method:
(1) Morphological identification:
morphological identification was performed by reference to the methods of the general bacterial systems identification handbook (Dongxiu beads and Cai Miaoying, 2001).
(2) And (3) physiological and biochemical identification:
according to the morphological characteristics of the target strain, common physiological and biochemical indexes are selected, and physiological and biochemical identification is carried out by referring to the method of the common bacterial System identification handbook (Dongxiu beads and Cai Miaoying, 2001).
(3) Molecular identification and phylogenetic analysis:
extracting bacterial genome DNA by using a bacterial genome DNA extraction kit (Bacterial DNA Kit), and preserving the bacterial genome DNA in a refrigerator at-20 ℃ for later use. PCR was performed using the extracted bacterial genomic DNA as a template, and the general primers (27F: 5 '-AGAGTTTGATCCGGCTCAG-3' and 142R: 5 '-TACGGCTACCTTGTTACGACTT-3') and the gyrB gene sequence primers (gyrB-F: 5'-GAAGTCATCATGA CCGTTCTGCAYGCNGGNGGNAARTTYGA-3' and gyrB-F-R:5 '-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3') for bacterial 16 SrDNA.
The total PCR system for 16S rDNA was 25. Mu.L: 10 XPCR reaction buffer 2.5. Mu.L, dNTPs (10 mmol/L) 2. Mu.L, primers (10 mmol/L) 1. Mu.L each, taq DNA polymerase (5U/. Mu.L) 0.5. Mu.L, DNA template 1. Mu.L, ddH 2 O makes up 25. Mu.L. Reaction conditions: pre-denaturation at 94℃for 5min;94 denaturation at 30s, annealing at 63 at 30s, extension at 72 ℃ for 2min,35 cycles; extending at 72℃for 7min.
GyrB gene sequence PCR population (25 μl): 10 XPCR Buffer 2.5. Mu.L, dNTP mix 0.5. Mu.L (10 mM), gyrB-F/gyrB-R (10. Mu. Mol/L) each 1. Mu.L, pro Taq enzyme (5U/. Mu.L) 0.2. Mu.L, DNA template 1. Mu.L, ddH 2 O to 25. Mu.L. Reaction conditions: pre-denaturation at 94 ℃ for 30s; denaturation at 98℃for 10s, annealing at 67℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72℃for 2min.
After the PCR was completed, 5. Mu.L of the PCR product was mixed with 1. Mu.L of 6×Loading Buffer, and the mixture was subjected to 120V electrophoresis on a 1% agarose gel for 30min using DNA marker 2000 as a molecular weight standard, and the length and concentration of the amplified product were detected in a gel imaging system.
And purifying the PCR products of the two primers by using the kit, detecting and recovering the purified PCR products by using 1% agarose gel electrophoresis after purification, and displaying a single band to show that the purification is successful. According to pMD TM 18-TVEctor instruction manual, product ligation and transformation were performed using pMD for single colonies TM PCR was performed with 18-T universal primer M13-47:5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and RV-M5'-GAGCGGATAACAATTTCAC ACACAGG-3'. The amplification reaction system and the reaction conditions are the same as described above. After the PCR was completed, 5. Mu.L of the PCR product was taken in 1% agarose gel Electrophoresis was performed at 120V for 30min, and the length of the amplified product was detected in a gel imaging system. If the length of the target fragment is about 1700bp, the corresponding colony is a positive clone, and the bacterial liquid is sent to Shanghai Biotechnology engineering Co.
The sequencing results were subjected to BLAST alignment on NCBI database, the 16S rDNA sequences of the standard strains of the strains in the alignment results were downloaded, and phylogenetic analysis was performed using MEGA5.05 software, and phylogenetic trees were established and cluster analysis was performed.
4. Identification of strains:
(1) Morphological identification results:
bacterial strain HMQAU20041 is cultured for 2d on LB solid medium at 28 ℃, bacterial colony bulges are milky white, opaque, round, irregular in edge and wrinkled on the surface, bacterial cells are gram-positive, rod-shaped (0.79+/-0.09) mu m- (2.09+/-0.11) mu m are arranged singly or in pairs, spores are produced, spores are oval, mesogenesis is achieved, and cysts are enlarged, as shown in figure 1.
(2) Physiological and biochemical identification results:
the physiological and biochemical measurement results show that the strain HMQAU20041 is anaerobic, has strong salt tolerance, can still grow in 10% NaCl culture solution, can not grow at the temperature of 4 ℃ and can grow at the temperature of 45 ℃. Acid and alkali resistance, and can grow at pH values of 5.5 and 9.0. Citrate, D-xylose, mannitol, glucose, sorbitol, and D-fructose may be used, and glucose cannot be oxidized and fermented. The physiological and biochemical tests of oxidase, indole reaction, denitrification, methyl red and the like are negative, and the physiological and biochemical tests of contact enzyme, nitrate reduction, V-P, starch hydrolysis and the like are positive, and are shown in Table 1. The bacterial strain HMQAU20041 can be primarily identified as Bacillus according to morphological characteristics and physiological and biochemical characteristics.
TABLE 1 results of physiological and biochemical tests of Bacillus bailii strain HMQAU20041
(3) Molecular identification and phylogenetic analysis results:
according to the sequencing result, the lengths of PCR products of the 16S rRNA gene and the gyrB gene sequences of the strain HMQAU20041 are 1514bp and 1259bp respectively, the 16SrDNA gene sequence obtained by sequencing is shown as SEQ ID No.1, and the gyrB gene sequence obtained by sequencing is shown as SEQ ID No. 2. BLAST comparison results in NCBI database show that the 16S rRNA gene similarity of the strains HMQAU20041 and Bacillus velezensis (EF 433407) is up to 99.87%, and phylogenetic tree analysis is established, and the results show that HMQAU20041 and bacillus belicus are on the same branch, see fig. 2. Further analysis of the gyrB gene sequence of HMQAU20041 and construction of phylogenetic tree showed that the homology of the strain HMQAU20041 with B.velezensis (KY 381949) was as high as 99.89% and on the same branch as Bacillus bailii, see FIG. 3. Based on morphological features, sequence alignment analysis, morphological and physiological biochemical characterization (Li Shengzhang et al, 2019; assisted icing et al, 2021; wang Qinghua et al, 2019; huang Hui, 2022), strain HMQAU20041 was determined to be Bacillus belicus (B.velezensis). The identification results are shown in Table 2.
TABLE 2 sequencing alignment results
The screened bacillus belgium (Bacillus velezensis) strain HMQAU20041 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 months and 14 days in 2022, wherein the preservation address is the national institute of microbiology, accession number of China academy of sciences: CGMCC No.24676. The requested collection unit is Qingdao university of agriculture.
5. Preparation of fermentation filtrate of strain HMQAU 20041:
(1) Culturing a seed bacterial liquid: inoculating the strain HMQAU20041 into LB culture solution, shaking culturing at 30deg.C and 200rpm for 8 hr until the OD value of seed bacterial solution is 0.5-0.8, and regulating the concentration of seed bacterial solution to 5×10 with blood cell counting plate 8 CFU/mL, seed bacterial liquid is prepared.
(2) And (3) preparing fermentation filtrate: the HMQAU20041 seed bacterial liquid was added to a 150mL conical flask containing 50mLLB culture liquid at a ratio of 1% by volume (in this example, 500. Mu.L was taken to have a concentration of 5X 10) 8 CFU/mL seed bacterial liquid is added into 50mL LB culture liquid), 30 ℃ and 200rpm shaking culture is carried out for 48 hours, then 8000rpm centrifugation is carried out for 15min, and 0.22 mu m microporous filter membrane filtration is carried out to prepare aseptic filter liquor of antagonistic bacteria. Wherein the live bacteria concentration of Bacillus bailii HMQAU20041 is 1×10 10 CFU/mL。
The formula of the LB culture solution is as follows: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride are used for regulating the pH value to 7.3.
6. Inhibition effect of bacterial strain HMQAU20041 fermentation filtrate on growth of different pathogenic bacteria hyphae
(1) The test method comprises the following steps:
the mycelium growth rate method (aged spring, 1991) is adopted, the fermentation filtrate stock solution and the PDA culture medium cooled after melting (45-50 ℃) are evenly mixed (diluted by 10 times), the mixture is poured into a flat plate, the PDA culture medium added with the equal volume of LB liquid culture medium is used as a reference, and the center of the flat plate is inoculated with germ cakes with the diameter of 5 mm. When the control grows up to 3/4 culture dishes, the colony diameters of the control group and the treatment group are measured by a crisscross method, the bacteriostasis rate is calculated, and 3 replicates are arranged for each treatment.
Inhibition (%) = (control colony radius (mm) -treated colony radius (mm))/control colony radius (mm) ×100%.
(2) Test results
The test results are shown in Table 3 and FIG. 4.
TABLE 3 inhibition of bacterial strain HMQAU20041 on the growth of mycelium of different pathogenic bacteria
Example 2 ratio screening of Strain HMQAU20041 and Strain HMQAU20091 biocontrol Agents
1. The test method comprises the following steps:
(1) Preparation of biocontrol agent fermentation filtrate
A. Culturing a seed bacterial liquid: respectively inoculating the strain HMQAU20041 and the strain HMQAU20091 into LB culture solution, shaking and culturing at 30deg.C and 200rpm for 8 hr until the OD value of each seed bacterial solution is 0.5-0.8, and regulating the concentration of seed bacterial solution to 5×10 by adopting a blood cell counting plate 8 CFU/mL, strain HMQAU20041 and strain HMQAU20091 seed bacterial solutions are respectively prepared.
B. And (3) preparing fermentation filtrate: mixing the seed bacterial liquid of the strain HMQAU20041 and the seed bacterial liquid of the strain HMQAU20091 according to different volumes, adding the mixed seed bacterial liquid into a 150mL conical flask filled with 50mL LB culture liquid according to the proportion of 1 percent by volume, carrying out shaking culture at 30 ℃ and 200rpm for 48 hours, centrifuging at 8000rpm for 15 minutes, and filtering with a 0.22 mu m microporous filter membrane to prepare the fermentation filtrate of the biocontrol agent.
The formula of the LB culture solution is as follows: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl are used for regulating the pH value to 7.3.
The preparation method of the bacterial preservation solution comprises the following steps: and diluting the glycerol with 0.9% physiological saline until the concentration is 30%, thus obtaining the bacterial preservation solution.
(2) Influence of biocontrol agent fermentation filtrate in different ratios on release of cucumber downy mildew sporangium
EC treated differently by concave slide method 50 Based on the values, the bacterial strain HMQAU20041 seed bacterial liquid and the bacterial strain HMQAU20091 seed bacterial liquid are subjected to mixed fermentation of biocontrol agents according to 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 biocontrol agent fermentation filtrate under different volume ratios to the cucumber downy mildew is measured. If the toxicity ratio (actual Inhibition rate/desired inhibition rate>1, a synergistic effect; if toxicity ratio is high<1, antagonism; the toxicity ratio is about 1, and is about the sum. Preparation of a concentration of 2 x 10 with sterilized tap water 5 Each/mL of the cucumber downy mildew sporangia suspension. Dripping 20 μl of sporangium suspension and fermentation filtrate onto concave glass slide, mixing with sterilized tap water and sporangium suspension at equal volume, placing concave glass slide into culture dish with wet filter paper, and placing at 4deg.C and RH>Culturing in 80% refrigerator in dark for 1 hr, and culturing in 20 deg.C incubator for 1 hr. And (3) observing the empty spore rate of 100 sporangia under a microscope when more than 80% of sporangia in the control release zoospores. The test was repeated twice with 3 replicates per treatment set. The actual inhibition rate of each proportion to the cucumber downy mildew is calculated by taking sterile water treatment as a control.
A. And B, calculating an expected inhibition rate after the two single agents are mixed, wherein the formula is as follows:
toxicity ratio = actual inhibition/desired inhibition
Actual inhibition rate: the inhibition rate of each proportion to cucumber downy mildew measured in the test;
the inhibition rate is expected: a against cucumber downy mildew EC 50 Actual inhibition rate x percentage in the mixture ratio+B vs. cucumber downy mildew EC 50 Actual inhibition x percentage in formulation.
3. Proportioning screening result of biocontrol mixture
The test results are shown in Table 4, EC 50 The strain HMQAU20041 and the strain HMQAU20091 are mixed at different dosages, and the effects of different proportions are different. Wherein, the toxicity ratio of the bacterial strain HMQAU20041 to the bacterial strain HMQAU20091 is highest in a volume ratio of 7:3, is 1.39 and is more than 1.2, and the toxicity ratio of other ratios is lower than 1.2.
TABLE 4 inhibition ratio of HMQAU20041 and HMQAU20091 to Peronospora Cucumidis
Example 3 determination of synergy of effective formulation of biocontrol Agents
1. The test method comprises the following steps:
according to example 2, the mixture ratio with the highest toxicity ratio was selected from the two single doses, and the EC of the effective mixture ratio to the cucumber downy mildew was determined by cotyledon spraying 50 The value was measured by the method described in grand Yunpei (1950) to determine the co-toxicity coefficient>120 is synergistic effect, and the co-toxicity coefficient is more than or equal to 80 and less than or equal to 120 is additive effect<80 is antagonism. The highest co-toxicity coefficient is the best proportion.
EC of actual drug toxicity index (ATI) =standard drug 50 EC of test agent 50 ×100
Theoretical toxicity index of mixture (TTI) =toxicity index of agent a ×content of agent a in mixture (%) +toxicity index of agent B ×content of agent B in mixture (%)
Co-toxicity coefficient of compound (CTC) =ati/tti×100
2. Synergistic effect determination result of effective proportion of biocontrol mixture
The results of the effective formulation synergy are shown in Table 5.
TABLE 5 determination of effective formulation synergy
Example 4 inhibition of biocontrol agent fermentation filtrate against different pathogenic bacteria
1. Plant disease and pathogenic bacteria for the test:
apicomplexation disease: sclerotinia sclerotiorum Sclerotinia sclerotiorum HMQAU170216; white rot of grape: white rot aschersonia Coniella diplodiella HMQAU170082; quinoa branch blight: siamese anthracnose Colletotrichum siamense HMQAU190267, phoma betanae Phoma betae HMQAU190266; sclerotinia rot of Chinese cabbage: rhizoctonia solani Rhizoctonia solani HMQAU180110; phytophthora blueberry root rot disease: phytophthora camphorax Phytophthora cinnamomi HMQAU180002; blueberry leaf spot: pestalotiopsis sp.HMQAU180119, phomopsis sp.HMQAU210069; tomato gray leaf spot: the process comprises the steps of (1) carrying out the process of preparing the phoma solanacearum Stemphylium solani HMQAU170209; cucumber scab: guacladosporium cucumerinum Cladosporium cucumerinum HMQAU180016; gray mold of kidney beans: botrytis cinerea Botrytis cinerea HMQAU170042; soft rot of kiwi fruit: mucor circinelloides Mucor circinelloides HMQAU150050; lettuce leaf spot disease: acidocella papyrifera Ulocladiumchartarum HMQAU210122, acidocella stolonifera Stemphylium botryosum HMQAU210121; garlic sclerotinia disease: rhizoctonia solani Rhizoctonia solani HMQAU210022; soft rot of carrot: rhizopus sp.HMQAU180037; lily gray mold: botrytis cinerea Botrytis cinerea HMQAU200037; brown spot of cucumber: the lentil is dried by a process of Corynespora cassiicola HMQAU 200087; black spot of soybean: alternaria alternata Alternaria alternata HMQAU210080; fusarium root rot of spinach: fusarium oxysporum Fusarium oxysporum HMQAU170173, fusarium equisetum Fusarium equiseti HMQAU170169; dry rot of garlic: fusarium oxysporum Fusarium oxysporum HMQAU210030 and HMQAU210034, fusarium solani Fusarium solani HMQAU210027; branch blight of blue berry hair color two spore: pseudo cocoa hair color two cells Lasiodiplodia pseudotheobromae HMQAU140073; pythium ultimum root rot of spinach: lin Qi Pythium ultimum Pythium sylvaticum HMQAU210092 and Pythium irregulare Pythium irregulare HMQAU210091; wheat scab: fusarium graminearum Fusarium graminearum HMQAU170037; fusarium root rot of Chinese yam: fusarium solani Fusarium solani HMQAU180201; all of the above were supplied by the Qingdao university of agriculture mycological laboratory.
2. Inhibiting effect result of biocontrol agent fermentation filtrate on different pathogenic bacteria
The inhibition effect of the bacterial strain HMQAU20041 and the bacterial strain HMQAU20091 on different pathogenic fungi is measured by a hypha growth rate method after LB fermentation filtrate of the mixture with the optimal volume ratio of 7:3 is diluted 10 times in PDA, and the results are shown in Table 6, and the fermentation filtrate has different antibacterial activities on 30 tested plant pathogenic fungi. Wherein the composition has complete inhibition effect on sclerotinia sclerotiorum causing celery sclerotinia sclerotiorum, white rot of grape aschersonia aleyrodis, chaetomium globosum causing quinoa branch blight and rhizoctonia solani of Chinese cabbage, and the inhibition rate is 100 percent; secondly, phytophthora camphorata causing phytophthora blueberry root rot, mucor pseudodisc of blueberry leaf blight, siamese anthracnose of quinoa branch blight, phomopsis of blueberry leaf blight, pythium solani of tomato gray leaf spot, melon branch spore of cucumber scab, beet stem point mold of quinoa branch blight and gray grape spore of bean gray mold, the inhibition rate is above 93.38%; the inhibition rate of 18 pathogenic bacteria such as Mucor circinelloides, mucor pulmonale and Mucor pulmonale causing kiwi soft rot is also between 46.5 percent and 89.67 percent; the antibacterial rate to Fusarium graminearum, pythium irregulare and Fusarium solani is relatively poor. The mixture has wide antibacterial spectrum, good antibacterial effect and high biocontrol potential.
TABLE 6 inhibition of biocontrol agent fermentation filtrate against different pathogenic bacteria
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Note that: letters following the same column of numbers indicate significant differences in P < 0.05 level (Duncan's new complex polar error method)
EXAMPLE 5 lethal effect of HMQAU20041, HMQAU20091 and biocontrol agent fermentation filtrate on Meloidogyne incognita
1. Test method
17 treatments are set in the test, namely, fermentation filtrate stock solutions of the HMQAU20041 and HMQAU20091 single agents and the mixture are diluted by 0 times, 5 times, 10 times, 25 times and 50 times for standby, 1mL of each treatment is taken and added into a 24-hole plate, and 200 mu l (about 300) root-knot nematodes are added into each hole; the drug control is abamectin (2 mug/mL), and the blank control is sterile water; each treatment was repeated 3 times. The 24-well plate is placed in an incubator at 25 ℃ for 12 hours and 24 hours. Nematode morphology was observed under dissecting glasses, nematode bending was considered to be alive and stiffness was considered to be dead. Mortality was statistically corrected and photographs were taken to record the different morphologies of the nematodes. The strain HMQAU20091 fermentation filtrate used in the present invention was prepared according to the preparation method of example 1 of the specification of Chinese patent CN 202210621801.4. Wherein, the living bacteria concentration of the strain HMQAU20091 is 1X 1010CFU/mL.
Corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ] ×100%
2. Lethal effect of treatments on Meloidogyne incognita
As shown in table 7, when the treatment time was 12 hours, the corrected mortality of the fermentation filtrate stock solution of HMQAU20041 reached 95.26%, the corrected mortality of the fermentation filtrate stock solution of HMQAU20091 was 81.82%, the corrected mortality of the mixture fermentation filtrate stock solution was 100%, and the difference between the three was significant; the corrected mortality rates were 47.13%, 33.72%, 54.94% when diluted 10-fold for each treatment, respectively, with significant differences between the three. When the treatment time is 24 hours, the corrected mortality of the 5-time dilutions of the fermentation filtrate of the HMQAU20041, the HMQAU20091 and the mixture are 97.58%,95.27% and 100% respectively, and the difference is not obvious; the corrected mortality rates were 31.97%, 25.58%, 49.51%, respectively, for each treatment diluted 50-fold, with significant differences between HMQAU20041 and HMQAU20091 and the cocktail.
TABLE 7 lethal effects of different treatments on Meloidogyne incognita
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Note that: letters following the same column of numbers indicate significant differences in P < 0.05 level (Duncan's new complex polar error method)
EXAMPLE 6 lethal effect of HMQAU20041, HMQAU20091 and biocontrol agent fermentation filtrate on rotten stem nematodes
1. Test method
The test sets 13 treatments, namely, diluting the fermentation filtrate stock solutions of the HMQAU20041 and HMQAU20091 single dose and the mixture by 0 time, 5 times, 10 times and 25 times, taking 1mL of each treatment, adding the treatments into a 24-hole plate, and adding 200 mu l (about 100) of rotten stem nematodes into each hole; avermectin is used as a medicament control (2 mug/mL), and sterile water is used as a blank control; each treatment was repeated 3 times and incubated in an incubator at 20℃for 6h and 10h. Nematode morphology was observed under dissecting glasses, nematode bending was considered to be alive and stiffness was considered to be dead. Mortality was statistically corrected and photographs were taken to record the different morphologies of the nematodes.
Corrected mortality (%) = [ (treatment mortality-control mortality)/(1-control mortality) ] ×100%
2. Lethal effect of treatments on rotting nematodes
As shown in table 8, when the treatment time was 6 hours, the corrected mortality rates of the fermentation filtrate stock solutions of HMQAU20041, HMQAU20091 and the mixture were 16.21%, 48.54% and 77.35%, respectively, and the differences between the three were significant; the corrected mortality rates of the 5-fold dilutions of the fermentation filtrates of HMQAU20041, HMQAU20091 and the mixture are 17.41%, 29.55% and 34.25%, respectively, and the differences between HMQAU20041 and HMQAU20091 and the mixture are significant; the corrected mortality of 10-fold dilution of the fermentation filtrate of the mixture, the HMQAU20041 and the HMQAU20091 is 25.72%, 8.74% and 18.47%, respectively, and the difference among the three is obvious; the corrected mortality rates for the 25-fold dilutions of the fermentation filtrates of the cocktail, HMQAU20041 and HMQAU20091 were 6.34%, 1.54% and 5.05%, respectively, with significant differences between HMQAU20041 and HMQAU20091 and cocktail, respectively.
When the treatment time is 10 hours, the corrected mortality of the fermentation filtrate stock solutions of the HMQAU20041, the HMQAU20091 and the mixture are 44.15 percent, 74.01 percent and 97.20 percent respectively, and the difference among the three is obvious; the corrected mortality of the 5-fold dilutions of the fermentation filtrates of HMQAU20041, HMQAU20091 and the mixture are 29.12%, 49.25% and 64.35%, respectively, with significant differences between the three; the corrected mortality rates of the HMQAU20041, the HMQAU20091 and the mixture 10 diluted times are 15.71%, 29.73% and 43.77%, respectively, and the difference between the HMQAU20041 and the HMQAU20091 and the mixture is obvious; the corrected mortality rates for the 25-fold dilutions of the fermentation filtrates of HMQAU20041, HMQAU20091 and the cocktail were 5.82%, 10.46% and 15.09%, respectively, with significant differences between HMQAU20041 and the cocktail.
TABLE 8 lethal effects of different treatments on rotting stem nematodes
Note that: letters following the same column of numbers indicate significant differences in P < 0.05 level (Duncan's new complex polar error method)
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A bacillus beleiensis (Bacillus velezensis) strain HMQAU20041, characterized in that the strain is deposited in the China general microbiological culture Collection center, accession number: CGMCC No.24676.
2. Use of bacillus beleimeris (Bacillus velezensis) strain HMQAU20041 according to claim 1 in (a 1) or (a 2) or (a 3) or (a 4) as follows, characterized in that (a 1) inhibits plant pathogenic bacteria; (a 2) preparing a product for inhibiting plant pathogenic bacteria; (a 3) controlling diseases caused by plant pathogenic bacteria; (a4) Preparing a product for controlling diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are Pythium gulum (Pseudoperonospora cubensis), pythium Lin Qi (Pythium sylvaticum), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), botrytis cinerea (Botryoshaeria dothidea), pacific white rot (Coniella diplodiella), paecilomyces graminis (Colletotrichum graminicola), botrytis cinerea (Botrytis cinerea), cladosporium cucumerium (Cladosporium cucumerinum), pacific cocoa hair two cell (Lasiodiplodia pseudotheobromae), paecilomyces pseudodisc (Pestalotiopsis sp.), phomopsis sp, pythium irregulare (Pythium irregulare), rhizoctonia solani (Rhizoctonia solani), fusarium oxysporum (Fusarium oxysporum), mucor circinelloides (Mucor circinelloides), pythium solani (Stemphylium lycopersici), fusarium asiaticum (Fusarium roseum), rhizopus sp, betula paradoxorum (Phoma betae), paecilomyces lividans (Corynespora cassiicola), alternaria (Alternaria alternata), paecilomyces pseudogracilomyces (Fusarium pseudograminearum), paecilomyces paper (Ubbelopsis sp.), fusarium oxysporum (53), fusarium roseum (52).
3. The biocontrol mixture is characterized in that the active ingredients of the biocontrol mixture comprise bacterial suspension, fermentation liquor or fermentation filtrate of two bacterial mixtures, namely bacillus belicus (Bacillus velezensis) strain HMQAU20041 and bacillus highland (Bacillus altitudinis) strain HMQAU 20091; the bacillus belicus (Bacillus velezensis) strain HMQAU20041 was deposited in the China general microbiological culture Collection center, accession number: CGMCC No.24676; the strain HMQAU20091 of Geobacillus (Bacillus altitudinis) was deposited in China general microbiological culture Collection center, accession number: CGMCC No.24677.
4. The biocontrol agent according to claim 3, wherein the active ingredients of the biocontrol agent are fermentation filtrates of two bacterial agents of bacillus beliensis strain HMQAU20041 and bacillus aloft strain HMQAU20091, wherein the total viable bacterial concentration of bacillus beliensis strain HMQAU20041 and bacillus aloft strain HMQAU20091 in the biocontrol agent is 1×10 8 ～2×10 8 cfu/mL。
5. The method for preparing the biocontrol agent as claimed in claim 4, wherein the specific steps comprise:
(1) Culturing bacterial strain HMQAU20041 and bacterial strain HMQAU20091 seed bacterial liquid: respectively inoculating the strain HMQAU20041 and the strain HMQAU20091 of claim 3 into LB culture solution for culture until the OD value of each seed bacterial solution is between 0.5 and 0.8, and obtaining the seed bacterial solutions of the strain HMQAU20041 and the strain HMQAU 20091;
(2) And (3) preparing fermentation filtrate: mixing the bacterial strain HMQAU20041 seed bacterial solution and the bacterial strain HMQAU20091 seed bacterial solution according to different volumes, adding the mixed seed bacterial solution into an conical flask filled with LB culture solution according to the volume percentage content of 1%, carrying out shaking culture, centrifuging, and filtering to prepare fermentation filtrate to obtain the biocontrol agent.
6. The method of producing a biocontrol agent as claimed in claim 5, wherein the cultivation conditions in the step (1) are shaking cultivation at 30℃and 200rpm for 8 to 10 hours.
7. The method for preparing a biocontrol agent according to claim 5, wherein the volume ratio of the bacterial strain HMQAU20041 seed bacterial solution to the bacterial strain HMQAU20091 seed bacterial solution in the step (2) is (1:9) - (9:1).
8. The use of a biocontrol agent according to claims 3-4 or a biocontrol agent prepared by the preparation method of any one of claims 5-7, characterized by the following (b 1) or (b 2) or (b 3) or (b 4), characterized by (b 1) inhibition of plant pathogenic bacteria; (b 2) preparing a product for inhibiting plant pathogenic bacteria; (b 3) controlling diseases caused by phytopathogens; (b4) Preparing a product for controlling diseases caused by plant pathogenic bacteria; the plant pathogenic bacteria are Pythium gulum (Pseudoperonospora cubensis), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), pachyrhizus reesei (Coniella diplodiella), portugal fungus (Botryoshaeria dothidea), rhizoctonia solani (Rhizoctonia solani), phytophthora camphorata (Phytophthora cinnamomi), mucor pseudodish (Pestalotiopsis sp.), leuconostoc (Colletotrichum siamense), phomopsis sp.), pythium solani (Stemphylium lycopersici), cladosporium cucumericum (Cladosporium cucumerinum), phoma betana (Phoma betae), botrytis cinerea (Botrytis cinerea), mucor circinelloides (Mucor circinelloides), purpureae (Stemphylium botryosum), rhizopus (Rhizopus sp.), rhizopus hypsojae (Corynespora cassiicola), alternaria paper (Ulocladium chartarum), alternaria (Alternaria alternata), fusarium oxysporum (Fusarium oxysporum), fusarium equi (Fusarium equiseti), fusarium solani (Fusarium solani), fusarium pseudoginseng (Lasiodiplodia pseudotheobromae), fusarium pseudocurvatum (7462), fuscoporia (Pythium sylvaticum), and Pythium gracilomyces (Pythium irregulare).
9. The use of the biocontrol agent according to claims 3 to 4 or the biocontrol agent prepared by the preparation method of claims 5 to 7 for controlling plant nematode disease, characterized in that the plant disease is any one of root knot nematode disease, rot stem nematode disease.
10. A method for controlling plant diseases, characterized in that the biocontrol agent according to claims 3 to 4 or the biocontrol agent prepared by the preparation method according to claims 5 to 7 is used by coating, dipping, spraying, atomizing, irrigating, volatilizing, dusting, broadcasting, foaming, coating or spraying the diseased part of a plant or a plant part organ when controlling plant diseases.
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| CN117603878A (en) * | 2023-12-01 | 2024-02-27 | 秦皇岛禾苗生物技术有限公司 | Composite microbial agent and preparation method and application thereof |
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| CN115851480A (en) * | 2022-07-21 | 2023-03-28 | 西北农林科技大学 | Bacillus belgii strain P87 and application thereof in prevention and treatment of pepper diseases |
| CN115851480B (en) * | 2022-07-21 | 2024-04-05 | 西北农林科技大学 | Bacillus bailii strain P87 and application thereof in pepper disease control |
| CN117603878A (en) * | 2023-12-01 | 2024-02-27 | 秦皇岛禾苗生物技术有限公司 | Composite microbial agent and preparation method and application thereof |
| CN117603878B (en) * | 2023-12-01 | 2024-04-30 | 秦皇岛禾苗生物技术有限公司 | Composite microbial agent and preparation method and application thereof |
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