Bacillus siamese, Bacillus velez, Serratia marcescens, microbial agents, pesticides and their applications

Abstract

The present application provides Bacillus siamensis, Bacillus velezensis, Serratia marcescens, and microbial agents and pesticides, and applications thereof. The present application has significant effects on early warning and prevention and control of pine wood nematode disease.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to Bacillus siamese, Bacillus velez, Serratia marcescens, microbial agents, pesticides and applications thereof. Background Art

[0002] Pine wilt disease (PWD), known as "pine cancer", is a forest-destroying disease that seriously endangers the safety of forestry in my country and the world. It is one of the four major forest diseases in the world and is considered to be the most serious threat to pine forests worldwide. Pine wood nematode (PWN, Bursaphelenchusxylophilus) is the main pathogenic nematode causing pine wilt disease. Due to its short incubation period, rapid onset, fast lethality, easy confusion with other diseases and incurable once infected, it has caused serious damage to the pine forest ecosystem in many countries in the world and caused huge economic losses. It is an important global quarantine object and is also listed as the largest foreign forest pest in my country.

[0003] At present, the prevention and control of pine wood nematode disease is mainly physical prevention, that is, cutting down infected trees. In recent years, some chemical agents have been used in forests, but such methods are costly and environmentally destructive and do not meet current requirements. At present, the forestry industry urgently needs to develop an environmentally friendly and long-lasting biological control method.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] The purpose of the present application is to address the above-mentioned problems, and the first purpose is to provide a siam Bacillus.

[0006] The second object of the present application is to provide a Bacillus Velezii.

[0007] The third object of the present application is to provide a microbial agent.

[0008] The fourth object of the present application is to provide a pesticide.

[0009] The fourth object of the present application is to provide an application of a pesticide.

[0010] To achieve the above objectives, this application adopts the following technical solutions:

[0011] 1. A Bacillus siamensis, whose deposit number is CGMCC24564.

[0012] 2. The Bacillus siamensis according to item 1, wherein the 16S rDNA gene sequence is shown in SEQ ID NO: 1.

[0013] 3. A Bacillus velezensis, whose accession number is CGMCC24565.

[0014] 4. The Bacillus velezensis according to item 3, wherein the 16S rDNA gene sequence is shown in SEQ ID NO: 2.

[0015] 5. A Serratia marcescens, whose deposit number is CGMCC24567.

[0016] 6. The Serratia marcescens according to item 5, wherein the 16S rDNA gene sequence thereof is shown in SEQ ID NO: 3.

[0017] 7. A microbial agent, comprising one or more of Bacillus siamese, Bacillus velezinii and Serratia marcescens;

[0018] Preferably, the bacterial agent includes Bacillus siamese, Bacillus velez and Serratia marcescens;

[0019] Further preferably, the bacterial agent consists of Bacillus siamese, Bacillus velezensii and Serratia marcescens.

[0020] More preferably, calculated by weight, the amount of the Bacillus siamese to the Serratia marcescens is 1-10 parts, and the amount of the Bacillus velez to the Serratia marcescens is 1-10 parts;

[0021] More preferably, the bacterial agent consists of Bacillus siamensis with a preservation number of CGMCC24564, Bacillus velezensis with a preservation number of CGMCC24565, and Serratia marcescens with a preservation number of CGMCC24567.

[0022] 8. A pesticide, comprising the microbial agent described in item 7.

[0023] 9. The pesticide according to item 8, characterized in that

[0024] The pesticide also includes an adjuvant;

[0025] Preferably, the auxiliary agent is selected from one or more of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract, and avermectin;

[0026] Further preferably, the adjuvant is 0.5-10wt% based on the mass percentage of the total weight of the pesticide; preferably 1-5wt%.

[0027] 10. A method for promoting plant growth or controlling pine wood nematodes or promoting the abundance of beneficial bacteria in plants, comprising applying the microbial agent described in item 7 or the pesticide described in item 8 or 9 to the plants.

[0028] 11. The method according to item 10,

[0029] The plant is selected from one or more of Pinus massoniana, Pinus koraiensis, and Pinus bungeana, preferably Pinus massoniana;

[0030] Preferably,

[0031] The application method is root irrigation and / or spraying, preferably root irrigation.

[0032] Effects of the Invention

[0033] 1. This application mainly uses microorganisms, supplemented by a variety of additives that can improve tree vigor and facilitate microbial colonization, to achieve ecological improvement of pine trees. This method has the advantages of low cost, environmental friendliness, and sustainable prevention and control effects. It is of great significance for early warning and prevention of pine wood nematode disease and preventing the spread of the disease.

[0034] 2. The protective effect of this application is long-lasting. Once the microorganisms are colonized in the plant microenvironment, they can reproduce on their own and provide continuous protection for the plants.

[0035] 3. The cost of this application is controllable, especially in large-scale control work, it has the characteristics of low cost and high efficiency; this application is highly compatible with other control methods, and the bacterial agents used can be freely adjusted according to actual conditions.

[0036] Collection Information

[0037] The Bacillus siamensis YG4-2 described in this application was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is YG4-2 and the accession number is CGMCC24564.

[0038] The Bacillus velezensis YY2P1 described in this application was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is YY2P1 and the accession number is CGMCC24565.

[0039] The Serratia marcescens AHPC29 described in this application was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is AHPC29 and the accession number is CGMCC24567. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the results of the nematicidal activity of the three bacteria described in Example 4;

[0041] Figure 2 This is a schematic diagram of the results of the microbial agent described in Example 5 reducing the amount of nematodes in plants;

[0042] Figure 3 This is the effect of the microbial agent described in Example 6 on the bacterial community structure. DETAILED DESCRIPTION

[0043] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0044] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0045] The application method of the pesticide of the present application can be appropriately selected according to the type of applied plant, the type of pest, the application site, the application period, the dosage form, etc. The application method in the present application is preferably root irrigation and / or spraying.

[0046] The pesticide of the present application can be directly applied as it is, or applied after dilution with water or a carrier. The application method can be listed as follows: spreading on plant stems and leaves, spreading on the base of the plant (plant element), spreading on the soil surface, soil mixing, soil irrigation, water surface application, seed powder coating (seed dressing), coating, dipping, root irrigation, spraying and other methods, but are not limited to these methods. Moreover, the pesticide formulation composition of the present application can also be mixed with other fungicides, insecticides, nematicides, herbicides, plant growth regulators, fertilizers, soil improvement materials, etc., applied alternately, or applied simultaneously, in which case it sometimes shows a more excellent effect.

[0047] The application site of the pesticide of the present application can be applied to nursery beds, farmlands, paddy fields, orchards, hydroponic culture facilities, etc. for cultivating agricultural and horticultural plants, but is not limited to these.

[0048] The application period of the pesticide of the present application is not limited to the planting period. In the case of a nursery, it can be applied at any time before, during, or after planting. In the case of the seedling raising period, it can be applied at any time before, during, or after sowing.

[0049] The application amount of the pesticide in this application varies according to the type of plant, the type of pest or weed, the state of the soil, the application period, the planting density, the dosage form, etc., so it cannot be generally specified. For example, in the case of the seedling stage, the application amount of the pesticide per 1m 2 You can use about 100 to 1000g of soil. In the case of a nursery, use about 100 to 1000g of soil per 1m 2 For soil of about 5 to 1000 g, about 1 to 1000 g can be used. In addition, when powder coating is formed on seeds (including seed potatoes, tubers, bulbs, bulbs, etc.), the crushed material can be used directly or after diluting with water, etc., at about 1 to 100 g per 1 kg of seeds.

[0050] In the present application, "control" means to avoid plant diseases and the like by preventing the infection of bacterial plant disease bacteria and the like that target crops (plants).

[0051] The present application provides a Siamese Bacillus siamensis, which is isolated from pine wood nematode diseased wood, and the Siamese Bacillus siamensis is named Siamese Bacillus siamensis YG4-2. The strain was deposited on March 22, 2022 at the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain name is Bacillus siamensis, and the deposit number is CGMCC24564.

[0052] Bacillus siamensis is a new species reported in 2010, first isolated from pickled crabs in Thailand. To date, there are still few reports of strains belonging to this species, and a considerable number of strains have been identified only based on 16SrRNA gene sequence homology analysis and morphological characteristics, so there is great uncertainty in the complex taxonomy of Bacillus. According to inquiries, in the GenBank database, only KCTC 13613T (AJVF00000000), XY18 (LAGT00000000), SRCM100169 (LYUE01000000) strains of Bacillus siamensis have been supported by whole genome data in taxonomy, and their core genes have high homology, which can serve as the most important basis for whether other strains are Bacillus siamensis.

[0053] In terms of biological control, Bacillus siamese exhibits the same excellent biological control ability as its related species Bacillus amyloliquefaciens and Bacillus velezensis. It is reported that the model strain of Siamese Bacillus KCTC 13613T can inhibit Rhizoctonia solani and Micrococcus luteus (J Bacteriol 2012, 194 (15): 4148-4149); the fermentation broth of Siamese Bacillus L13 is used to prepare microbial seaweed fertilizer, which has a disinfecting effect on tobacco mosaic virus and a growth-promoting effect on mung bean (CN201310165929.5); Siamese Bacillus LYLB4 can prevent and control pear ring rot and soft rot (CN201610048328.X); Siamese Bacillus FC12-05 can antagonize tomato Phytophthora root rot (Journal of Northwest Agriculture and Forestry University 2012, 40: 107-114); or Siamese Bacillus is combined with other Bacillus to prepare a microbial compound fertilizer preparation (CN201610718598.7). In addition, there are few reports on the use of Bacillus siamese to prepare active products. For example, Bacillus siamese FC12-05 can produce an antibacterial active product (possibly a protein) that is precipitated by a 20% ammonium sulfate saturation solution, and Bacillus siamese FJAT-28592 can produce an antifungal lipopeptide with a retention time consistent with iturin A at HPLC (Journal of Agricultural Biotechnology 2016, 24: 261-269).

[0054] According to the present application, the method for isolating the Siamese Bacillus can be a conventional method for screening new strains in the art. For example, the isolation method may include: Siamese Bacillus (Bacillus siamensis) YG4-2 can be screened and obtained by the following method:

[0055] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0056] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNA Polymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0057] The DNA template for full-length identification of bacterial strain 16S involved in this embodiment is prepared quickly and with high quality by Mighty Prep reagent for DNA (Takara). The PCR amplification primers for the full length of 16S rDNA use 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (denoted as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (denoted as SEQ ID NO: 5), and the primers are synthesized by Beijing Qingke Biotechnology Co., Ltd. The PCR product is detected using 1% agarose gel, and the fragment size is 1500bp.

[0058] The 16SrDNA gene sequence of Bacillus siamensis YG4-2 is shown in SEQ ID NO: 1:

[0059] SEQ ID NO: 1

[0060] GCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAA ACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGT GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTG GCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCC CCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAA CCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGA CCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCC AGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACG CGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAG CCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGTCCCCATCTGTCC GCTCGACTTGC

[0061] The present application provides a Bacillus velezensis, which is isolated from pine wood nematode diseased wood, and the Bacillus velezensis is named Bacillus velezensis YY2P1. The strain was deposited on March 22, 2022 at the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain name is (Bacillus velezensis), and the deposit number is CGMCC24565.

[0062] Bacillus velezensis is a Gram-positive, rod-shaped, off-white colony with a wrinkled, opaque, aerobic, chemoheterotrophic bacterium.

[0063] According to the present application, the method for isolating the Bacillus velezensis may be a conventional method for screening new strains in the art. For example, the isolation method may include: Bacillus velezensis YY2P1 may be screened and obtained by the following method:

[0064] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0065] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNAPolymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0066] The DNA template for full-length identification of bacterial strain 16S involved in this embodiment is prepared quickly and with high quality by Mighty Prep reagent for DNA (Takara). The PCR amplification primers for the full length of 16Sr DNA are 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (denoted as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (denoted as SEQ ID NO: 5), and the primers are synthesized by Beijing Qingke Biotechnology Co., Ltd. The PCR product is detected using 1% agarose gel, and the fragment size is 1500bp.

[0067] The 16S rDNA gene sequence of Bacillus velezensis YY2P1 is shown in SEQ ID NO: 2:

[0068] SEQ ID NO: 2

[0069] GCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACT CTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTA GCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTC CGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCA CGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGA AGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC GGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCC CTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACC GCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAA GAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTT CCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGC CTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCAC CCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCA CCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCC CGGTT

[0070] The present application provides a Serratia marcescens, which is isolated from pine wood nematode diseased wood, and the Serratia marcescens is named Serratia marcescens AHPC29. The strain was deposited on March 22, 2022 at the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain name is Bacillus siamensis, and the deposit number is CGMCC24567.

[0071] The "Serratia marcescens" in this article, also known as Bacillus thuringiensis, belongs to the genus Serratia of the family Enterobacteriaceae and is a Gram-negative facultative anaerobic bacillus. Serratia marcescens is a non-demanding microorganism that can utilize a variety of carbon sources, such as chitosan, glucose, sucrose, mannose, cellobiose, citric acid glycerol, etc. Such a wide spectrum of carbon sources provides favorable conditions for its fermentation production. The nuclease from Serratia marcescens consists of 266 amino acids. Unlike other Gram-negative bacteria, the signal peptide composed of 1-21 amino acids helps the nuclease to be secreted into the culture medium. The proteins of other Gram-negative bacteria are secreted into the plasma membrane space rather than the surrounding culture medium. During the secretion process, the signal peptide (1-21 amino acids) is removed; the 22-266 amino acid part with hydrolytic nuclease activity is secreted into the culture medium, with a molecular weight of about 26.7kDa.

[0072] According to the present application, the method for isolating the Serratia marcescens may be a conventional method for screening new strains in the art. For example, the separation method may include: Serratia marcescens AHPC29 may be screened and obtained by the following method:

[0073] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0074] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNA Polymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0075] The DNA template for full-length identification of bacterial strain 16S involved in this embodiment is prepared quickly and with high quality by Mighty Prep reagent for DNA (Takara). The PCR amplification primers for the full length of 16Sr DNA are 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (denoted as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (denoted as SEQ ID NO: 5), and the primers are synthesized by Beijing Qingke Biotechnology Co., Ltd. The PCR product is detected using 1% agarose gel, and the fragment size is 1500bp.

[0076] The 16SrDNA gene sequence of Serratia marcescens AHPC29 is shown in SEQ ID NO: 3:

[0077] SEQ ID NO: 3

[0078] TTCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTT CTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGTACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATACGCCATT GTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCA TCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTT AACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGAT GTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTG CGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATC TCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTTCACCACCTTCCTCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCT TCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAAATT CCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGGATCGTCGCCTAGGTGAG CCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAG CTACCGTTTCCAGTAGTTATCCCCTCCATCAGGCAGTTTCCCAGACATT ACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCCGTGCT ACCGCTCGACTTGCATGTGTTAAAGGCCCCCCCG

[0079] The present application provides a microbial agent, which includes one or more of Bacillus siamese, Bacillus velezensis and Serratia marcescens.

[0080] In some embodiments of the present application, the bacterial agent includes Bacillus siamese, Bacillus velezinii and Serratia marcescens.

[0081] In some embodiments of the present application, the bacterial agent consists of Bacillus siamese, Bacillus Velezii and Serratia marcescens.

[0082] In some embodiments of the present application, calculated by weight, the amount of the Bacillus siamese to the amount of the Serratia marcescens is 1-10 parts, and the amount of the Bacillus velez to the amount of the Serratia marcescens is 1-10 parts;

[0083] For example, the amount of the Bacillus siamensis relative to the Serratia marcescens may be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or any range therebetween;

[0084] The amount of the Bacillus Velezii relative to the Serratia marcescens may be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or any range therebetween.

[0085] In some embodiments of the present application, the bacterial agent is composed of Bacillus siamensis with a preservation number of CGMCC24564, Bacillus velezensis with a preservation number of CGMCC24565, and Serratia marcescens with a preservation number of CGMCC24567.

[0086] The present application provides a pesticide, which includes the above-mentioned microbial agent. In addition, the pesticide of the present application may also include other suitable pesticides.

[0087] Other suitable pesticides are biologically active compounds used to control agricultural pests and include, for example, herbicides, plant growth regulators, crop desiccants, fungicides, bactericides, bacteriostats, insecticides, and repellents, as well as their water-soluble salts and esters. Suitable pesticides include, for example, aryloxyphenoxy-propionate herbicides such as haloxyfop-butyl, cyhalofop-butyl, and quizalofop-butyl, triazine herbicides such as metribuzin, hexaxinone, or atrazine; sulfonylurea herbicides such as chlorsulfuron; uracils such as cypermethrin, cypermethrin, or terbacil; urea herbicides such as linuron, diuron, cypermethrin, or chloranil; acetanilide herbicides such as alachlor or isopropyl metolachlor; thiocarbamate herbicides such as cypermethrin and cypermethrin; oxadiazolone herbicides such as oxadiazolone; isoxathiocarb herbicides, phenoxycarboxylic acid herbicides such as dichlorophenoxyacetic acid ("2,4-D"), dichlorophenoxybutyric acid, ("2,4-DB"), 2-methyl-4-chlorophenoxyacetic acid ("MCPA"), 4-(4-chloro-2-methylphenoxy)butanoic acid ("MCPB"), 2,4-dichlorprop, and 2-methyl-4-chlorophenoxypropionic acid, diphenyl ether herbicides such as fluazifop, acifluorfen, chlorfenapyr, or oxyfluorfen; dinitroaniline herbicides such as trifluralin; organophosphonate herbicides such as glufosinate salts and esters and glyphosate salts and esters; dihalobenzonitrile herbicides such as bromoxynil or ioxynil, benzoic acid herbicides such as dicamba, bipyridinium herbicides such as paraquat, and pyridine and pyridyloxycarboxylic acid herbicides such as clopyralid, fluazifop, piclopram, triclopyr, and aminopyralid. Suitable fungicides include, for example, nitrilo-oxime fungicides such as cymoxanil; imidazole fungicides such as benomyl, carbendazim, or thiophanate-methyl; triazole fungicides such as triadimefon; sulfenamide fungicides such as captan; dithiocarbamate fungicides such as maneb, mancozeb, or thiram; chlorinated aromatic fungicides such as chlorpyrifos; dichloroaniline fungicides such as isoprodinil, strobilurin fungicides such as kresoxim-methyl, trifloxystrobin, or azoxystrobin; thiophanate-methyl; copper salt fungicides such as copper oxychloride; sulfur; aniline; and amide fungicides such as metalaxyl or metalaxyl-M. Suitable insecticides include, for example, carbamate insecticides such as methomyl, carbaryl, furadan, or aldicarb; organothiophosphate insecticides such as EPN, isopropylphos, isoxathiophos, chlorpyrifos, or chlormethoate; organophosphate insecticides such as terbufos, monocrotophos, or stirofos; fully chlorinated organic insecticides such as methoxychlor; synthetic pyrethroid insecticides such as fenvalerate, avermectin or emamectin benzoate, neonicotinoid insecticides such as thiamethoxam or imidacloprid; pyrethroid insecticides such as lambda-cyhalothrin, cypermethrin or bifenthrin, and oxadiazine insecticides such as indoxacarb, imidacloprid, or fipronil.Suitable miticides include, for example, propargyl sulfite miticides, such as propargyl; triazapentadiene miticides, such as amitraz; chlorinated aromatic hydrocarbon miticides, such as ethyl chlorfenapyr, or tetrachlorofenapyr; and dinitrophenol miticides, such as binachlor. Suitable nematicides include carbamate nematicides, such as oxamyl.

[0088] Pesticide compounds are generally referred to herein by the names assigned by the International Organization for Standardization (ISO). ISO common names can be cross-referenced with the International Union of Pure and Applied Chemistry ("IUPAC") and Chemical Abstracts Service ("CAS") names through a number of sources.

[0089] In some embodiments of the present application, the pesticide further comprises an adjuvant. Preferably, the adjuvant is selected from one or more of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract, and avermectin.

[0090] In some embodiments of the present application, the adjuvant is 0.5-10wt% in terms of mass percentage of the total weight of the pesticide; preferably 1-5wt%;

[0091] For example, the adjuvant may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 wt % or any range therebetween, measured as a percentage by mass of the total weight of the pesticide.

[0092] The present application provides a method for promoting plant growth, comprising applying the above-mentioned microbial agent or the above-mentioned pesticide on the plants.

[0093] In some embodiments of the present application, the plant is selected from one or more of Pinus massoniana, Pinus koraiensis, and Pinus bungeana, preferably Pinus massoniana.

[0094] In some embodiments of the present application, the application method is root irrigation and / or spraying, preferably root irrigation.

[0095] The present application provides a method for controlling pine wood nematodes, comprising applying the above-mentioned microbial agent or the above-mentioned pesticide on plants.

[0096] In some embodiments of the present application, the plant is selected from one or more of Pinus massoniana, Pinus koraiensis, and Pinus bungeana, preferably Pinus massoniana.

[0097] In some embodiments of the present application, the application method is root irrigation and / or spraying, preferably root irrigation.

[0098] The present application provides a method for promoting the abundance of beneficial bacteria in plants, comprising applying the above-mentioned microbial agent or the above-mentioned pesticide on the plants.

[0099] In some embodiments of the present application, the plant is selected from one or more of Pinus massoniana, Pinus koraiensis, and Pinus bungeana, preferably Pinus massoniana.

[0100] In some embodiments of the present application, the application method is root irrigation and / or spraying, preferably root irrigation.

[0101] The weight of different strains can be calculated based on methods known to those skilled in the art, such as based on the same OD 600 It is calculated based on the volume ratio.

[0102] Example

[0103] Example 1 Screening and identification process of Bacillus siamensis YG4-2

[0104] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0105] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNAPolymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0106] The 16SrDNA gene sequence of Bacillus siamensis YG4-2 is shown in SEQ ID NO:1.

[0107] The Bacillus siamensis YG4-2 was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is Bacillus siamensis and the preservation number is CGMCC24564.

[0108] Example 2 Screening and Identification Process of Bacillus velezensis YY2P1

[0109] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0110] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNA Polymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0111] The 16S rDNA gene sequence of Bacillus velezensis YY2P1 is shown as SEQ ID NO:2.

[0112] The Bacillus velezensis YY2P1 was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is (Bacillus velezensis) and the accession number is CGMCC24565.

[0113] Example 3 Screening and identification process of Serratia marcescens AHPC29

[0114] Strain isolation: The pupal chamber of the infected wood was taken from the forest farm of Qingyang County, Anhui Province. The wood tissue on the surface of the pupal chamber was scraped with a sterilized blade, transferred to PBST buffer solution, and placed in a 150rpm shaker for 20 minutes. After the culture, 10μL of PBST bacterial suspension was taken with a pipette, evenly spread on the surface of TSA culture medium, and then placed in a 37℃ incubator for 12 hours. When a single colony can be observed on the surface, a single colony is picked up with a pipette tip and purified and cultured.

[0115] Strain identification: Take 1.3mL of bacterial culture solution and place it in a centrifuge. Centrifuge at 1000rpm for 1 minute, then take 1.0-1.2mL of supernatant and recover it in a new centrifuge tube. Then centrifuge the supernatant at 15000rpm for 3 minutes. After centrifugation, discard the supernatant and add 100μL MightyPrep reagent for DNA. After mixing, heat it at 95℃ for 10 minutes. Finally, centrifuge it at 15000rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5μL; 1μL each of primers (27F, 1492R); template: 1μL; MightyAmp DNA Polymerase: 0.5μL; 10×Additive forHigh Specificity: 2.5μL; ddH2O is supplemented to 25μL.

[0116] The 16SrDNA gene sequence of Serratia marcescens AHPC29 is shown in SEQ ID NO:3.

[0117] Serratia marcescens AHPC29 was deposited on March 22, 2022 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is Bacillus siamensis and the accession number is CGMCC24567.

[0118] Example 4 In vitro nematicidal test

[0119] Pine wood nematode (PWN) was cultured and isolated by the Institute of Zoology, Chinese Academy of Sciences. Before testing, PWN were washed with 3% hydrogen peroxide for 10 minutes and then with sterile 0.03M MgSO4. The effect of bacteria on the reproductive capacity of PWN was tested in PDA plates cultured with B. cinerea. The control group 1 was 300 μL PWN suspension (about 100 pine nematodes resuspended in 0.03M MgSO4); the treatment groups were 300 μL PWN suspension (about 100 pine nematodes resuspended in 0.03M MgSO4) and the siam Bacillus suspension obtained in Example 1 (the siam Bacillus suspension had an OD600 of 0.5, and 700 μL of the mixture was taken), 300 μL PWN suspension (about 100 nematodes resuspended in 0.03M MgSO4) and the Velez Bacillus suspension obtained in Example 2 (the Velez Bacillus suspension had an OD600 of 0.5, and 700 μL of the mixture was taken), 300 μL PWN suspension (about 100 nematodes resuspended in 0.03M MgSO4) and the Serratia marcescens suspension obtained in Example 3 (OD600 of the Serratia marcescens suspension = 0.5, 700 μL) were taken to form a mixture. After 7 days, the nematodes were extracted by the Baermann funnel technique and counted under a stereomicroscope.

[0120] in, Figure 1 In the above, Bacillus siamensis represents a mixture of 300 μL of PWN suspension (about 100 pine nematodes resuspended in 0.03 M MgSO4) and the siam Bacillus suspension obtained in Example 1 (the siam Bacillus suspension has an OD600 of 0.5, and 700 μL is taken);

[0121] Bacillus velezensis refers to a mixture of 300 μL of PWN suspension (about 100 nematodes resuspended in 0.03 M MgSO 4 ) and the Bacillus velezensis suspension obtained in Example 2 (the OD600 of the Bacillus velezensis suspension is 0.5, and 700 μL is taken);

[0122] Serratia marcescens refers to a mixture of 300 μL of a PWN suspension (about 100 nematodes resuspended in 0.03 M MgSO 4 ) and the Serratia marcescens suspension obtained in Example 3 (the OD 600 of the Serratia marcescens suspension is 0.5).

[0123] Figure 1 The nematode killing rate refers to the ratio of the number of dead pine wood nematodes (PWN) to the total number of pine wood nematodes (PWN).

[0124] The results are as follows Figure 1The results showed that Bacillus siamensis, Bacillus velezensis and Serratiamarcescens had significant activity in killing pine wood nematodes (PWN).

[0125] Example 5 In vivo nematode inhibition study

[0126] Pine wood nematodes (PWN) were cultured and isolated by the Institute of Zoology, Chinese Academy of Sciences. The greenhouse temperature of Masson pine was 30°C, the relative humidity was 80%, and the photoperiod was 16 / 8h (light / dark). The average height of the pine trees was 50cm.

[0127] Two treatments were set up in the experiment: Treatment 1: PWN and root irrigation pesticide were inoculated at the same time; Treatment 2: PWN was inoculated only. The control group was Masson pine that was not irrigated with bacterial fermentation liquid and not inoculated with PWN. After one month, the number of PWN was counted.

[0128] The PWN inoculation method is as follows: use a sterile blade to make a T-shaped incision on the stem 10 cm above the ground, insert a small cotton ball, add 20 μL of PWN suspension on it, and finally seal the wound with a parafilm.

[0129] The preparation method of the pesticide is as follows: the purified Siamese Bacillus in Example 1, the purified Bacillus Velezii in Example 2, and the purified Serratia marcescens in Example 3 are added to the culture medium respectively; the culture time is 48 hours, and the rotation speed is 200 rpm; after the culture, the fermentation liquid of Siamese Bacillus in Example 1, the fermentation liquid of Bacillus Velezii in Example 2, and the fermentation liquid of Serratia marcescens in Example 3 are heated to OD 600 =0.5 and mixed according to a volume ratio of 1:1:1, and then an aqueous solution of an auxiliary agent is added, wherein the auxiliary agent consists of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract and avermectin, wherein, in terms of mass percentage of the total weight of the auxiliary agent aqueous solution, the molasses powder is 1%, the sodium nitrite is 0.1%, the potassium sulfate is 0.1%, the monoammonium phosphate is 0.1%, the urea is 1%, the wettable powder cuprous oxide is 0.1%, the soapberry extract is 1%, and the avermectin is 0.01%.

[0130] The culture medium compositions of the purified Bacillus siamese in Example 1, the purified Bacillus velez in Example 2, and the purified Serratia marcescens in Example 3 are all as follows: in terms of the mass percentage of each component to the total weight of the culture medium, yeast extract is 1%, glucose is 1%, ammonium sulfate is 0.02%, amino acids are 0.1%, potassium dihydrogen phosphate is 0.03%, magnesium sulfate is 0.05%, ferrous sulfate is 0.03%, and shrimp powder is 0.03%.

[0131] The root irrigation method is as follows: Use a shovel to gently expose part of the roots. Use a measuring cup to water 200mL of fermentation solution, then backfill with soil. Water all plants as needed.

[0132] The nematode count method is as follows: the above-ground part of the sapling is dug out and cut into 2 cm long segments. It is then placed in a funnel pre-placed with paper towels and sterile water is added to cover the wood segments. The nematodes will automatically transfer to the water and sink to the bottom of the funnel. After 12 hours, the PWN suspension is collected and counted. At the same time, the wood slices are dried and the weight is calculated.

[0133] Figure 2 In the table, root irrigation + inoculation represents the results of treatment 1; inoculation only represents the results of treatment 2.

[0134] Figure 2 The amount of nematodes in wood refers to the number of pine wood nematodes (PWN) contained in each gram of wood.

[0135] The results are as follows Figure 2 As shown, pesticides can significantly reduce the number of nematodes in plants.

[0136] Example 6 Effect of microbial agents on the bacterial community structure of plant endophytes.

[0137] The root irrigation operation is as described in Example 5:

[0138] Two treatments were set up in the experiment: Treatment 1: PWN and root irrigation pesticide were inoculated at the same time; Treatment 2: PWN was inoculated only. The control group was Masson pine that was not irrigated with bacterial fermentation liquid and not inoculated with PWN. After one month, the number of PWN was counted.

[0139] The PWN inoculation method is as follows: use a sterile blade to make a T-shaped incision on the stem 10 cm above the ground, insert a small cotton ball, add 20 μL of PWN suspension on it, and finally seal the wound with a parafilm.

[0140] The preparation method of the pesticide is as follows: the purified Siamese Bacillus in Example 1, the purified Bacillus Velezii in Example 2, and the purified Serratia marcescens in Example 3 are added to the culture medium respectively; the culture time is 48 hours, and the rotation speed is 200 rpm; after the culture, the fermentation liquid of Siamese Bacillus in Example 1, the fermentation liquid of Bacillus Velezii in Example 2, and the fermentation liquid of Serratia marcescens in Example 3 are heated to OD 600=0.5 and mixed according to a volume ratio of 1:1:1, and then an aqueous solution of an auxiliary agent is added, wherein the auxiliary agent consists of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract and avermectin, wherein, in terms of mass percentage of the total weight of the auxiliary agent aqueous solution, the molasses powder is 1%, the sodium nitrite is 0.1%, the potassium sulfate is 0.1%, the monoammonium phosphate is 0.1%, the urea is 1%, the wettable powder cuprous oxide is 0.1%, the soapberry extract is 1%, and the avermectin is 0.01%.

[0141] The culture medium compositions of the purified Bacillus siamese in Example 1, the purified Bacillus velez in Example 2, and the purified Serratia marcescens in Example 3 are all as follows: in terms of the mass percentage of each component to the total weight of the culture medium, yeast extract is 1%, glucose is 1%, ammonium sulfate is 0.02%, amino acids are 0.1%, potassium dihydrogen phosphate is 0.03%, magnesium sulfate is 0.05%, ferrous sulfate is 0.03%, and shrimp powder is 0.03%.

[0142] The root irrigation method is as follows: Use a shovel to gently expose part of the roots. Use a measuring cup to water 200mL of fermentation solution, then backfill with soil. Water all plants as needed.

[0143] The steps for microbial community analysis are as follows:

[0144] Weigh 1g of Masson pine stem sample, first wash in 80% ethanol and 0.25% NaOCl to remove surface microorganisms, then wash in sterile water 3 times (1 minute each time). Use sterile scalpel and tweezers to scrape sawdust from the wood. All samples are placed in 2mL sterile centrifuge tubes and sequenced and analyzed by Meiji Biotechnology.

[0145] The results are as follows Figure 3 As shown in the figure, in treatment 1, the abundance of Bacillus increased significantly, proving that root irrigation with microbial agents can significantly increase the number of beneficial bacteria in plants. At the same time, the number of pine wood nematode-associated bacteria represented by Pantoea decreased significantly, indicating that the structure of endophytic bacterial communities in plants shifted in a direction that was beneficial to plant growth.

[0146] After root irrigation, the abundance of beneficial bacteria in the plants increased significantly.

[0147] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. Sequence Listing <110> Institute of Zoology, Chinese Academy of Sciences <120> Bacillus siamese, Bacillus velez, Serratia marcescens, microbial agents, pesticides and their applications <130> PF02138 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 1416 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 1 gcggctggct cctaaaggtt acctcaccga cttcgggtgt tacaaactct cgtggtgtga 60 cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 120 agcgattcca gcttcacgca gtcgagttgc agactgcgat ccgaactgag aacagatttg 180 tgggattggc ttaacctcgc ggtttcgctg ccctttgttc tgtccattgt agcacgtgtg 240 tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300 accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa gggttgcgct 360 cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420 tcactctgcc cccgaagggg acgtcctatc tctaggattg tcagaggatg tcaagacctg 480 gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540 gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt 600 tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt ttacggcgtg 660 gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag cgtcagttac 720 agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat ttcaccgcta 780 cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa tgaccctccc 840 cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgagcc cttacgccc 900 aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960 cgtggctttc tggttaggta ccgtcaaggt gccgccctat ttgaacggca cttgttcttc 1020 cctaacaaca gagctttacg atccgaaaac cttcatcact cacgcggcgt tgctccgtca 1080 gactttcgtc cattgcggaa gattccctac tgctgcctc cgtaggagtc tgggccgtgt 1140 ctcagtccca gtgtggccga tcaccctctc aggtcggcta cgcatcgtcg cttggtgag 1200 ccgttacctc accaactagc taatgcgccg cgggtccatc tgtaagtggt agccgaagcc 1260 accttttatg tctgaaccat gcggttcaaa caaccatccg gtattagccc cggtttcccg 1320 gagttatccc agtcttacag gcaggttacc cacgtgttac tcacccgtcc gccgctaaca 1380 tcagggagca agctcccatc tgtccgctcg acttgc 1416 <210> 2 <211> 1312 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic sequence <400> 2 gctggctcct aaaggttacc tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg 60 gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120 gattccagct tcacgcagtc gagttgcaga ctgcgatccg aactgagaac agatttgtgg 180 gattggctta acctcgcggt ttcgctgccc tttgttctgt ccattgtagc acgtgtgtag 240 cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc 300 ggcagtcacc ttagagtgcc caactgaatg ctggcaacta agatcaaggg ttgcgctcgt 360 tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca 420 ctctgccccc gaaggggacg tcctatctct aggattgtca gaggatgtca agacctggta 480 aggttcttcg cgtgcttg aattaaacca catgctccac cgcttgtgcg ggccccgtc 540 aattccttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt aatgcgttag 600 ctgcagcact aaggggcgga aaccccctaa cacttagcac tcatcgttta cggcgtggac 660 taccagggta tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt cagttacaga 720 ccagagagtc gccttcgcca ctggtgttc tccacatctc tacgcatttc accgctacac 780 gtggaattcc actctcctct tctgcactca agttccccag tttccaatga ccctccccgg 840 ttgagccggg ggctttcaca tcagacttaa gaaaccgcct gcgagccctt tacgcccaat 900 aattccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt 960 ggctttctgg ttaggtaccg tcaaggtgcc gccctatttg aacggcactt gttcttccct 1020 aacacagag cttacgatc cgaaaacctt catcactcac gcggcgttgc tccgtcagac 1080 tttcgtccat tgcggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140 agtcccagtg tggccgatca ccctctcagg tcggctacgc atcgttgcct tggtgagccg 1200 ttacctcacc aactagctaa tgcgccgcgg gtccatctgt aagtggtagc cgaagccacc 1260 ttttatgtct gaaccatgcg gttcaaacaa ccatccggta ttagccccgg tt 1312 <210> 3 <211> 1442 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic sequence <400> 3 ttcacaaagt ggtaagcgcc ctcccgaagg ttaagctacc tacttctttt gcaacccact 60 cccatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgt agcattctga 120 tctacgatta ctagcgattc cgacttcatg gagtcgagtt gcagactcca atccggacta 180 cgacgtactt tatgaggtcc gcttgctctc gcgaggtcgc ttctctttgt atacgccatt 240 gtagcacgtg tgtagcccta ctcgtaaggg ccatgatgac ttgacgtcat ccccaccttc 300 ctccagttta tcactggcag tctcctttga gttcccggcc gaaccgctgg caacaaagga 360 taagggttgc gctcgttgcg ggacttaacc caacatttca caacacgagc tgacgacagc 420 catgcagcac ctgtctcaga gttcccgaag gcaccaatcc atctctggaa agttctctgg 480 atgtcaagag taggtaaggt tcttcgcgtt gcatcgaatt aaaccacatg ctccaccgct 540 tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc ggccgtactc cccaggcggt 600 cgatttaacg cgttagctcc ggaagccacg cctcaagggc acaacctcca aatcgacatc 660 gtttacagcg tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgcacctg 720 agcgtcagtc ttcgtccagg gggccgcctt cgccaccggt attcctccag atctctacgc 780 atttcaccgc tacacctgga attctacccc cctctacgag actctagctt gccagtttca 840 aatgcagttc ccaggttgag cccggggatt tcacatctga cttaacaaac cgcctgcgtg 900 cgctttacgc ccagtaattc cgattaacgc ttgcaccctc cgtattaccg cggctgctgg 960 cacggagtta gccggtgctt cttctgcgag taacgtcaat tgatgaacgt attaagttca 1020 ccaccttcct cctcgctgaa agtgctttac aacccgaagg ccttcttcac acacgcggca 1080 tggctgcatc aggcttgcgc ccattgtgca atattcccca ctgctgcctc ccgtaggagt 1140 ctggaccgtg tctcagttcc agtgtggctg gtcatcctct cagaccagct agggatcgtc 1200 gcctaggtga gccattaccc cacctactag ctaatcccat ctgggcacat ctgatggcaa 1260 gaggcccgaa ggtccccctc tttggtcttg cgacgttatg cggtattagc taccgtttcc 1320 agtagttatc cccctccatc aggcagtttc ccagacatta ctcacccgtc cgccgctcgt 1380 cacccaggga gcaagctccc ccgtgctacc gctcgacttg catgtgttaa aggccccccc 1440 cg 1442 <210> 4 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 4 gcggatccgc ggccgctgca gagtttgatc ctggctcag 39 <210> 5 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 5 gctcgagcgg ccgcccgggt taccttgtta cgactt 36

Claims

1. A Bacillus siamensis, whose deposit number is CGMCC24564.

2. A microbial agent, comprising Bacillus siamese, Bacillus velezinii and Serratia marcescens; The deposit number of the Siamese Bacillus is CGMCC24564.

3. The microbial agent according to claim 2, wherein: The bacterial agent consists of Bacillus siamese, Bacillus velez and Serratia marcescens.

4. The microbial agent according to claim 2, wherein Calculated by weight, the amount of the siamese Bacillus is 1-10 parts relative to the Serratia marcescens, and the amount of the Velez subtilis is 1-10 parts relative to the Serratia marcescens.

5. The microbial agent according to claim 2, wherein The accession number of the Bacillus Velezii is CGMCC24565, and / or the accession number of the Serratia marcescens is CGMCC24567.

6. A pesticide, comprising the microbial agent according to any one of claims 2 to 5.

7. The pesticide according to claim 6, characterized in that The pesticide also includes an adjuvant.

8. The pesticide according to claim 7, wherein The auxiliary agent is selected from one or more of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract, and avermectin.

9. The pesticide according to claim 7, wherein Calculated by mass percentage based on the total weight of the pesticide, the adjuvant is 0.5-10wt%.

10. The pesticide according to claim 9, wherein Calculated by weight percentage based on the total weight of the pesticide, the adjuvant is 1-5wt%.

11. A method for promoting plant growth or controlling pine wood nematodes, wherein: The method comprises applying the microbial agent according to any one of claims 2 to 5 or the pesticide according to any one of claims 6 to 10 to plants; The plants are selected from one or more of Pinus massoniana, Pinus koraiensis and Pinus bungeana.

12. The method according to claim 11, wherein: The plant is Pinus massoniana.

13. The method according to claim 11, wherein: The application method is root irrigation and / or spraying.

14. The method according to claim 13, wherein: The application method is root irrigation.

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