CN117165463A - Siamese bacillus, bacillus belicus, serratia marcescens, microbial agent, pesticide and application thereof - Google Patents

Abstract

The application provides a Siamese bacillus (Bacillus siamensis), a Bacillus bailii (Bacillus velezensis), serratia marcescens (Serratia marcescens), a microbial agent and a pesticide, and application thereof. The application has remarkable effect on early warning, prevention and control of pine wood nematode disease.

Description

Bacillus siamese, Bacillus Velez, Serratia marcescens, microbial agents, Pesticides and their applications

Technical field

This application belongs to the field of microbial technology and specifically relates to Bacillus siamese, Bacillus veleis, Serratia marcescens, microbial agents, pesticides and their applications.

Background technique

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

At present, the control of pine wood nematode disease is mainly based on physical control, which means cutting down the infected trees. In recent years, some chemicals have been used in forests, but such methods are costly, environmentally destructive, and do not meet today's requirements. At present, the forestry industry urgently needs to develop an environmentally friendly and long-lasting biological control method.

In view of this, this application is filed.

Contents of the invention

The purpose of this application is to address the above problems, and the first purpose is to provide a Bacillus siamese bacterium.

The second object of the present application is to provide a Bacillus veleis.

The third object of this application is to provide a microbial inoculant.

The fourth object of this application is to provide a pesticide.

The fourth object of this application is to provide an application of pesticides.

In order to achieve the above purpose, this application adopts the following technical solutions:

1. Bacillus siamensis, whose deposit number is CGMCC24564.

2. Bacillus siamensis (Bacillussiamensis) according to item 1, whose 16SrDNA gene sequence is shown in SEQ ID NO: 1.

3. Bacillus velezensis, whose deposit number is CGMCC24565.

4. Bacillus velezensis according to item 3, whose 16SrDNA gene sequence is shown in SEQ ID NO: 2.

5. Serratia marcescens, the deposit number of which is CGMCC24567.

6. Serratia marcescens according to item 5, whose 16SrDNA gene sequence is shown in SEQ ID NO: 3.

7. A microbial agent, which includes one or more of Bacillus siamese, Bacillus veleis and Serratia marcescens;

Preferably, the bacterial agent includes Bacillus siamese, Bacillus veleis and Serratia marcescens;

Further preferably, the bacterial agent is composed of Bacillus siamese, Bacillus veleis and Serratia marcescens.

More preferably, in terms of parts by weight, the Bacillus siameseum is 1-10 parts relative to the Serratia marcescens, and the Bacillus veleis is 1-10 parts relative to the Serratia marcescens. 1-10 servings;

More preferably, the bacterial agent is composed of Bacillus siamensis with the deposit number CGMCC24564, Bacillus velezensis with the deposit number CGMCC24565 and Serratia marcescens with the deposit number CGMCC24567 marcescens) composition.

8. A pesticide, which includes the microbial inoculant described in item 7.

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

The pesticide also includes auxiliaries;

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 abamectin. ;

Further preferably, the auxiliary agent is 0.5-10wt%; preferably 1-5wt%, based on the mass percentage of the total weight of the pesticide.

10. A method for promoting plant growth or controlling pine wood nematode or promoting the abundance of beneficial bacterial flora in plants, including applying the microbial inoculant described in Item 7 or the pesticide described in Item 8 or 9 on the plant.

11. According to the method described in item 10,

The plant is selected from one or more of masson pine, red pine, and whitebark pine, and is preferably masson pine;

Preferably,

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

Effect of the invention

1. This application mainly uses microorganisms, supplemented by a variety of auxiliary agents that can increase 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 control effects. It is of great significance for early warning and prevention of pine wood nematode disease and preventing the spread of the disease.

2. The control effect of this application is long-lasting. When microorganisms colonize in the plant microenvironment, they can reproduce on their own and provide continuous protection to the plants.

3. The cost of this application is controllable, especially in large-area eradication work. It has the characteristics of low cost and high efficiency; this application is highly compatible with other eradication methods, and the fungicides used can be freely adjusted according to the actual situation.

deposit information

Bacillus siamensis YG4-2 described in this application was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the name of the strain is YG4-2, and the accession number is CGMCC24564.

Bacillus velezensis YY2P1 described in this application was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, the strain name is YY2P1, and the accession number is CGMCC24565.

Serratia marcescens AHPC29 described in this application was deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on March 22, 2022. The address is 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.

Description of drawings

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

Figure 2 is a schematic diagram of the results of reducing the amount of nematodes in plants using the microbial inoculants described in Example 5;

Figure 3 shows the effect of the microbial inoculants described in Example 6 on the bacterial community structure.

Detailed ways

The present application will be further described below with reference to the examples. It should be understood that the examples are only used to further illustrate and illustrate the present application and are not intended to limit the present application.

Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control. Additionally, materials, methods, and examples are illustrative only and not limiting. The present application will be further described below with reference to specific embodiments, but shall not be used to limit the scope of the present application.

The application method of the pesticide of the present application can be appropriately selected according to the type of plants to be used, types of pests and diseases, application location, application period, dosage form, etc. The application method in this application is preferably root irrigation and/or spraying.

The pesticide of this application can be applied directly as it is, or can be applied after dilution with water or carrier. Application methods can be listed: spreading on plant stems and leaves, spreading on the base of the plant (plant element), spreading on the soil surface, soil mixing, soil filling, 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 and applied, alternately or simultaneously with other fungicides, insecticides, nematicides, herbicides, plant growth regulators, fertilizers, soil improvement materials, etc. as needed. , sometimes showing better results in this case.

The application site of the pesticide of the present application can be applied to seedbeds for cultivating agricultural and horticultural plants, farmland, paddy fields, orchards, liquid cultivation facilities, etc., but is not limited to these.

The application period of the pesticide of this application is not limited to the planting period. In the case of a nursery, it can be applied at any period before, during, or after planting. In the case of a seedling period, it can be applied before or at the same time as sowing. , applied at any time after sowing.

The application amount of pesticides in this application varies depending on the type of plants, types of pests, diseases, or weeds, soil conditions, application period, planting density, dosage form, etc., so it cannot be stipulated uniformly. For example, in the seedling stage, every 1m About 100-1000g of ² soil can be used, and in the case of a nursery, about 5-1000g can be used per ¹ m of soil. In addition, when a powder coating is formed on seeds (including seed potatoes, tubers, bulbs, bulbs, etc.), the crushed product can be used directly or after dilution with water, etc., at an amount of about 1 to 100 g per 1 kg of seeds.

In addition, in this application, "prevention and control" refers to avoiding infection of bacterial plant disease fungi and the like targeting crops (plants) and avoiding the plant diseases and the like.

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

Bacillus siamensis is a new species reported in 2010 and was first isolated from pickled crabs in Thailand. So far, there are still not many 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. Therefore, there is great uncertainty in the complex taxonomy of Bacillus. According to the query, in the GenBank database, only KCTC 13613T (AJVF00000000), XY18 (LAGT00000000), SRCM100169 (LYUE01000000) are supported by whole-genome data in taxonomy, and their core genes have high homology. , can be used as the most important basis for whether other strains are Bacillus siamese.

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

According to the present application, the method for isolating the Bacillus siamensis can be a conventional method in the art for screening new bacterial species. For example, the isolation method can include: Bacillus siamensis YG4-2 can be screened and obtained by the following method :

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL of each primer (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10×Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

The DNA template for full-length identification of 16S bacterial strain involved in this embodiment was prepared quickly and with high quality by MightyPrep reagent for DNA (Takara). The PCR amplification primers for the full length of 16SrDNA used 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (recorded as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (recorded as SEQ ID NO: 5). The primers were provided by Beijing Qingke Biotechnology Co., Ltd. synthesis. PCR products were detected using 1% agarose gel, and the fragment size was 1500 bp.

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

SEQ ID NO: 1

GCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAA ACTCTCGTGGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAA GGGGCATGATGATTTGACGTCATCCCCACCTTC CTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTG GCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCC CGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATG CTCCACCGCTTG TGCGGGCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCC CCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAA CCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTAT CTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGA CCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTT CACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCC AGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACG CTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGC TTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGT TCTTCCCTAACAACAGA GCTTTACGATCCGAAAACCTTCATCACTCACG CGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGAT CACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAG CCACCTTTTATGTCTGAACCATGCGGTTCAA ACAACCATCCGGTATTAG CCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTG TTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGTCCCCATCTGTCCGCTCGACTTGC

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

Bacillus velezensis is a Gram-positive, rod-shaped, off-white colony with wrinkled surface, opaque, aerobic, and chemoheterotrophic bacteria.

According to the present application, the method for isolating the Bacillus velezensis can be a conventional method in the art for screening new bacterial species. For example, the isolation method can include: Bacillus velezensis YY2P1 can be screened by the following method :

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL each of primers (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10× Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

The DNA template for full-length identification of 16S bacterial strain involved in this embodiment was prepared quickly and with high quality by MightyPrep reagent for DNA (Takara). The PCR amplification primers for the full length of 16Sr DNA used 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (recorded as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (recorded as SEQ ID NO: 5). The primers were provided by Beijing Qingke Biotechnology Co., Ltd. Company Synthesis. PCR products were detected using 1% agarose gel, and the fragment size was 1500 bp.

The 16SrDNA gene sequence of Bacillus velezensis YY2P1 is shown in SEQ IDNO: 2:

SEQ ID NO: 2

GCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACT CTCGTGGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACC GCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTA ACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTA GCCCAGGTCATAAGG GGCATGATGATTTGACGTCATCCCCACCTTCCTC CGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCA ACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCA CGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGA AGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTA AGGTTCTTCGCGTTGCTTCGAATTAAACCACATG CTCCACCGCTTGTGC GGGCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCA GGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCC CTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAG AGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTC ACC GCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTT TCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAA GAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTG CCACCTACGATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTC TGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTT CCCTAACAACAGA GCTTTACGATCCGAAAACCTTCATCACTCACGCGG CGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGC CTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCAC CCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACC AACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCA CCTTTTATGTCTGAACCATGCGG TTCAAACAACCATCCGGTATTAGCCC CGGTT

The present application provides a kind of Serratia marcescens (Serratia marcescens), which is isolated from pine wood nematode infected wood, and the Serratia marcescens (Serratia marcescens) is named Serratia marcescens AHPC29. The strain was deposited at the General Microbiology Center of the China Microbial Culture Collection and Management Committee on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the name of the strain is Bacillus siamensis. The deposit number is CGMCC24567.

The "Serratia marcescens" in this article, also known as Lepiobacter, belongs to the genus Serratia of the Enterobacteriaceae family and is a Gram-negative facultative anaerobe. Serratia marcescens is a non-fastidious microorganism that can utilize a variety of carbon sources, such as chitosan, glucose, sucrose, mannose, cellobiose, citrate glycerin, etc. Such a wide spectrum of carbon sources enables its fermentation production Favorable conditions are provided. The nuclease derived from Serratia marcescens consists of 266 amino acids. Unlike other Gram-negative bacteria, the signal peptide consisting of 1-21 amino acids helps the nuclease to be secreted into the culture medium. Other Gram-negative bacteria The proteins of C. 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 portion with hydrolytic nuclease activity is secreted into the culture medium. , molecular weight is approximately 26.7kDa.

According to the present application, the method for isolating the Serratia marcescens can be a conventional method in the art for screening new bacterial species. For example, the isolation method can include: Serratia marcescens AHPC29 can be used by the following method Filter to get:

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL of each primer (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10×Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

The DNA template for full-length identification of 16S bacterial strain involved in this embodiment was prepared quickly and with high quality by MightyPrep reagent for DNA (Takara). The PCR amplification primers for the full length of 16Sr DNA used 8F 5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (recorded as SEQ ID NO: 4) and 1492R 5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (recorded as SEQ ID NO: 5). The primers were provided by Beijing Qingke Biotechnology Co., Ltd. Company Synthesis. PCR products were detected using 1% agarose gel, and the fragment size was 1500 bp.

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

SEQ ID NO: 3

TTCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTT CTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCC GGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTTGCAGACTCCAATCCGGACTACGACGTACTTT ATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATACGCCATT GTAGCACGTGTGTA GCCCTACTCGTAAGGGCCATGATGACTTGACGTCA TCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGG CCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTT AACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGT CTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGAT GTCAAGAGTAGGTAAGGTTCTTCGCGTTG CATCGAATTAAACCACATG CTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTG CGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCC ACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCG TCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGA TC TCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGAC TCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTT CACATCTGACTTAACAAACCGCCTGCGTGCTTTACGCCCAGTAATTC CGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTT AGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTT CACCACC TTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATT CCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGT GTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAG CCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCTCTT TGGTCTTGCGACGTTATGCGGTATTAG CTACCGTTTCCAGTAGTTATCCCCTCCATCAGGCAGTTTCCCAGACATT ACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCCGTGCT ACCGCTCGACTTGCATGTGTTAAAGGCCCCCCCG

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

In some embodiments of the present application, the bacterial agent includes Bacillus siamese, Bacillus veleis and Serratia marcescens.

In some embodiments of the present application, the bacterial agent is composed of Bacillus siamese, Bacillus veleis and Serratia marcescens.

In some embodiments of the present application, calculated in parts by weight, the Bacillus siameseum is 1-10 parts relative to the Serratia marcescens, and the Bacillus veleisi is 1-10 parts relative to the Serratia marcescens. Serratia is 1-10 parts;

For example, the Bacillus siamese may be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or more relative to the Serratia marcescens. Any range therebetween;

The Bacillus veleis can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or other parts thereof relative to the Serratia marcescens. any range in between.

In some embodiments of the present application, the bacterial agent is composed of Bacillus siamensis with the deposit number CGMCC24564, Bacillus velezensis with the deposit number CGMCC24565, and Bacillus velezensis with the deposit number CGMCC24567 Composed of Serratia marcescens.

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

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 Insect repellents, together with their water-soluble salts and esters. Suitable pesticides include, for example, aryloxyphenoxy-propionate herbicides, such as clofeniod, cyhalofop, and quizalofop, triazine herbicides, such as mefenacet, cyclazine Ketone (hexaxinone), or atrazine; sulfonylurea herbicides, such as chlorsulfuron; uracil, such as cycllopyr, herbicide, or terclofen; urea herbicides, such as linuron, diuron, Cyclopyramide, or cyclofenac; acetanilide herbicides, such as alachlor, or metolachlor; thiocarbamate herbicides, such as fenfosulfan, dicamba; oxadiazolones Herbicides, such as oxadiazon; isoxatrione herbicides, phenoxycarboxylic acid herbicides, such as dichlorophenoxyacetic acid ("2,4-D"), dichlorophenoxybutyric acid ("2,4-D") 2,4-DB”), 2-methyl-4-chlorophenoxyacetic acid (“MCPA”), 4-(4-chloro-2-methylphenoxy)butyric acid (“MCPB”), 2 , 4-D propionic acid, and 2-methyl-4-chlorophenoxypropionic acid, diphenyl ether herbicides, such as flufenopop, acifluorfen, oxyfluorfen, or oxyfluorfen; Dinitroaniline herbicides, such as trifluralin; organophosphonate herbicides, such as glufosinate ammonium salts and esters and glyphosate salts and esters; dihalobenzonitrile herbicides, such as bromochloride, or iodine Benzonitrile, benzoic acid herbicides such as dicamba, dipyridinium herbicides such as paraquat, and pyridine and pyridinoxycarboxylic acid herbicides such as clopyridin, fluopyr, picloram, and triclopyr , and clopyralid. Suitable fungicides include, for example, nitriloxime fungicides, such as cymoxanil; imidazole fungicides, such as benomyl, carbendazim, or thiophanate-methyl; triazole fungicides, such as triadimefon ; Sulfenamide fungicides, such as captan; disulfide carbamate fungicides, such as maneb, mancozeb, or thiram; chlorinated aromatic fungicides, such as Dimosan; Chloroaniline fungicides, such as iprodione, strobilurin fungicides, such as tristrostrobin, trifloxystrobin, or azoxystrobin; chlorothalonil; copper salt fungicides, such as copper oxychloride; sulfur; aniline; and amide-based fungicides such as metalaxyl or metalaxyl. Suitable insecticides include, for example, carbamate insecticides such as methionine, carbofuran, carbofuran, or aldicarb; organophosphorothioate insecticides such as EPN, isopropamidophos, isoxam Zophos, chlorpyrifos, or chlormethoate; organophosphate insecticides such as terbuphos, monocrotophos, or sterophos; fully chlorinated organic insecticides such as methoxychlor; synthetic pyrethroids Ester insecticides, such as fenvalerate, abamectin, or emamectin benzoate; neonicotinoid insecticides, such as thiamethoxam or imidacloprid; pyrethroid insecticides, such as lambda-chloride Cyfluthrin, cypermethrin, or bifenthrin, and an oxadiazine insecticide such as indoxacarb, imidacloprid, or fipronil. Suitable acaricides include, for example, propynyl sulfite acaricides, such as profenate; triazoprene acaricides, such as mitamidine; chlorinated aromatic hydrocarbon acaricides, such as dicofol ethyl ester , or tetrachloromiticidal sulfone; and dinitrophenol acaricides, such as Lexafen. Suitable nematicides include carbamate nematicides such as oxalyl.

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

In some embodiments of the present application, the pesticide further includes an auxiliary agent. Preferably, the auxiliary agent is selected from the group consisting of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, etc. One or more of soapberry extract and abamectin.

In some embodiments of the present application, the auxiliary agent is 0.5-10wt% based on the mass percentage of the total weight of the pesticide; preferably 1-5wt%;

For example, based on the mass percentage of the total weight of the pesticide, the auxiliary agent can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 wt% or any range therebetween. .

The present application provides a method for promoting plant growth, which includes applying the above-mentioned microbial inoculants or the above-mentioned pesticides on plants.

In some embodiments of the present application, the plant is selected from one or more of masson pine, red pine, and whitebark pine, and is preferably masson pine.

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

The present application provides a method for preventing and controlling pine wood nematode, which includes applying the above-mentioned microbial inoculants or the above-mentioned pesticides on plants.

In some embodiments of the present application, the plant is selected from one or more of masson pine, red pine, and whitebark pine, and is preferably masson pine.

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

The present application provides a method for promoting the abundance of beneficial bacterial flora in plants, which includes applying the above-mentioned microbial agents or the above-mentioned pesticides on the plants.

In some embodiments of the present application, the plant is selected from one or more of masson pine, red pine, and whitebark pine, and is preferably masson pine.

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

The weight parts of different strains can be calculated based on methods known to those skilled in the art, for example, calculated based on the volume ratio under the premise of equivalent OD ₆₀₀ .

Example

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

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL each of primers (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10× Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

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

The Bacillus siamensis YG4-2 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, the name of the strain is Bacillus siamensis, and the accession number is CGMCC24564.

Example 2 Screening and identification process of Bacillus velezensis YY2P1

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL of each primer (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10×Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

The 16SrDNA gene sequence of Bacillus velezensis YY2P1 is shown in SEQ ID NO: 2.

The Bacillus velezensis YY2P1 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Microbiology Research, Chinese Academy of Sciences The name of the strain is (Bacillus velezensis) and the accession number is CGMCC24565.

Example 3 Screening and identification process of Serratia marcescens AHPC29

Isolation of strains: The pupal chambers of Phytophthora blight were obtained from the forest farm in Qingyang County, Anhui Province. The xylem tissue on the surface of the pupal chambers was scraped with a sterilized blade, transferred into PBST buffer solution, and cultured on a 150 rpm shaker for 20 minutes. After the culture is completed, use a pipette to absorb 10 μL PBST bacterial suspension, spread it evenly on the surface of the TSA culture medium, and place it in a 37°C incubator for 12 hours. When a single colony can be observed growing on the surface, use a pipette tip to pick up a single colony and purify and culture it.

Strain identification: Place 1.3mL of bacterial culture liquid in a centrifuge, centrifuge at 1000rpm for 1 minute, take 1.0-1.2mL of supernatant and recover it into a new centrifuge tube, then centrifuge the supernatant at 15000rpm for 3 minutes, and centrifuge Discard the supernatant and add 100 μL MightyPrep reagent for DNA. Mix and heat at 95°C for 10 minutes. Finally, centrifuge at 15,000 rpm for 2 minutes. The supernatant is the PCR template. The PCR reaction system is: 2×MightyAmp Buffer: 12.5 μL; 1 μL of each primer (27F, 1492R); template: 1 μL; MightyAmp DNA Polymerase: 0.5 μL; 10×Additive for High Specificity: 2.5 μL; ddH2O is added to 25 μL.

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

Serratia marcescens (Serratia marcescens) AHPC29 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 22, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Microbiology Research, Chinese Academy of Sciences The name of the strain is Bacillus siamensis, and the accession number is CGMCC24567.

Example 4 In vitro nematicidal test

Pine wood nematode (PWN) was cultured and isolated from the Institute of Zoology, Chinese Academy of Sciences. Before testing, pine wood nematodes were cleaned with 3% hydrogen peroxide for 10 minutes and then with sterilized _0.03 M MgSO. The effect of the bacteria on the reproductive capacity of pine wood nematode was tested in PDA dishes cultured with B. cinerea. Control group 1 is 300 μL PWN suspension (about 100 pine wood nematodes resuspended in 0.03M MgSO ₄ ); treatment groups are respectively 300 μL PWN suspension (about 100 pine wood nematodes resuspended in 0.03M MgSO ₄ ) and The Bacillus siamese suspension obtained in Example 1 (the OD600 of the Bacillus siamese suspension is 0.5, take a mixture composed of 700 μL, 300 μL PWN suspension (about 100 nematodes are resuspended in 0.03M MgSO ₄ ) and A mixture consisting of the B. veleis suspension obtained in Example 2 (the OD600 of the B. veleis suspension was 0.5, take 700 μL) and 300 μL PWN suspension (about 100 nematodes were resuspended in 0.03M MgSO ₄ (medium) and the Serratia marcescens suspension obtained in Example 3 (the OD600 of the Serratia marcescens suspension was 0.5, take 700 μL,). After 7 days, the nematodes were extracted using the Bellman funnel technique and Count under a stereomicroscope.

Among them, in Figure 1, Bacillus siamensis represents 300 μL PWN suspension (about 100 pine wood nematodes resuspended in 0.03M MgSO ₄ ) and the Bacillus siamese suspension obtained in Example 1 (the Bacillus siamese suspension) OD600=0.5, take 700μL) mixture;

Bacillus velezensis represents 300 μL of PWN suspension (about 100 nematodes resuspended in 0.03MMgSO ₄ ) and the Bacillus velezensis suspension obtained in Example 2 (the OD600 of the Bacillus velezensis suspension = 0.5, take 700 μL) Mixed liquid composed of;

Serratia marcescens represents the composition of 300 μL PWN suspension (about 100 nematodes resuspended in 0.03MMgSO ₄ ) and the Serratia marcescens suspension obtained in Example 3 (the OD600 of the Serratia marcescens suspension = 0.5) of mixture.

In 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).

The results show in Figure 1 that three bacteria, Bacillus siamensis, Bacillus velezensis, and Serratiamarcescens, have significant activity against pine wood nematode (PWN).

Example 5 In vivo study on nematode suppression

Pine wood nematode (PWN) was cultured and isolated from the Institute of Zoology, Chinese Academy of Sciences. The average temperature of the masson pine greenhouse is 30°C, the relative humidity is 80%, and the photoperiod is 16/8h (light/dark). The average height of pine trees is 50cm.

The experiment set up two treatments: treatment 1: inoculated with PWN and root irrigation pesticides at the same time; treatment 2: only inoculated with PWN. The control group was masson pine that was not irrigated with bacterial fermentation broth and not inoculated with PWN. After one month, count the number of PWNs.

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

The preparation method of the pesticide is as follows: respectively add the purified Bacillus siamese in Example 1, the purified Bacillus veleis in Example 2, and the purified Serratia marcescens in Example 3 into the culture medium; culture The time is 48 hours, the rotation speed is 200 rpm; after cultivation, the Bacillus siamese fermentation broth of Example 1, the Bacillus veleis fermentation broth of Example 2, and the Serratia marcescens fermentation broth of Example 3 are at OD ₆₀₀ = 0.5 After mixing according to the volume ratio of 1:1:1, add the aqueous solution of the additive, which consists of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry The extract is composed of avermectin, in which, based on the mass percentage of the total weight of the auxiliary agent aqueous solution, molasses powder is 1%, sodium nitrite is 0.1%, potassium sulfate is 0.1%, and monoammonium phosphate is 0.1% , urea is 1%, wettable powder cuprous oxide is 0.1%, soapberry extract is 1%, and abamectin is 0.01%.

Among them, the culture medium compositions of Bacillus siamese purified in Example 1, Bacillus veleis purified in Example 2, and Serratia marcescens purified in Example 3 are: each composition accounts for the culture medium In terms of mass percentage of the total weight, yeast extract is 1%, glucose is 1%, ammonium sulfate is 0.02%, amino acid is 0.1%, dipotassium hydrogen phosphate is 0.03%, magnesium sulfate is 0.05%, ferrous sulfate is 0.03%, Shrimp powder is 0.03%.

Root irrigation method is as follows: Use a shovel to gently expose part of the roots. Use a measuring cup to pour 200mL of fermentation liquid, then backfill with soil. Water all plants as needed.

The method for counting nematodes is as follows: dig out the above-ground part of the sapling and cut it into 2cm long sections. Then place it into a funnel pre-placed with paper towels and add sterile water that covers the wood section. The nematodes will automatically transfer to the water and sink to the bottom of the funnel. After 12 hours, the PWN suspension was collected and counted. Meanwhile, the wood slices are dried and the weight is calculated.

In Figure 2, root irrigation + inoculation represents the results of treatment 1; inoculation alone represents the results of treatment 2.

The nematode count in Figure 2 refers to the number of pine wood nematodes (PWN) contained per gram of wood.

The results are shown in Figure 2. Pesticides can significantly reduce the amount of nematodes in plants.

Example 6 Effect of microbial inoculants on plant endophyte bacterial community structure.

The root irrigation operation is as described in Example 5:

The experiment set up two treatments: treatment 1: inoculated with PWN and root irrigation pesticides at the same time; treatment 2: only inoculated with PWN. The control group was masson pine that was not irrigated with bacterial fermentation broth and not inoculated with PWN. After one month, count the number of PWNs.

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

The preparation method of the pesticide is as follows: respectively add the purified Bacillus siamese in Example 1, the purified Bacillus veleis in Example 2, and the purified Serratia marcescens in Example 3 into the culture medium; culture The time is 48 hours, the rotation speed is 200 rpm; after cultivation, the Bacillus siamese fermentation broth of Example 1, the Bacillus veleis fermentation broth of Example 2, and the Serratia marcescens fermentation broth of Example 3 are at OD ₆₀₀ = 0.5 After mixing according to the volume ratio of 1:1:1, add the aqueous solution of the additive, which consists of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry The extract is composed of avermectin, in which, based on the mass percentage of the total weight of the auxiliary agent aqueous solution, molasses powder is 1%, sodium nitrite is 0.1%, potassium sulfate is 0.1%, and monoammonium phosphate is 0.1% , urea is 1%, wettable powder cuprous oxide is 0.1%, soapberry extract is 1%, and abamectin is 0.01%.

Among them, the culture medium compositions of Bacillus siamese purified in Example 1, Bacillus veleis purified in Example 2, and Serratia marcescens purified in Example 3 are: each composition accounts for the culture medium In terms of mass percentage of the total weight, yeast extract is 1%, glucose is 1%, ammonium sulfate is 0.02%, amino acid is 0.1%, dipotassium hydrogen phosphate is 0.03%, magnesium sulfate is 0.05%, ferrous sulfate is 0.03%, Shrimp powder is 0.03%.

Root irrigation method is as follows: Use a shovel to gently expose part of the roots. Use a measuring cup to pour 200mL of fermentation liquid, then backfill with soil. Water all plants as needed.

The steps for microbial community analysis are as follows:

Weigh 1 g of Pinus massoniana stem sample, first wash it in 80% ethanol and 0.25% NaOCl to remove surface microorganisms, and then wash it three times in sterile water (1 minute each time). Use a sterile scalpel and tweezers to scrape sawdust from the xylem. All samples were put into 2mL sterile centrifuge tubes and sequenced and analyzed by Meji Biotech.

The results are shown in Figure 3. 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 the plant. At the same time, the number of associated bacteria of pine wood nematodes represented by Pantoea increased significantly. The decrease indicates that the plant endophyte community structure is changing in a direction beneficial to plant growth.

The abundance of beneficial bacteria in the plants increased significantly after root irrigation.

The above descriptions are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included in the protection scope of this application.

sequence list

<110> Institute of Zoology, Chinese Academy of Sciences

<120> Bacillus siamese, Bacillus bellesii, Serratia marcescens, microbial agents, pesticides and their applications

<130> PF02138

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<170> PatentIn version 3.5

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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 (11)

1. Bacillus siamensis, whose deposit number is CGMCC24564. 2. Bacillus siamensis (Bacillus siamensis) according to claim 1, whose 16SrDNA gene sequence is shown in SEQ ID NO: 1. 3. Bacillus velezensis, whose deposit number is CGMCC24565. 4. Bacillus velezensis according to claim 3, whose 16SrDNA gene sequence is shown in SEQ ID NO: 2. 5. Serratia marcescens, whose deposit number is CGMCC24567. 6. Serratia marcescens according to claim 5, whose 16SrDNA gene sequence is shown in SEQ ID NO: 3. 7. A microbial agent, which includes one or more of Bacillus siamese, Bacillus veleis and Serratia marcescens; Preferably, the bacterial agent includes Bacillus siamese, Bacillus veleis and Serratia marcescens; Further preferably, the bacterial agent is composed of Bacillus siamese, Bacillus veleis and Serratia marcescens. More preferably, in terms of parts by weight, the Bacillus siameseum is 1-10 parts relative to the Serratia marcescens, and the Bacillus veleis is 1-10 parts relative to the Serratia marcescens. 1-10 servings; More preferably, the bacterial agent is composed of Bacillus siamensis with a deposit number of CGMCC24564, Bacillus velezensis with a deposit number of CGMCC24565, and Serratia marcescens with a deposit number of CGMCC24567. Serratia marcescens). 8. A pesticide, said pesticide comprising the microbial inoculant according to claim 7. 9. The pesticide according to claim 8, characterized in that, The pesticide also includes auxiliaries; 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 abamectin. ; Further preferably, the auxiliary agent is 0.5-10 wt%; preferably 1-5 wt%, based on the mass percentage of the total weight of the pesticide. 10. A method for promoting plant growth or preventing and controlling pine wood nematode or promoting the abundance of beneficial bacterial flora in the plant, which is characterized in that it includes applying the microbial agent of claim 7 or the microbial agent of claim 8 or 9 on the plant. the pesticides mentioned above. 11. The method according to claim 10, characterized in that, The plant is selected from one or more of masson pine, red pine, and whitebark pine, and is preferably masson pine; Preferably, The application method is root irrigation and/or spraying, preferably root irrigation.