CN117165463B

CN117165463B - Bacillus siamese, Bacillus velez, Serratia marcescens, microbial agents, pesticides and their applications

Info

Publication number: CN117165463B
Application number: CN202210577417.9A
Authority: CN
Inventors: 赵莉蔺; 孙江华; 单少杰; 韩懂博
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2025-02-11
Anticipated expiration: 2042-05-25
Also published as: CN117165463A

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

Siamese bacillus, bacillus belicus, serratia marcescens, microbial agent, pesticide and application thereof

Technical Field

The application belongs to the technical field of microorganisms, and particularly relates to bacillus siamensis, bacillus behenryis, serratia marcescens, a microbial agent, a pesticide and application thereof.

Background

Pine wilting disease (PINE WILT DISEASE, PWD), known as "pine cancer", is a forest destructive disease that seriously jeopardizes the safety of forestry in China and the world, is one of four forest diseases in the world currently, and has been considered as the most serious threat to pine worldwide. Pine wood nematodes (pine wood nematode, PWN, bursaphelenchus xylophilus) are the major causative nematodes responsible for pine wilting. Because of the characteristics of short incubation period, rapid onset, rapid death speed, easy confusion with other diseases, incurability in case of infection, and the like, the ecological system of pine in many countries in the world is seriously damaged, and huge economic loss is caused, and the ecological system is an important worldwide quarantine object and is also listed as a first forestry foreign pest in China.

At present, the prevention and treatment of pine wood nematode disease is mainly physical prevention and treatment, namely, the epidemic wood is cut off. In recent years, some chemical agents are applied to forests, but the method has high cost and great environmental destruction and does not meet the current requirements. At present, the development of an environment-friendly biological control means with lasting effect is needed in forestry.

In view of this, the present application has been made.

Disclosure of Invention

The present application aims to solve the above problems and provides a first object to provide a bacillus siamensis.

The second object of the present application is to provide bacillus beijerinus.

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

A fourth object of the present application is to provide a pesticide.

A fourth object of the present application is to provide a pesticide application.

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

1. a Siamese bacillus (Bacillus siamensis) has a preservation number of CGMCC24564.

2. The Siamella siamensis Siamensis (Bacillus siamensis) (Bacillus siamensis) of item 1 having a 16SrDNA gene sequence as set forth in SEQ ID NO. 1.

3. Bacillus bailii (Bacillus velezensis) with a preservation number of CGMCC24565.

4. The Bacillus bailii (Bacillus velezensis) according to item 3, wherein the 16SrDNA gene sequence is shown in SEQ ID NO. 2.

5. Serratia marcescens (SERRATIA MARCESCENS) with a preservation number of CGMCC24567.

6. The Serratia marcescens (SERRATIA MARCESCENS) according to item 5, wherein the 16SrDNA gene sequence is shown in SEQ ID NO. 3.

7. A microbial agent, wherein the microbial agent comprises one or more than two of bacillus siamensis, bacillus behenensis and serratia marcescens;

preferably, the microbial inoculum comprises bacillus siamensis, bacillus behenensis and serratia marcescens;

further preferably, the microbial inoculum is composed of bacillus siamensis, bacillus behenensis and serratia marcescens.

More preferably, the bacillus siamensis is 1 to 10 parts by weight relative to the serratia marcescens and the bacillus beijensis is 1 to 10 parts by weight relative to the serratia marcescens;

More preferably, the microbial inoculum is composed of bacillus siamensis (Bacillus siamensis) with the preservation number of CGMCC24564, bacillus beijerinus (Bacillus velezensis) with the preservation number of CGMCC24565 and Serratia marcescens (SERRATIA MARCESCENS) with the preservation number of CGMCC 24567.

8. A pesticide comprising the microbial agent of claim 7.

9. The agricultural chemical according to item 8, characterized in that,

The pesticide also comprises an auxiliary agent;

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

further preferably, the adjuvant is 0.5-10wt%, preferably 1-5wt%, based on the total weight of the pesticide.

10. A method of promoting plant growth or controlling pine wood nematodes or promoting the abundance of beneficial bacterial flora in plants comprising applying to the plants the microbial inoculant of claim 7 or the pesticide of claim 8 or 9.

11. The method according to item 10,

The plant is selected from one or more of Pinus massoniana, pinus koraiensis and Pinus alba, preferably Pinus massoniana;

Preferably, the method comprises the steps of,

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

ADVANTAGEOUS EFFECTS OF INVENTION

1. The application takes microorganisms as the main material and is supplemented with a plurality of auxiliary agents which can improve the tree vigor and facilitate the field planting of the microorganisms, thereby realizing the ecological improvement of pine. The method has the advantages of low cost, environmental protection, sustainable control effect and the like, and has important significance for early warning, prevention and control of pine wood nematode diseases and prevention of spread and spread of diseases.

2. The application has lasting control effect, can automatically reproduce after the microorganism is planted in the plant microenvironment, and can continuously protect the plant.

3. The application has the advantages of controllable cost, low cost, high efficiency and the like, particularly in large-area treatment and removal work, and has high compatibility with other treatment and removal means, and the used microbial inoculum can be freely adjusted according to actual conditions.

Preservation information

The Siamese bacillus (Bacillus siamensis) YG4-2 is stored in the common microorganism center of China Committee for culture Collection of microorganisms at the date of 22.3 of 2022, the address is North Star Xiyu No. 1,3 of the Korean area of Beijing, the strain name is YG4-2 of the institute of microorganisms of the national academy of sciences of China, and the storage number is CGMCC24564.

The bacillus belicus (Bacillus velezensis) YY2P1 is stored in the common microorganism center of China Committee for culture Collection of microorganisms at the 3 rd month 22 year 2022, the address is North Star Xway No. 1, 3 rd, the institute of microorganisms of the national academy of sciences of Beijing, the strain name is YY2P1, and the storage number is CGMCC24565.

Serratia marcescens (SERRATIA MARCESCENS) AHPC29 is stored in the China general microbiological culture Collection center (CGMCC) at the 3 rd month 22 of 2022, and has the address of North Star XILU No. 1 and 3 rd of the Korean area of Beijing, the strain name of AHPC29 is China academy of sciences microbiological study, and the storage number of CGMCC24567.

Drawings

FIG. 1 is a graph showing the results of the nematicidal activity of three bacteria described in example 4;

FIG. 2 is a graph showing the results of microbial agents described in example 5 for reducing nematode levels in plants;

FIG. 3 shows the effect of the microbial agents described in example 6 on the structure of bacterial communities.

Detailed Description

The application will be further illustrated with reference to the following examples, which are to be understood as merely further illustrating and explaining the application and are not to be construed as limiting the application.

Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the materials and methods are described herein below. In case of conflict, the present specification, including definitions therein, will control and materials, methods, and examples, will control and be in no way limiting. The application is further illustrated below in connection with specific examples, which are not intended to limit the scope of the application.

The method of applying the agricultural chemical of the present application may be appropriately selected depending on the type of plant to be applied, 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.

The agricultural chemical of the present application may be applied as it is or after diluted with water or a carrier or the like. Examples of the application method include, but are not limited to, methods of spreading on plant stems and leaves, spreading on plant bases (plant members), spreading on soil surface layers, soil mixing, soil pouring, water surface application, seed powder coating (seed dressing), coating, dipping, root irrigation, spraying, and the like. The pesticidal composition of the present application may be applied in combination with other bactericides, pesticides, nematicides, herbicides, plant growth regulators, fertilizers, soil improvement materials and the like, alternately or simultaneously, if necessary, and may exhibit more excellent effects in this case.

The agricultural chemical application site of the present application may be applied to a seed bed, a farmland, a paddy field, a fruit tree garden, a hydroponic plant, etc. for cultivating agricultural and horticultural plants, but is not limited thereto.

The period of application of the agricultural chemical of the present application is not limited to the period of planting, and in the case of a nursery, the agricultural chemical may be applied at any period before planting, at the time of planting, or after planting, and in the case of a seedling stage, the agricultural chemical may be applied at any period before sowing, at the same time of sowing, or after sowing.

The amount of the agricultural chemical of the present application to be applied varies depending on the type of plant, plant diseases and insect pests, the type of weed, the state of soil, the application period, the planting density, the dosage form, etc., and therefore cannot be specified at all, for example, about 100 to 1000g can be used per 1m ² of soil in the case of seedling stage, and about 5 to 1000g can be used per 1m ² of soil in the case of nursery. In the case of forming a coating on seeds (including potato seeds, tubers, bulbs, and bulbs), the pulverized product may be used as it is or after being diluted with water or the like, at about 1 to 100g per 1kg of seeds.

In the present application, "control" means to prevent a crop (plant) from being infected with a bacterial plant disease fungus or the like that targets the plant, thereby avoiding the plant disease.

The application provides a Siamese bacillus (Bacillus siamensis) which is separated from pine wood nematode epidemic, wherein the Siamese bacillus (Bacillus siamensis) is named as Siamese bacillus (Bacillus siamensis) YG4-2. The strain is stored in China general microbiological culture Collection center (CGMCC) at the 3 rd month 22 of 2022, and has the address of North Star Xili No. 1,3 rd, china academy of sciences microbiological study, and the strain name of Bacillus siamensis and the storage number of CGMCC24564.

The Siamese bacillus (Bacillus siamensis) is a new species reported in 2010 and is first isolated from crabs salted in Thailand. Up to now there are few reports of strains belonging to this species, and a significant part of strains have been identified only by 16SrRNA gene sequence homology analysis and morphological features, thus having a large uncertainty in the complex taxonomy of bacillus. Only KCTC 13613T (AJVF 00000000), XY18 (LAGT 00000000), SRCM100169 (LYUE 01000000) strains of Siamella are inquired in a GenBank database, the whole genome data are supported in taxonomies, the homology of core genes is higher, and the method can be used as the most important basis for judging whether other strains are Siamella or not.

In terms of biocontrol, the Siamese bacillus exhibits the same excellent biocontrol ability as the Bacillus amyloliquefaciens and Bacillus bailii (Bacillus velezensis) which are similar to the Bacillus amyloliquefaciens and Bacillus bailii. The Siamese bacillus model strain KCTC 13613T is reported to inhibit rhizoctonia solani and micrococcus luteus (J Bacteriol 2012,194 (15): 4148-4149), the fermentation broth of Siamese bacillus L13 is reported to prepare a microbial seaweed fertilizer, the fermentation broth has a disinfecting effect on tobacco mosaic virus and a mung bean growth promoting effect (CN 201310165929.5), siamese bacillus LYLB4 is reported to prevent and treat pear ring rot and soft rot (CN201610048328. X), siamese bacillus FC12-05 is reported to antagonize phytophthora solani root rot (Siamese agriculture university 2012, 40:107-114), or Siamese bacillus is compounded with other bacillus to prepare a microbial compound fertilizer preparation (CN 201610718598.7). Furthermore, the use of Bacillus siamensis for the preparation of active products has been recently reported, e.g., bacillus siamensis FC12-05 produced an antimicrobial active product (possibly a protein) precipitated by a 20% ammonium sulfate saturation solution, and Bacillus siamensis FJAT-28592 produced antifungal lipopeptides consistent with iturin A at HPLC retention times (agricultural biotechnology journal 2016, 24:261-269).

According to the application, the method for separating the Siamese bacillus can be a method for screening new strains which is conventional in the art, for example, the separation method can comprise the steps that the Siamese bacillus (Bacillus siamensis) YG4-2 can be screened by the following method:

Separating strain, namely taking the epidemic wood pupa room from the forest farm of Qingyang county of Anhui province, scraping the xylem tissue on the surface of the pupa room by using a sterilizing blade, transferring the wood pupa room into PBST buffer solution, and placing the wood pupa room in 150rpm shaking culture for 20min. After the cultivation, 10. Mu.L of PBST bacterial suspension is sucked by a pipettor, uniformly smeared on the surface of a TSA culture medium and then placed in a 37 ℃ incubator for cultivation for 12 hours. When single colony growth is observed on the surface, a pipette head is used for picking the single colony, and the single colony is purified and cultured.

And (3) strain identification, namely putting 1.3mL of thallus culture solution into a centrifuge, centrifuging at 1000rpm for 1 minute, taking 1.0-1.2mL of supernatant, recovering the supernatant into a new centrifuge tube, centrifuging the supernatant at 15000rpm for 3 minutes, centrifuging, discarding the supernatant, adding 100 mu L MIGHTYPREP REAGENT for DNA, mixing, heating at 95 ℃ for 10 minutes, and centrifuging at 15000rpm for 2 minutes, wherein the supernatant is the PCR template. The PCR reaction was carried out in a 2X MIGHTYAMP BUFFER:12.5. Mu.L manner, 1. Mu.L of each of the primers (27F, 1492R), 1. Mu.L of the template, 0.5. Mu.L of MIGHTYAMP DNA Polymerase, 2.5. Mu.L of 10X Additive for HIGH SPECIFICITY, and 25. Mu.L of ddH 2O.

The DNA template identified by the bacterial strain 16S according to the present embodiment was prepared from MIGHTYPREP REAGENT for DNA (Takara) rapidly and with high quality. The 16SrDNA full length PCR amplification primers were synthesized by Beijing engine biosciences Co., ltd using 8F 5 '-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (SEQ ID NO: 4) and 1499R 5' -GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (SEQ ID NO: 5). PCR products were detected using a 1% agarose gel with a fragment size of 1500bp.

The 16SrDNA gene sequence of the Siamese bacillus (Bacillus siamensis) YG4-2 is shown as SEQ ID NO: 1:

SEQ ID NO:1

GCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAA ACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGT CGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGT GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTG GCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCC CCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAA CCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGA CCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCC AGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGC TTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACG CGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAG CCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCC GCTCGACTTGC

The application provides bacillus beleidstock (Bacillus velezensis), which bacillus beleidstock (Bacillus velezensis) is isolated from pine wood nematode epidemic wood, and the bacillus beleidstock (Bacillus velezensis) is named bacillus beleidstock (Bacillus velezensis) YY2P1. The strain is stored in China general microbiological culture Collection center (CGMCC) at day 22 of 3 months of 2022, the address is North Star Xili No.1, 3 of the Korean area of Beijing, the strain name is Bacillus velezensis, and the storage number is CGMCC24565.

Bacillus belicus (Bacillus velezensis) is a gram positive, rod-shaped, off-white bacterial colony, surface wrinkling, opaque, aerobic, chemoheterotrophic bacterium.

According to the present application, the method for isolating Bacillus belicus may be a method for screening a novel strain conventional in the art, for example, the isolation method may include that Bacillus belicus (Bacillus velezensis) YY2P1 may be screened by the following method:

And (3) strain identification, namely putting 1.3mL of thallus culture solution into a centrifuge, centrifuging at 1000rpm for 1 minute, taking 1.0-1.2mL of supernatant, recovering the supernatant into a new centrifuge tube, centrifuging the supernatant at 15000rpm for 3 minutes, centrifuging, discarding the supernatant, adding 100 mu L MIGHTYPREP REAGENT for DNA, mixing, heating at 95 ℃ for 10 minutes, and centrifuging at 15000rpm for 2 minutes, wherein the supernatant is the PCR template. The PCR reaction system was 2X MIGHTYAMP BUFFER:12.5. Mu.L, 1. Mu.L of each primer (27F, 1492R), 1. Mu.L of template, MIGHTYAMP DNAPOLYMERASE:0.5. Mu.L, 10X Additive for HIGH SPECIFICITY:2.5. Mu.L, and ddH2O to 25. Mu.L.

The DNA template identified by the bacterial strain 16S according to the present embodiment was prepared from MIGHTYPREP REAGENT for DNA (Takara) rapidly and with high quality. The PCR amplification primers for the full length of 16Sr DNA were 8F 5 '-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (SEQ ID NO: 4) and 1499R 5' -GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (SEQ ID NO: 5), and were synthesized by Beijing qing family Biotechnology Co., ltd. PCR products were detected using a 1% agarose gel with a fragment size of 1500bp.

The 16SrDNA gene sequence of the bacillus bailii (Bacillus velezensis) YY2P1 is shown as SEQ ID NO: 2:

SEQ ID NO:2

GCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACT CTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGA GTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTA GCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTC CGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCA CGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGA AGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC GGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCC CTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACC GCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAA GAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTG CCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTT CCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGC CTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCAC CCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCA CCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCC CGGTT

The application provides Serratia marcescens (SERRATIA MARCESCENS), which Serratia marcescens (SERRATIA MARCESCENS) is isolated from pine wood nematode epidemic, and the Serratia marcescens (SERRATIA MARCESCENS) is named Serratia marcescens (SERRATIA MARCESCENS) AHPC29. The strain is stored in China general microbiological culture Collection center (CGMCC) at the 3 rd month 22 of 2022, and has the address of North Star Xili No. 1,3 rd, china academy of sciences microbiological study, and the strain name of Bacillus siamensis and the storage number of CGMCC24567.

The "Serratia marcescens", also known as LING-BI, belongs to the genus Serratia of the family Enterobacteriaceae, and is a gram-negative facultative anaerobic bacillus. Serratia marcescens is a non-demanding microorganism, and various carbon sources such as chitosan, glucose, sucrose, mannose, cellobiose, citric acid glycerol and the like can be utilized, so that a wide carbon source spectrum provides favorable conditions for the fermentation production of the Serratia marcescens. The nuclease derived from Serratia marcescens (SERRATIA MARCESCENS) consists of 266 amino acids, unlike other gram-negative bacteria in which the signal peptide consisting of 1-21 amino acids helps the nuclease to secrete into the medium, other gram-negative bacteria proteins are secreted into the plasma membrane space rather than the surrounding medium, during secretion the signal peptide (1-21 amino acids) is cleaved off, and the 22-266 amino acid portion with hydrolytic nuclease activity is secreted into the medium with a molecular weight of about 26.7kDa.

The method for separating Serratia marcescens according to the present application may be a method for screening a novel strain conventionally used in the art, for example, the separation method may include that Serratia marcescens (SERRATIA MARCESCENS) AHPC29 is screened by the following method:

The 16SrDNA gene sequence of the serratia marcescens (SERRATIA MARCESCENS) AHPC29 is shown in SEQ ID NO: 3:

SEQ ID NO:3

TTCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTT CTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCG ACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGTACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATACGCCATT GTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCA TCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTT AACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGAT GTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTG CGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATC TCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTT CACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTT AGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTTCACCACCTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCT TCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATT CCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAG CCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAG CTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATT ACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCCGTGCT ACCGCTCGACTTGCATGTGTTAAAGGCCCCCCCCG

The application provides a microbial agent, which comprises one or more than two of bacillus siamensis, bacillus beijensis and serratia marcescens.

In some embodiments of the application, the bacterial agents include bacillus siamensis, bacillus behenensis and serratia marcescens.

In some embodiments of the application, the microbial inoculum consists of bacillus siamensis, bacillus behenensis and serratia marcescens.

In some embodiments of the present application, the bacillus siamensis is 1 to 10 parts by weight relative to the serratia marcescens and the bacillus behenryis is 1 to 10 parts by weight relative to the serratia marcescens;

for example, the siamese bacillus 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, relative to the serratia marcescens;

The bacillus belicus 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 with respect to the serratia marcescens.

In some embodiments of the application, the microbial inoculum is composed of bacillus siamensis (Bacillus siamensis) with a collection number of CGMCC24564, bacillus beijerinus (Bacillus velezensis) with a collection number of CGMCC24565 and Serratia marcescens (SERRATIA MARCESCENS) with a collection number of CGMCC 24567.

The application provides a pesticide, which comprises the microbial agent. In addition, the pesticide of the present application may also include other additional suitable pesticides.

Other suitable pesticides are bioactive compounds for controlling agricultural pests and include, for example, herbicides, plant growth regulators, crop desiccants, fungicides, bactericides, bacteriostats, insecticides, and insect repellents, as well as water soluble salts and esters thereof. Suitable pesticides include, for example, aryloxyphenoxy-propionate herbicides such as fluazifop-p-butyl, cyhalofop-butyl, and quizalofop-butyl, triazine herbicides such as metribuzin, hexazinone (hexaxinone), or atrazine, sulfonylurea herbicides such as chlorsulfuron, uracil such as cyprodinil, triclopyr, or terfenacet, urea herbicides such as linuron, diuron, cyclouron, or meturon, acetanilide herbicides such as alachlor, or metolachlor, thiocarbamate herbicides such as carbobenzoxazole, dicamba, oxadiazole (oxadiazolone) herbicides such as oxadiazon, isoxaflutole herbicides, Phenoxy carboxylic 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) butyric acid ("MCPB"), 2,4-D propionic acid, and 2-methyl-4-chlorophenoxypropionic acid, diphenyl ether herbicides such as fluazifop-p-butyl, acifluorfen, oxyfluorfen, dinitroaniline herbicides such as trifluralin, organophosphate herbicides such as glufosinate 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, Fluroxypyr, picloram, triclopyr, and aminopyralid. Suitable fungicides include, for example, nitriloxime fungicides, such as cymoxanil, imidazole fungicides, such as benomyl, carbendazim, or thiophanate-methyl, triazole fungicides, such as triazolone, sulfenamide fungicides, such as captan, dithiocarbamate fungicides, such as mancozeb, or thiram, chlorinated aromatic fungicides, such as triclosan, dichloroaniline fungicides, such as iprodione, strobilurin fungicides, such as kresoxim-methyl, trifloxystrobin, or azoxystrobin, chlorothalonil, copper salt fungicides, such as copper oxychloride, sulfur, aniline, and amide-based fungicides, such as metalaxyl or mefenoxam. suitable insecticides include, for example, carbamate insecticides, such as methoprene, amoenan, furadan, or aldicarb, organic thiophosphate insecticides, such as EPN, isopropamide, isoxazole phosphorus, chlorpyrifos, or chlorpyrifos, organic phosphate insecticides, such as terbufos, monocrotophos, or settop Luo Lin, perchlorinated organic insecticides, such as methoprene, 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 acaricides include, for example, propynyl sulfite acaricides such as propargite, triazapentadiene acaricides such as amitraz, chlorinated aromatic acaricides such as ethylacet acaricidal alcohol or tetracloxasulfone, and dinitrophenol acaricides such as le acaricides. Suitable nematicides include carbamate nematicides such as oxamyl.

Pesticidal compounds are generally referred to herein by the designation specified by the international standardization association (ISO). ISO common names may be cross-referenced with international union of pure and applied chemistry ("IUPAC") and chemical abstract service ("CAS") names by a number of sources.

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

In some embodiments of the application, the adjuvant is 0.5-10wt%, preferably 1-5wt%, based on the total weight of the pesticide;

for example, the adjuvant may be 0.5, 1,2, 3,4, 5,6, 7, 8, 9, 10wt% or any range therebetween, based on the total weight of the pesticide.

The application provides a method for promoting plant growth, which comprises applying the microbial agent or the pesticide to plants.

In some embodiments of the application, the plant is selected from one or more of pinus massoniana, pinus koraiensis, pinus alba, preferably pinus massoniana.

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

The application provides a method for preventing and controlling pine wood nematodes, which comprises the step of applying the microbial agent or the pesticide to plants.

The application provides a method for promoting the abundance of beneficial bacteria in plants, which comprises the step of applying the microbial agent or the pesticide to plants.

Wherein the parts by weight of the different strains can be calculated on the basis of methods known to the person skilled in the art, for example on the basis of the volume ratio with the same OD ₆₀₀.

Examples

Example 1 Siamese bacillus (Bacillus siamensis) YG4-2 screening and identification procedure

The 16SrDNA gene sequence of the Siamese bacillus (Bacillus siamensis) YG4-2 is shown as SEQ ID NO. 1.

The Siamese bacillus (Bacillus siamensis) YG4-2 is stored in the China general microbiological culture Collection center (CGMCC) at the date of 22, 3, 2022, and has the address of North Star Xiyu No. 1,3 in the Korean area of Beijing, the national academy of sciences of microbiology, strain name Bacillus siamensis and the storage number of CGMCC24564.

EXAMPLE 2 Bacillus bailii (Bacillus velezensis) YY2P1 screening and identification procedure

The 16SrDNA gene sequence of the Bacillus bailii (Bacillus velezensis) YY2P1 is shown as SEQ ID NO. 2.

The Bacillus belicus (Bacillus velezensis) YY2P1 is stored in the China general microbiological culture Collection center (CGMCC) at the position of North Star Xiyu No.1, 3 in the Korean area of Beijing, the strain name is (Bacillus velezensis) and the preservation number is CGMCC24565.

Example 3 Serratia marcescens (SERRATIA MARCESCENS) AHPC29 screening and identification procedure

The 16SrDNA gene sequence of the serratia marcescens (SERRATIA MARCESCENS) AHPC29 is shown in SEQ ID NO. 3.

Serratia marcescens (SERRATIA MARCESCENS) AHPC29 is stored in China general microbiological culture Collection center (CGMCC) at day 22 of 3 and 3 of 2022, and has the address of North Star Xidelu No. 1,3 of the Korean region of Beijing, the institute of microorganisms of the national academy of sciences, strain name Bacillus siamensis and the storage number of CGMCC24567.

Example 4 in vitro nematicidal test

Pine Wood Nematodes (PWN) were cultured and isolated by animal institute of China academy of sciences. Prior to testing, the pine wood nematodes were washed with 3% hydrogen peroxide for 10 minutes, then with sterilized 0.03M MgSO ₄. The effect of bacteria on the reproductive capacity of pine wood nematodes was tested in PDA plates incubated with b.cinerea. Control 1 was 300. Mu.L of PWN suspension (about 100 pieces of pine wood nematodes resuspended in 0.03M MgSO ₄), treatment was a mixture of 300. Mu.L of PWN suspension (about 100 pieces of pine wood nematodes resuspended in 0.03M MgSO ₄) with the Siamella Siamensis suspension obtained in example 1 (OD 600 = 0.5 of which) and a mixture of 700. Mu.L of PWN suspension (about 100 pieces of nematodes resuspended in 0.03M MgSO ₄) and the B.beijensis suspension obtained in example 2 (OD 600 = 0.5 of which) and 700. Mu.L of PWN suspension (about 100 pieces of nematodes resuspended in 0.03M ₄) were taken, and Serratia marcescens suspension (OD 600 = 0.5 of which) obtained in example 3 was taken, and after 7 days was extracted by a Belmannan technique of three-dimensional microscopic MgSO.

Wherein, bacillus siamensis in FIG. 1 represents a mixture of 300. Mu.L of PWN suspension (about 100 pine wood nematodes resuspended in 0.03M MgSO ₄) and the Siamese Bacillus suspension obtained in example 1 (OD 600 = 0.5 of the Siamese Bacillus suspension, 700. Mu.L);

Bacillus velezensis represents a mixture of 300 μl PWN suspension (about 100 nematodes resuspended in 0.03M MgSO ₄) and bacillus bailii suspension obtained in example 2 (this bacillus bailii suspension od600=0.5, 700 μl);

SERRATIA MARCESCENS represents a mixture of 300 μl PWN suspension (about 100 nematodes resuspended in 0.03M MgSO ₄) and serratia marcescens suspension obtained in example 3 (OD 600 = 0.5).

The nematicidal rate in fig. 1 refers to the ratio of the number of dead Pine Wood Nematodes (PWNs) to the total number of Pine Wood Nematodes (PWNs).

The results are shown in fig. 1, where Bacillus siamensis, bacillus velezensis, SERRATIA MARCESCENS bacteria have significant Pine Wood Nematode (PWN) killing activity.

EXAMPLE 5 in vivo nematode inhibition study

Pine Wood Nematodes (PWN) were cultured and isolated by animal institute of China academy of sciences. The temperature of the masson pine planting greenhouse is 30 ℃, the relative humidity is 80%, and the photoperiod is 16/8h (light/dark). The average height of pine is 50cm.

The experiment sets two treatments, namely treatment 1, inoculating PWN and root-irrigation pesticide simultaneously, and treatment 2, inoculating PWN only. The control group was a non-irrigated bacterial broth, a non-PWN inoculated masson pine. After 1 month, the PWN number was counted.

The PWN inoculation method is that a T-shaped incision is made at the stem 10cm away from the ground by a sterile knife blade, a small absorbent cotton ball is inserted, and 20 mu L of PWN suspension is dripped on the small absorbent cotton ball. Finally, the wound is sealed by a sealing film.

The preparation method of the pesticide comprises the steps of respectively adding the purified Siamese bacillus in example 1, the purified Bacillus belicus in example 2 and the purified Serratia marcescens in example 3 into a culture medium, wherein the culture time is 48 hours, the rotation speed is 200rpm, the Siamese bacillus fermentation broth in example 1, the Bacillus belicus fermentation broth in example 2 and the Serratia marcescens fermentation broth in example 3 after the culture are mixed according to the volume ratio of 1:1:1 when OD ₆₀₀ =0.5, and then adding an aqueous solution of an auxiliary agent, wherein the auxiliary agent consists of molasses powder, sodium nitrite, potassium sulfate, monoammonium phosphate, urea, wettable powder cuprous oxide, soapberry extract and avermectin, wherein the total weight of the aqueous solution of the auxiliary agent is calculated by mass percent, the molasses powder is 1%, the sodium nitrite is 0.1%, the potassium sulfate is 0.1%, the urea is 1%, the wettable powder is 0.1%, the soapberry extract is 0.01%.

The compositions of the culture mediums of the Siamese bacillus purified in the example 1, the Bacillus belicus purified in the example 2 and the Serratia marcescens purified in the example 3 are 1% of yeast extract, 1% of glucose, 0.02% of ammonium sulfate, 0.1% of amino acid, 0.03% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 0.03% of ferrous sulfate and 0.03% of shrimp meal, wherein the mass percentages of the compositions are calculated based on the total weight of the culture medium.

The root irrigation method is that a shovel is used to lightly expose part of root. 200mL of the fermentation broth was poured in a measuring cup and then backfilled. All plants were watered as needed.

The nematode amount counting method includes digging out the aerial part of tree seedling and cutting into 2cm long sections. It was then placed in a funnel with paper towels pre-placed and sterile water was added that had been diffused through the wood segments. The nematodes will automatically transfer into the water and sink to the bottom of the funnel. After 12 hours, PWN suspensions were collected and counted. At the same time, the wood slices were dried and the weight was calculated.

In FIG. 2, root irrigation+inoculation indicates the results of treatment 1, and inoculation alone indicates the results of treatment 2.

The amount of nematodes in fig. 2 refers to the number of Pine Wood Nematodes (PWN) per gram of wood.

The results are shown in figure 2, the pesticide can significantly reduce the amount of nematodes in plants.

Example 6 effect of microbial agents on plant endophyte bacterial community structure.

Root irrigation operations were as described in example 5:

The microbial community analysis steps are as follows:

1g of a stem sample of Pinus massoniana was weighed, washed first in 80% ethanol and 0.25% NaOCl to remove surface microorganisms, and then washed 3 times (1 minute each) in sterile water. Sawdust was scraped from the xylem using a sterile scalpel and forceps. All samples were placed into 2mL sterile centrifuge tubes and sequenced by the metage biology company.

As shown in FIG. 3, in treatment 1, the abundance of Bacillus was significantly increased, demonstrating that root irrigation by microbial agents significantly increased the number of beneficial bacteria in plants, while the number of associated pine wood nematodes, represented by Pantoea, was significantly decreased, indicating a shift in endophyte colony structure in the direction beneficial to plant growth.

The abundance of beneficial bacteria in the plant body is obviously increased after root irrigation.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Sequence listing

<110> Animal institute of China academy of sciences

<120> Siamese bacillus, bacillus bailii, serratia marcescens, microbial agent, pesticide and application thereof

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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 adjuvants.

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, comprising 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 the plant;

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.