CN117126757B

CN117126757B - Serratia marcescens, microbial agent, pesticide and application thereof

Info

Publication number: CN117126757B
Application number: CN202210577426.8A
Authority: CN
Inventors: 赵莉蔺; 张红霞; 王欣晨
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: 2025-01-28
Anticipated expiration: 2042-05-25
Also published as: CN117126757A

Abstract

本申请提供了一种粘质沙雷氏菌(Serratia marcescens)、一种肠球菌(Enterococcus sp.)、一种摩根菌(Morganella morganii)，本申请还提供了一种微生物菌剂，所述菌剂包括粘质沙雷氏菌、肠球菌和摩根菌中的一种或两种以上。可以适用于玉米苗期对草地贪夜蛾的预防和直接杀灭防控技术。The present application provides a Serratia marcescens, an Enterococcus sp., and a Morganella morganii. The present application also provides a microbial agent, which includes one or more of Serratia marcescens, Enterococcus sp., and Morganella morganii. The microbial agent can be applied to the prevention and direct killing of fall armyworms in the corn seedling stage.

Description

Serratia marcescens, microbial agent, pesticide and application thereof

Technical Field

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

Background

Spodoptera frugiperda (Spodoptera frugiperda), a major agricultural pest that newly invades our country, belongs to the genus spodoptera of the family spodoptera, also known as fall armyworm, is evaluated by international CABI as a ten-plant pest in the world. The pests are originally produced in tropical and subtropical areas of america, and rapidly spread and spread to 1366 counties (city and district) of 24 provinces in China after first discovery in Yunnan of China from 1 month 11 in 2019.

Spodoptera frugiperda has strong host adaptation, and larvae of spodoptera frugiperda can eat more than 80 crop species plants such as corn, rice, peanut and the like, so that serious damage is caused. In the prior art, the prevention and control of spodoptera frugiperda mainly adopts a chemical prevention and control mode, has the risks of pesticide residue, drug resistance and the like, and can provide an effective way for realizing efficient, green and continuous prevention and control of spodoptera frugiperda by screening more novel strain resources for inhibiting spodoptera frugiperda egg hatching and larva death. The bacterial microbial agents screened at present are mainly bacillus thuringiensis and cryptobacter breve, and the demands for production prevention and control application are far from enough.

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

Disclosure of Invention

The present application aims to solve the above problems, and a first object is to provide Serratia marcescens SERRATIA MARCESCENS.

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

A third object of the present application is to provide a pesticide or plant growth promoting agent.

A fourth object of the present application is to provide a pesticide or plant growth promoting agent for use.

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

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

2. The Serratia marcescens according to item 1, wherein the 16S rDNA gene sequence is shown in SEQ ID NO. 1.

3. An Enterococcus sp, with a preservation number of CGMCC24566.

4. The enterococcus strain according to item 3, wherein the 16S rDNA gene sequence is shown in SEQ ID NO. 2.

5. Morganella morganii (Morganella morganii) has a preservation number of CGMCC24593.

6. The Morganella according to item 5, wherein the 16S rDNA gene sequence is shown in SEQ ID NO. 3.

7. A microbial agent is prepared from the raw materials including edible fungus, the microbial inoculum comprises one or more than two of Serratia marcescens, enterococcus and Morganella morganii;

preferably, the microbial agents include Serratia marcescens, enterococci and Morganella morganii;

Further preferably, the microbial agent consists of Serratia marcescens, enterococcus and Morganella morganii;

more preferably, the Serratia marcescens is 1-10 parts by weight relative to the enterococcus and the Morganella morganii is 1-10 parts by weight relative to the enterococcus;

more preferably, the microbial inoculum is composed of Serratia marcescens with the preservation number of CGMCC24567, enterococcus with the preservation number of CGMCC24566 and Morganella morganii with the preservation number of CGMCC 24593.

8. A pesticide or plant growth promoting agent comprising the microbial agent of claim 7.

9. The agricultural chemical or plant growth promoter according to item 8,

The pesticide or plant growth promoting agent also comprises an auxiliary agent;

Preferably, the auxiliary agent is selected from one or more than two of methyl methacrylate and methacrylic acid copolymer nano-particles, nano-gold particles, urea, wettable powder cuprous oxide, ferrous sulfate, sodium alkyl sulfonate and soapberry extract, preferably methyl methacrylate and methacrylic acid copolymer nano-particles;

Further preferably, the auxiliary agent is 0.1-10wt% based on the total weight of the pesticide or plant growth promoting agent.

10. The pesticide or plant growth promoting agent according to item 8 or 9, which comprises Serratia marcescens.

11. A method of promoting plant growth or controlling spodoptera frugiperda or promoting the self-phase stunting of spodoptera frugiperda larvae, comprising applying the microbial agent of item 7 or the pesticide of item 8 or 9 to a plant or promoting plant growth;

preferably, the plant is selected from one or more of corn, rice, sorghum, barley, cotton, canola, leguminous plants, preferably corn;

the application method is root irrigation and/or spraying.

ADVANTAGEOUS EFFECTS OF INVENTION

1. The application separates and obtains a serratia marcescens, researches and discovers that the serratia marcescens has preventive and insecticidal effects on spodoptera frugiperda, tests and synergism improvement are carried out on the effect of directly killing spodoptera frugiperda on the basis, and finally the insecticidal effects of enterococcus and Morganella morganii, the synergism of serratia marcescens and the optimal preparation method and application mode of microbial agents are determined.

2. Serratia marcescens also has certain effect of promoting plant growth. The microbial pesticide disclosed by the application is nontoxic and pollution-free, is not easy to generate drug resistance, has high biological safety, and is suitable for the technology of preventing and directly killing spodoptera frugiperda in the seedling stage of crops.

3. The application confirms that Serratia marcescens strain extracted from insect intestinal tracts can improve the growth of corn and the prevention effect of the Serratia marcescens strain on spodoptera frugiperda, and on the basis, the effect of directly killing spodoptera frugiperda is tested and synergistically improved, and finally, the configuration of microbial agents, methyl methacrylate and methacrylic acid copolymer nano particles is determined.

4. The application provides a microbial agent or pesticide with remarkable insecticidal effect. The application is suitable for the technology of preventing and directly killing spodoptera frugiperda in the maize seedling stage.

Preservation information

Serratia marcescens (SERRATIA MARCESCENS) AHPC29 of the application is preserved in China general microbiological culture Collection center (CGMCC) at the 3 rd month 22 year 2022, and has the address of North Star XILU No. 1,3 rd, the institute of microbiology, academy of sciences of Beijing, the strain name SERRATIA MARCESCENS AHPC, and the preservation number of CGMCC24567.

The Enterococcus (Enterococcus sp.) 10A is preserved in China general microbiological culture Collection center (CGMCC) at day 3 and day 22 of 2022, and has the address of the No. 3 of West-way No.1 of the Korean area North Star of Beijing, the strain name of the institute of microorganisms of the national academy of sciences of China is Enterococcus sp.10A, and the preservation number of the strain is CGMCC24566.

Morganella morganii (Morganella morganii) CDHu is preserved in China general microbiological culture Collection center (CGMCC) in 3 and 25 days of 2022, and has an address of China national academy of sciences microbiological study, strain name Morganella morganii CDHu and a preservation number of CGMCC24593, and is North Star, west Lu No.1, no. 3, in the Korean area of Beijing.

Drawings

FIG. 1 is a graph showing the effect of different temperatures on Serratia marcescens strain growth in example 2;

FIG. 2 is a graph showing the effect of different incubation times on Serratia marcescens strain growth in example 2;

FIG. 3 is a graph showing the effect of Serratia marcescens on maize seedling height in example 5;

FIG. 4 is a graph showing the effect of Serratia marcescens on leaf number in maize seedlings in example 5;

FIG. 5 is a graph showing the effect of Serratia marcescens on leaf surface width of maize seedlings in example 5;

FIG. 6 is a graph showing the ratio of the number of leaves taken by Spodoptera frugiperda from maize seedlings treated with Serratia marcescens in example 6;

FIG. 7 is a graph showing the ratio of the leaf surfaces of corn seedlings treated with Serratia marcescens to that of spodoptera frugiperda in example 6;

FIG. 8 is a graph showing that Serratia marcescens reduces mortality of spodoptera frugiperda on corn seedlings in example 6;

FIG. 9 shows the synergistic effect of various adjuvants on Serratia marcescens for crop growth in example 7;

FIG. 10 shows the synergistic effect of different concentrations of methyl methacrylate and methacrylic acid copolymer nanoparticles on the microbial agent in example 8;

FIG. 11 is a graph showing the direct insecticidal effect of Serratia marcescens, enterococcus and Morganella on Spodoptera frugiperda in example 9;

FIG. 12 is a graph showing the effect of various formulation ratios of the microbial agents of example 10 on mortality of Spodoptera frugiperda;

FIG. 13 shows the effect of different formulation ratios of the microbial agents of example 11 on the rate of autoclaving of Spodoptera frugiperda.

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.

Specific examples of plants to be applied with the agricultural chemical of the present application include: cereal (e.g., rice, wheat, barley, rye, oat, corn, sorghum, millet, yu gu, finger, buckwheat), potato (e.g., potato, sweet potato, taro, yam, konjac), bean (e.g., soybean, red bean, kidney bean, pea, broad bean, peanut, cowpea, chickpea, pigeon pea), vegetable (e.g., eggplant, tomato, green pepper, capsicum, cucumber, melon, watermelon, pumpkin, zucchini, melon, cucurbit, winter melon, balsam pear, cabbage, broccoli, cauliflower, white radish, turnip, chinese cabbage, pinus koraiensis, waterweed, onion, leek, garlic (GARLIC CHIVE), asparagus, lettuce, burdock, garland chrysanthemum, petunia, carrot, cress, celery, parsley, strawberry, spinach, okra, perilla, basil, peppermint, taro, mioga), fruit tree (e.g., apples, pears, american pears, quince, papaya, sweet cherries, peaches, plums, apricots, chestnuts, walnuts, almonds, pecans, grapes, kiwi fruits, akebia stem, persimmons, figs, pomegranates, raspberries, blackberries, blueberries, cranberries, oranges, loquat, olives, waxberries, mangoes, guava, avocado, date palm, coconut, bananas, pineapple, papaya, passion fruit, acerola), specialty crops (e.g., cotton, flax, rush, rapeseed, sunflower, sesame, palm oil, beet, sugarcane, tea, coffee, cocoa, hops, tobacco), flowers (e.g., bust, morning glory, marigold, hops, tobacco), garden balsam, star-of-the-world, vanilla, falcate, chrysanthemum, carnation, tulip, lily, narcissus, gladiolus, cyclamen, begonia, water lily, marble, rose, cymbidium, jidelia), turf grass (e.g., zoysia gracilis, korean grass, zoysia japonica, bermuda grass, furfuryl, festuca, ryegrass, bluegrass), trees (e.g., cherry, azalea, oak, beech, cypress, beech), and the like, but are not limited to these examples. In addition, it can be applied to plants which have been imparted with resistance to plant diseases and insect pests, resistance to herbicides, resistance to environmental stresses such as desiccation, etc. by conventional breeding methods, genetic recombination techniques, etc.

In the present application, the plant is preferably one or more of corn, rice, sorghum, barley, cotton, canola, and leguminous plants, and most preferably corn.

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 present application provides Serratia marcescens (SERRATIA MARCESCENS) isolated from an insect gut microorganism, designated Serratia marcescens (SERRATIA MARCESCENS) as Serratia marcescens (SERRATIA MARCESCENS) AHPC29. The strain is preserved 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 SERRATIA MARCESCENS AHPC and the preservation 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:

Sequentially rinsing larvae of intestinal bacteria in 75% alcohol and sterile water for about 3 seconds in an ultra-clean bench, slowly dissecting the larva body wall by using sterile dissecting scissors, taking out the intestinal tracts, putting into a sterile centrifuge tube, adding 1mL of sterile water for grinding, diluting the grinding liquid by 10 ⁶, taking 10 mu L of diluent, coating the diluent, and placing the flat plate in a 28 ℃ incubator for dark culture for 2-3 days.

The 16SrDNA gene sequence of the serratia marcescens (SERRATIA MARCESCENS) AHPC29 is shown in SEQ ID NO: 1:

SEQ ID NO:1

TTCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGTACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATACGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTTCACCACCTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCCGTGCTACCGCTCGACTTGCATGTGTTAAAGGCCCCCCCCG

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 full length PCR amplification primers of 16S rDNA were synthesized by Beijing engine biotechnology Co., ltd using 8F 5 '-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (SEQ ID NO: 4) and 1499rR 5' -GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (SEQ ID NO: 5) (Weisburg et al, 1991). PCR products were detected using a 1% agarose gel with a fragment size of 1500bp.

The application provides an Enterococcus sp, which is isolated from the intestinal tract of insects, the Enterococcus sp is named Enterococcus sp 10A. The strain is preserved in China general microbiological culture Collection center (CGMCC) at the 3 rd month 22 of 2022, and has the address of North Star Xiyu No. 1,3 rd, china academy of sciences microbiological study, and the name of Enterococcus sp.10A, and the preservation number of CGMCC24566.

The method of isolating the enterococci according to the present application may be a method of screening a novel strain conventional in the art, and for example, the isolation method may include that enterococci (Enterococcus sp.) 10A may be screened by:

The 16S rDNA gene sequence of the Enterococcus sp.10A is shown in SEQ ID NO. 2:

SEQ ID NO:2

AAAAAATCCAGTGCGGGTGCTATACATGCAAGTCGAACGCTTCTTTCCTGCCGAGTGCTTGCACTCACTTGGAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAATAGTCGACACCGCATGGTGTTGATTTGAAAGACGCTTTCGGGTGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCATGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGTAGGAGAGTAACTGCTCTTGCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCTAGGCCGCGAGGTCATGCAAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGAAGAAAATTCTC

The present application provides a Morganella (Morganella morganii), said Morganella (Morganella morganii) being isolated from the insect gut, the Morganella (Morganella morganii) being designated Morganella (Morganella morganii) CDHu. The strain is stored in China general microbiological culture Collection center (CGMCC) at 25 days of 3 months of 2022, and has the address of North Star Xiyu No. 1, 3 of the Korean area of Beijing, the national academy of sciences of China, and the strain name of Morganella morganii CDHu and the storage number of CGMCC24593.

Morganella morganii (Morganella morganii for short) is a facultative anaerobe, and is oxidase negative. The colony is in an off-white opaque color, and the Morganella morganii is in a straight rod shape when grown on an agar plate, and has a diameter of about 06-07 micrometers and a length of about 10-17 micrometers. It moves by means of Zhou Mao flagella but some strains do not form flagella at 30 ℃.

The method for isolating Morganella according to the present application may be a method for screening a novel strain conventional in the art, for example, the isolation method may comprise that Morganella (Morganella morganii) CDHu may be screened by the following method:

The 16S rDNA gene sequence of the Morganella morganii (Morganella morganii) CDHu is shown in SEQ ID NO: 3:

SEQ ID NO:3

ACCGCTTTTTATCACAAAGTGGTAAGCGCCCTCCCGGAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGTACTTTATGAGTTCCGCTTGCCCTCGCGGGGTCGCTTCCCTTTGTATACGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCGCCATCACGCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGGTGCATCGAATTAAACCACAAGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCACATCTCTACGCATTTCACCGCTACACATGGAATTCTACCCCCCTCTACAAGACTCTAGCTGACCAGTATCAGATGCAATTCCCGGGTTAAGCCCGGGGATTTCACATCTGACTCAATCAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCCGATTAACGCTTGCAACCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGTCGGTAACGTCAATTGCCAAGGTTATTAACCTTGACACCTTCCTCCCGACTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTAAGCCGTTACCTCACCTACTAGCTAATCCCATATGGGTTCATCTGATGGCGCGAGGCCCGGAGGTCCCCCGCTTTGGTCCGAAGACATTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCGCCATCAGGCAGATCCCCATACATTACTCACCCGTCCGCCGCTCGTCAGCAGAGAAGCAAGCTTCTCCCTGTTACCGCCCGACTTGCATGTGTTAGCTGCCGCAAGTTCC

The DNA template identified by the bacterial strain 16S according to the present example was prepared from MIGHTYPREP REAGENT for DNA (Takara) rapidly and with high quality. The full length PCR amplification primers of 16S rDNA were synthesized by Beijing engine biotechnology Co., ltd using 8F 5 '-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG) (SEQ ID NO: 4) and 1499rR 5' -GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT) (SEQ ID NO: 5) (Weisburg et al, 1991). PCR products were detected using a 1% agarose gel with a fragment size of 1500bp.

The application provides a microbial agent, which comprises one or more than two of Serratia marcescens, enterococcus and Morganella.

In some embodiments of the application, the bacterial agents include Serratia marcescens, enterococci and Morganella morganii.

In some embodiments of the application, the microbial agent consists of Serratia marcescens, enterococcus and Morganella morganii.

In some embodiments of the application, calculated as parts by weight, wherein parts by weight are calculated based on the volume ratio of the equivalent OD ₆₀₀, the serratia marcescens is 1-10 parts relative to the enterococcus and the morganella morganii is 1-10 parts relative to the enterococcus;

For example, the Serratia marcescens may be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any range therebetween, relative to the enterococcus;

The Morganella 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 enterococcus.

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.

In some specific embodiments of the application, the microbial inoculum consists of Serratia marcescens with the preservation number of CGMCC24567, enterococcus with the preservation number of CGMCC24566 and Morganella morganii with the preservation number of CGMCC 24593.

The application also 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 application, the pesticide further comprises an adjuvant.

In some embodiments of the present application, the auxiliary agent is selected from one or more than two of methyl methacrylate and methacrylic acid copolymer nanoparticles, gold nanoparticles, urea, wettable powder cuprous oxide, ferrous sulfate, sodium alkyl sulfonate, soapberry extract, preferably methyl methacrylate and methacrylic acid copolymer nanoparticles.

In some embodiments of the application, the adjuvant is 0.1-10wt% based on the total weight of the pesticide;

for example, the adjuvant may be 0.1, 1, 2, 3,4, 5, 6, 7, 8, 9, 10wt% or any range therebetween, based on the total weight of the pesticide.

The application also provides a plant growth promoting agent, which comprises the microbial agent.

In some embodiments of the application, the plant growth promoting agent further comprises an adjuvant.

In some embodiments of the application, the adjuvant is 0.1-10wt% based on the total weight of the plant growth promoting agent;

For example, the adjuvant may be 0.1, 1,2, 3,4,5, 6, 7, 8, 9, 10wt% or any range therebetween, based on the total weight of the plant growth promoting agent.

In some embodiments of the application, the plant growth promoting agent comprises Serratia marcescens.

The application also provides a preparation method of the Serratia marcescens, which comprises the following steps:

Fermenting Serratia marcescens strain in culture medium by liquid fermentation method to obtain fermentation liquid, controlling fermentation temperature at 28-30deg.C for 36-48 hr, and preparing the fermentation liquid into water aqua.

The application also provides a preparation method of the enterococcus, which comprises the following steps:

The method comprises the steps of fermenting enterococcus strain in a culture medium by using a liquid fermentation method to obtain fermentation liquor, controlling the fermentation temperature to be 28 ℃ and the fermentation time to be 48 hours, and preparing the fermentation liquor into a water agent.

The application also provides a preparation method of the Morganella morganii, which comprises the following steps:

Fermenting Morganella morganii strain in a culture medium by using a liquid fermentation method to obtain fermentation liquor, controlling the fermentation temperature to be 28 ℃ and the fermentation time to be 48 hours, and then preparing the fermentation liquor into a water agent.

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 corn, rice, sorghum, barley, cotton, canola, leguminous plants, preferably corn.

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

The application provides a method for controlling spodoptera frugiperda, which comprises applying the microbial agent or the pesticide to plants.

The application provides a method for promoting the self-phase killing of spodoptera frugiperda larvae, which comprises the step of applying the microbial agent or the pesticide to plants.

Examples

EXAMPLE 1 Serratia marcescens (SERRATIA MARCESCENS) AHPC29 screening and identification procedure

When single colony grows on the culture medium, a small amount of hypha is picked up by a sterile toothpick to be continuously cultured on a flat plate to obtain a purified strain. The obtained pure strains were classified, numbered and counted according to colony size, color, thickness, transparency and texture on the same medium. The single colony of the strain is red in color. After purification, red single colonies are picked up, genomic DNA is extracted and used as a template for PCR amplification and sequencing. The sequencing results were aligned with the model strain in EzBioCloud database and the sequences in GenBank database, respectively, and phylogenetic tree construction was performed to make sure that the isolated strain belongs to Serratia marcescens (SERRATIA MARCESCENS).

The strain obtained by the screening is named as Serratia marcescens (SERRATIA MARCESCENS) AHPC29.

The 16S rDNA gene sequence of the strain obtained by screening is shown as SEQ ID NO. 1.

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 edition of the Korean area of Beijing, the strain name of SERRATIA MARCESCENS AHPC, and the storage number of CGMCC24567.

Example 2

The optimal fermentation temperature and fermentation time of the Serratia marcescens strain isolated in example 1 were determined.

Culturing Serratia marcescens strain at 25deg.C, 28deg.C, 30deg.C, 35deg.C and 37deg.C for 48 hr, OD600 was measured. The results in FIG. 1 show that the fermentation temperature directly affects the fermentation rate, and that all of 25 ℃, 35 ℃ and 37 ℃ have an inhibitory effect on Serratia growth, the most intense of which is 35 ℃, the OD600 value at this temperature is only 0.228, while the most favorable Yu Sha Rayleigh growth occurs at a fermentation temperature of 30 ℃, at which the OD600 value reaches 0.479. The optimum fermentation temperature thus selected was 30 ℃.

Wherein the culture medium comprises 10g/L tryptone, 5g/L yeast extract and 5g/L sodium chloride.

Serratia marcescens strains are cultured for 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours in LB culture medium at 30 ℃, bacterial liquid OD600 is measured, and the influence of different fermentation time on bacterial growth is detected. As shown in FIG. 2, the growth of Serratia marcescens is regulated slowly in 12-24 hr, and the growth of Serratia marcescens is started slowly after 48 hr in logarithmic growth phase in 24-48 hr. Therefore, in the actual production process, the optimal fermentation time is selected to be 36h and 48h for saving the fermentation time.

EXAMPLE 3 Enterococcus (Enterococcus sp.) 10A screening and identification procedure

When single colony grows on the culture medium, a small amount of hypha is picked up by a sterile toothpick to be continuously cultured on a flat plate to obtain a purified strain. The single colony of the strain is yellowish, moist, opaque and circular. After purification, single colony is selected, genomic DNA is extracted and used as a template for PCR amplification and sequencing. The sequencing results were aligned with the model strain in EzBioCloud database and the sequences in GenBank database, respectively, and phylogenetic tree construction was performed to see that the isolated strain belongs to Enterococcus sp.

The strain obtained by screening was designated as 10A.

The 16S rDNA gene sequence of the strain obtained by screening is shown as SEQ ID NO. 2.

The Enterococcus (Enterococcus sp.) 10A is preserved in China general microbiological culture Collection center (CGMCC) at day 22 of 3 and 2022, and has the address of the Ministry of Kogyo North Star, west Lu No. 1 and 3 of Beijing, the institute of microbiology, china academy of sciences, and the strain name of the Enterococcus sp.10A, and the preservation number of the Enterococcus strain CGMCC24566.

EXAMPLE 4 Morganella morganii (Morganella morganii) CDHu screening and identification procedure

When single colony grows on the culture medium, a small amount of hypha is picked up by a sterile toothpick to be continuously cultured on a flat plate to obtain a purified strain. The single colony of the strain is a wet, convex, non-spreading growth and gray colony. After purification, single colony is selected, genomic DNA is extracted and used as a template for PCR amplification and sequencing. The sequencing results were aligned with the model strain in EzBioCloud database and the sequences in GenBank database, respectively, and phylogenetic tree construction was performed to see that the isolated strain belongs to Morganella (Morganella morganii).

The strain obtained by screening is named CDHu.

The 16S rDNA gene sequence of the strain obtained by screening is shown as SEQ ID NO. 3.

The Morganella morganii (Morganella morganii) CDHu is preserved in China general microbiological culture Collection center (CGMCC) at 3/25/2022, and has an address of North Star Xiyu No. 1/3 in the Korean area of Beijing, a strain name of Morganella morganii CDHu and a preservation number of CGMCC24593.

Example 5

The growth promoting effect of Serratia marcescens (SERRATIA MARCESCENS) (designated Serratia marcescens treatment) isolated in example 1 on maize seedlings was studied, and a control strain was made with the isolated pale bacteria (Pseudochrobactrum sp.) (designated pale bacteria treatment), and control group 1 was distilled water sprayed or root irrigated on maize seedlings. The pseudopallidum (Pseudochrobactrum sp.) is commercially available.

Culturing Serratia marcescens and the pseudo pallor fungus of the comparison strain separated in the example 1 in LB culture medium at 28 ℃ for 48 hours, respectively taking 20mL of fungus liquid, adding 200mL of distilled water to spray or root-irrigate the maize seedlings which have just germinated, spraying 10mL of each maize seedling which has just germinated for 10 times, repeating the steps, counting the plant height, the leaf number and the leaf width of the maize seedlings every other week, and continuously counting for three weeks. Fig. 3 shows the effect of spraying or root-drenching serratia marcescens on the plant height of the maize seedlings after 1-3 weeks, fig. 4 shows the effect of spraying or root-drenching serratia marcescens on the leaf number of the maize seedlings, and fig. 5 shows the effect of spraying or root-drenching serratia marcescens on the leaf width of the maize seedlings.

In fig. 3 to 5, serratia marcescens spray treatment is denoted serratia marcescens spray, serratia marcescens root irrigation treatment is denoted serratia marcescens root irrigation, pseudo-pallia marcescens spray treatment is denoted pseudo-pallia marcescens spray, pseudo-pallia marcescens root irrigation treatment is denoted pseudo-pallia marcescens root irrigation, and control group 1 is distilled water spray and root irrigation.

As can be seen from FIG. 3, the plant heights of the corn seedlings after spraying or root irrigation Serratia marcescens for 1-3 weeks are higher than those of the control group 1 and the pseudo-pale bacteria treatment group, as can be seen from FIG. 4, the leaf numbers of the corn seedlings sprayed or root irrigation Serratia marcescens are obviously higher than those of the control group 1 and the pseudo-pale bacteria treatment group, and as can be seen from FIG. 5, the leaf widths of the corn seedlings of root irrigation Serratia marcescens are obviously higher than those of the control group 1 and the pseudo-pale bacteria treatment group.

In conclusion, serratia marcescens strain has potential of being developed into a crop fattening microbial preparation and can be used as a plant growth promoting agent.

Example 6

The control effect and effect of Serratia marcescens (SERRATIA MARCESCENS) (designated Serratia marcescens treatment) on spodoptera frugiperda isolated in example 1 was compared with that of the control strain (Pseudochrobactrum sp.) (designated as pseudo-pallidum treatment), and the control group 2 was corn seedling spray or root irrigation distilled water.

After Serratia marcescens and pseudo-pale bacteria separated in the example 1 are cultured in LB culture medium at 28 ℃ for 48 hours, 20mL of bacterial liquid is respectively taken, 200mL of distilled water is added to carry out spraying or root irrigation on maize seedlings which are just germinated, 10mL of each maize seedling which are just germinated is sprayed, 10 times of repetition are carried out, after three weeks, 5 heads of spodoptera frugiperda are connected, the damage degree of the maize seedlings, including the number of leaves fed by spodoptera frugiperda, the leaf surface size and the death rate of the maize seedlings, is observed. Fig. 6 shows the leaf number to whole leaf ratio indicating that corn leaf is eaten, fig. 7 shows the leaf area to whole leaf area ratio of corn leaf is eaten, and fig. 8 shows mortality after spodoptera frugiperda is inoculated to a corn plant inoculated with serratia marcescens.

In fig. 6 to 8, serratia marcescens spray treatment is designated serratia marcescens spray, serratia marcescens root irrigation treatment is designated serratia marcescens root irrigation, pseudo-pallidum spray treatment is designated pseudo-pallidum spray, pseudo-pallidum root irrigation treatment is designated pseudo-pallidum root irrigation, and control group 2 is distilled water spray and root irrigation.

As shown in FIG. 6, the ratio of the number of the leaves taken by the corn leaves inoculated with Serratia marcescens to the number of the whole leaves is smaller than that of the treated group and the control group 2, as shown in FIG. 7, the ratio of the leaf area taken by the corn leaves inoculated with Serratia marcescens to the whole leaf area is smaller than that of the treated group and the control group 2, and as shown in FIG. 8, the death rate of the corn plants inoculated with Serratia marcescens after being inoculated with spodoptera frugiperda is significantly reduced compared with that of the treated group and the control group 2.

In conclusion, the damage of spodoptera frugiperda to corn plants inoculated with Serratia marcescens is significantly reduced, and the Serratia marcescens strain has the potential to be developed into a microbial pesticide for preventing and controlling spodoptera frugiperda.

As can be seen from examples 5 and 6, under two different application measures of root irrigation and spraying of the maize seedlings which have just germinated with the bacterial liquid, the growth condition of the maize seedlings is compared with the damage condition of spodoptera frugiperda after the seedlings are inoculated, and the results show that the maize plant height, the leaf number and the leaf width of the serratia marcescens root irrigation treatment group are larger than those of the serratia marcescens spraying group, and the harm reduction condition of the spodoptera frugiperda is better than that of the spraying group. Therefore, serratia marcescens is preferably root irrigation application mode in the application of promoting crop growth and preventing spodoptera frugiperda.

Example 7

After Serratia marcescens strain is cultured for 48 hours in LB culture medium at 30 ℃, distilled water is added according to the proportion of 1:10 (according to the volume ratio of Serratia marcescens solution to water being 1:10), then methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (marked as aqueous solution of auxiliary 1) or gold nanoparticle aqueous solution (marked as aqueous solution of auxiliary 2) is added to prepare pesticide, and the final concentration of the auxiliary 1 or the auxiliary 2 is 1% based on the total weight of the pesticide by mass percent, and the control group 3 is only added with distilled water.

In FIG. 9, the mixture containing only Serratia marcescens liquid and water is designated Serratia marcescens+water, the mixture containing Serratia marcescens liquid, water and adjuvant 1 is designated Serratia marcescens+adjuvant 1, and the mixture containing Serratia marcescens liquid, water and adjuvant 2 is designated Serratia marcescens+adjuvant 2, and the control group 3 contains distilled water only.

10ML of the root of each maize seedling which has just germinated is irrigated, the method is repeated 5 times, the maize plant height is compared statistically after 20 days, and the effect of the auxiliary agent in the application of promoting the growth of crops is shown in FIG. 9.

As can be seen from fig. 9, the best adjuvant for Serratia in the crop growth promotion application is methyl methacrylate and methacrylic acid copolymer nanoparticles (i.e., adjuvant 1).

Example 8

After Serratia marcescens strains are cultured for 48 hours in LB culture medium at 30 ℃, distilled water (marked as Serratia marcescens plus water) is directly added according to the proportion of 1:10 (the volume ratio of Serratia marcescens liquid to water is 1:10), aqueous solutions of methyl methacrylate and methacrylic acid copolymer nano particles with different concentrations (namely aqueous solution of auxiliary 1) are added to prepare pesticides, and the final concentrations of the methyl methacrylate and methacrylic acid copolymer nano particles are respectively 10% (marked as Serratia marcescens plus auxiliary 1 percent), 1% (marked as Serratia marcescens plus auxiliary 11 percent) and 0.1% (marked as Serratia marcescens plus auxiliary 1.1 percent) based on the total weight of the pesticides, and the control group 4 is only added with distilled water. 10mL of corn seedling roots which are just germinated are irrigated for 5 times, and the corn plant heights are counted and compared after 20 days. Figure 10 shows the effect of different methyl methacrylate and methacrylic acid copolymer nanoparticle concentrations on promotion of crop growth.

In fig. 10, the mixture containing only Serratia marcescens solution and water is referred to as Serratia marcescens+water, the mixture containing Serratia marcescens solution, water and adjuvant 1 (the final concentration of adjuvant 1 is 10% based on the total weight of the pesticide) is referred to as Serratia marcescens+adjuvant 1.10%, the mixture containing Serratia marcescens solution, water and adjuvant 1 (the final concentration of adjuvant 1 is 1% based on the total weight of the pesticide) is referred to as Serratia marcescens+adjuvant 1 1%, the mixture containing Serratia marcescens solution, water and adjuvant 1 (the final concentration of adjuvant 1 is 0.1% based on the total weight of the pesticide) is referred to as Serratia marcescens+adjuvant 1.1%, and the control group 4 contains only distilled water.

As can be seen from fig. 10, the concentration of the methyl methacrylate and methacrylic acid copolymer nanoparticles is 10%, 1% and 0.1% by mass based on the total weight of the pesticide, and the concentration of the methyl methacrylate and methacrylic acid copolymer nanoparticles is 1% by mass based on the total weight of the pesticide.

Example 9

The control potential of Spodoptera frugiperda, i.e., the direct insecticidal effect on Spodoptera frugiperda, was studied using Serratia marcescens (SERRATIA MARCESCENS) isolated in example 1 and Enterococcus sp isolated in example 3 and Morganella morganii (Morganella morganii) isolated in example 4.

Three bacteria of Serratia marcescens (SERRATIA MARCESCENS), enterococcus sp and Morganella morganii Morganella morganii are respectively cultured for a plurality of days under the condition of LB culture medium of 30 ℃, and then are respectively prepared into bacterial liquid with a ratio of 99:1 with aqueous solution of methyl methacrylate and methacrylic acid copolymer nano particles (auxiliary 1) (the concentration of the methyl methacrylate and the methacrylic acid copolymer nano particles is 1 mg/mL) to prepare pesticide, wherein the final concentration of the auxiliary 1 is 1 percent based on the total weight of the pesticide. Spodoptera frugiperda larvae were sprayed, 100 μl of each spodoptera frugiperda was sprayed, and three groups of experiments were repeated for each strain, each group of spodoptera frugiperda larvae, 6 heads. The control group 5 was sprayed with an aqueous solution of clear water and a mixture of methyl methacrylate and methacrylic acid copolymer nanoparticles in a mass ratio of 99:1. After 12 hours of spraying treatment, the mortality rate of spodoptera frugiperda was counted. FIG. 11 shows mortality of spodoptera frugiperda larvae after spraying with Serratia marcescens (SERRATIA MARCESCENS), enterococcus sp. And Morganella morganii Morganella morganii.

In FIG. 11, serratia marcescens and aid 1 spray-treated Serratia marcescens, corresponding control group indicated enterococcus with water and aid 1 spray-treated, corresponding control group indicated Morganella morganii with aid 1 spray-treated, and corresponding control group 5 indicated Morganella morganii with water and aid 1 spray-treated.

As can be seen from fig. 11, the mortality of spodoptera frugiperda larvae after spraying with serratia marcescens, enterococcus and morganella was significantly higher than that of control group 5.

Example 10

Example 1 three bacteria of Serratia marcescens (SERRATIA MARCESCENS), enterococcus sp. Isolated from example 3 and Morganella morganii Morganella morganii isolated from example 4 were cultured in LB medium at 30℃for several days, calculated as parts by weight based on the equivalent OD ₆₀₀ by volume ratio, three sets of experiments were set, wherein the first set of Serratia marcescens was 1 part by weight, the Enterococcus was 1 part by weight, the Morganella morganii was 1 part by weight, the second set of Serratia marcescens was 1 part by weight, 1 part by weight of Enterococcus, 10 parts by weight of Morganella morganii, 10 parts by weight of Serratia marcescens, 1 part by weight of Enterococcus, 10 parts by weight of Morganella morganii, and then respectively spraying mixed bacterial liquid of aqueous solutions of methyl methacrylate and methacrylic acid copolymer nano particles (aqueous solution of auxiliary 1, wherein the final concentration of the auxiliary 1 is 1% respectively in terms of mass percent of the total weight of the pesticide) into pesticide, and calculating the death rate of spodoptera frugiperda after 12 hours. Three experiments were repeated for each proportion, 6 spodoptera frugiperda larvae were repeated for each experiment, 100 μl was sprayed on each spodoptera frugiperda, and an aqueous solution of water and methyl methacrylate and methacrylic acid copolymer nanoparticles at a ratio of 99:1 was sprayed on control group 6. Fig. 12 shows mortality of spodoptera frugiperda larvae after spraying with the microbial agent.

In FIG. 12, the first group of pesticides is designated 1:1:1, the second group of pesticides is designated 1:1:10, the third group of pesticides is designated 10:1:10, and control group 6 represents spraying of an aqueous solution of the same adjuvant 1 concentration.

As can be seen from FIG. 12, the mortality of the spodoptera frugiperda larvae after spraying, which is the microbial agent, is a mixture of Serratia marcescens, enterococcus and Morganella morganii, was significantly higher than that of the control group 6, and the three bacteria had the best insecticidal effect at a ratio of 10:1:10.

Example 11

After three bacteria of Serratia marcescens separated in example 1, enterococcus separated in example 3 and Morganella morganii separated in example 4 are cultured in LB medium for several days, respectively adding aqueous solutions of methyl methacrylate and methacrylic acid copolymer nanoparticles (the aqueous solution of the auxiliary 1 is 1% based on the total weight of the pesticide), spraying on spodoptera frugiperda larvae, wherein the first group of Serratia marcescens is 10 parts by weight, the enterococcus is 1 part by weight, the Morganella morgana is 10 parts by weight, then adding aqueous solutions of methyl methacrylate and methacrylic acid copolymer nanoparticles (the aqueous solution of the auxiliary 1 is 1% based on the total weight of the pesticide), spraying 100uL of each spodoptera frugiperda, the second group of Serratia marcescens is the aqueous solution of the auxiliary 1 on the premise that the equivalent weight percentage of the pesticide is 1, the first group of Serratia marcescens is sprayed on the basis of the equivalent weight of the pesticide, the aqueous solution of the auxiliary 1% of the second group of Serratia marcescens is the pesticide, the aqueous solution of the auxiliary 1% based on the equivalent weight of the pesticide, the aqueous solution of the auxiliary 53 is prepared by adding the aqueous solution of the auxiliary 1% of the methacrylic acid copolymer, the aqueous solution of the auxiliary 1% based on the equivalent weight of the pesticide, and the aqueous solution of the auxiliary 1% of the aqueous solution of the auxiliary 1% based on the total weight of the pesticide, and the aqueous solution of the auxiliary 1% based on the total weight of the aqueous solution of the auxiliary 1% of the aqueous solution of the auxiliary 1, spraying 100 mu L of spodoptera frugiperda on each, adding methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (1% of the aqueous solution of the auxiliary agent) into the Morganella morganii on the premise of the same OD ₆₀₀, spraying 100 mu L of the pesticide prepared by the aqueous solution of the auxiliary agent 1 accounting for the total weight of the pesticide, and spraying the aqueous solution of the clear water, the methyl methacrylate and the methacrylic acid copolymer nanoparticle prepared according to the proportion of 99:1 on each spodoptera frugiperda on the control group 7. Each treatment was repeated 3 times, each experiment was repeated 6 spodoptera frugiperda larvae, each spodoptera frugiperda was sprayed 100uL, and the spodoptera frugiperda self-phase killing probability was counted after 6 hours of treatment. Fig. 13 shows the probability of spodoptera frugiperda larvae self-phase killing after spraying with the microbial inoculant.

In FIG. 13, 10:1:10 represents a pesticide prepared by mixing Serratia marcescens 10 parts by weight, enterococcus 1 part by weight and Morganella morganii 10 parts by weight and adding a nanoparticle aqueous solution of methyl methacrylate and methacrylic acid copolymer (aqueous solution of adjuvant 1);

Serratia marcescens represents a pesticide prepared by adding methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (aqueous solution of an auxiliary agent 1) into Serratia marcescens on the premise of the same OD ₆₀₀;

Enterococcus refers to a pesticide prepared by adding methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (aqueous solution of an auxiliary agent 1) into enterococcus under the premise of equal OD ₆₀₀;

Morganella is a pesticide prepared by adding methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (aqueous solution of an auxiliary agent 1) into Morganella under the premise of the same OD ₆₀₀;

control 7 represents an aqueous solution of 1% methyl methacrylate and methacrylic acid copolymer nanoparticle aqueous solution (aqueous solution of adjuvant 1).

As can be seen from FIG. 13, the probability of the spodoptera frugiperda larvae being self-phase disabled increases after the three bacteria of Serratia marcescens, enterococcus and Morganella are mixed and sprayed.

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> Serratia marcescens, microbial agent, pesticide and application thereof

<130> PF02093

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1442

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence, artificial sequence

<400> 1

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tctacgatta ctagcgattc cgacttcatg gagtcgagtt gcagactcca atccggacta 180

cgacgtactt tatgaggtcc gcttgctctc gcgaggtcgc ttctctttgt atacgccatt 240

gtagcacgtg tgtagcccta ctcgtaaggg ccatgatgac ttgacgtcat ccccaccttc 300

ctccagttta tcactggcag tctcctttga gttcccggcc gaaccgctgg caacaaagga 360

taagggttgc gctcgttgcg ggacttaacc caacatttca caacacgagc tgacgacagc 420

catgcagcac ctgtctcaga gttcccgaag gcaccaatcc atctctggaa agttctctgg 480

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tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc ggccgtactc cccaggcggt 600

cgatttaacg cgttagctcc ggaagccacg cctcaagggc acaacctcca aatcgacatc 660

gtttacagcg tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgcacctg 720

agcgtcagtc ttcgtccagg gggccgcctt cgccaccggt attcctccag atctctacgc 780

atttcaccgc tacacctgga attctacccc cctctacgag actctagctt gccagtttca 840

aatgcagttc ccaggttgag cccggggatt tcacatctga cttaacaaac cgcctgcgtg 900

cgctttacgc ccagtaattc cgattaacgc ttgcaccctc cgtattaccg cggctgctgg 960

cacggagtta gccggtgctt cttctgcgag taacgtcaat tgatgaacgt attaagttca 1020

ccaccttcct cctcgctgaa agtgctttac aacccgaagg ccttcttcac acacgcggca 1080

tggctgcatc aggcttgcgc ccattgtgca atattcccca ctgctgcctc ccgtaggagt 1140

ctggaccgtg tctcagttcc agtgtggctg gtcatcctct cagaccagct agggatcgtc 1200

gcctaggtga gccattaccc cacctactag ctaatcccat ctgggcacat ctgatggcaa 1260

gaggcccgaa ggtccccctc tttggtcttg cgacgttatg cggtattagc taccgtttcc 1320

agtagttatc cccctccatc aggcagtttc ccagacatta ctcacccgtc cgccgctcgt 1380

cacccaggga gcaagctccc ccgtgctacc gctcgacttg catgtgttaa aggccccccc 1440

cg 1442

<210> 2

<211> 1475

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence, artificial sequence

<400> 2

aaaaaatcca gtgcgggtgc tatacatgca agtcgaacgc ttctttcctg ccgagtgctt 60

gcactcactt ggaaagagga gtggcggacg ggtgagtaac acgtgggtaa cctgcccatc 120

agagggggat aacacttgga aacaggtgct aataccgcat aatagtcgac accgcatggt 180

gttgatttga aagacgcttt cgggtgtcac tgatggatgg acccgcggtg cattagctag 240

ttggtgaggt aacggctcac caaggccatg atgcatagcc gacctgagag ggtgatcggc 300

cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcgg 360

caatggacga aagtctgacc gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa 420

aactctgttg ttagagaaga acaagtagga gagtaactgc tcttgccttg acggtatcta 480

accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540

tgtccggatt tattgggcgt aaagcgagcg caggcggttt cttaagtctg atgtgaaagc 600

ccccggctca accggggagg gtcattggaa actgggagac ttgagtgcag aagaggagag 660

tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg aggaacacca gtggcgaagg 720

cgactctctg gtctgtaact gacgctgagg ctcgaaagcg tggggagcaa acaggattag 780

ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttggagggt ttccgccctt 840

cagtgctgca gcaaacgcat taagcactcc gcctggggag tacgaccgca aggttgaaac 900

tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960

gcgaagaacc ttaccaggtc ttgacatcct ttgaccactc tagagataga gctttccctt 1020

cggggacaaa gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080

taagtcccgc aacgagcgca acccttattg ttagttgcca tcattcagtt gggcactcta 1140

gcgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200

tatgacctgg gctacacacg tgctacaatg ggaagtacaa cgagtcgcta ggccgcgagg 1260

tcatgcaaat ctcttaaagc ttctctcagt tcggattgta ggctgcaact cgcctacatg 1320

aagccggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt 1380

gtacacaccg cccgtcacac cacgaaagtt tgtaacaccc gaagtcggtg aggtaacctt 1440

ttggagccag ccgcctaagg tggaagaaaa ttctc 1475

<210> 3

<211> 1457

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence, artificial sequence

<400> 3

accgcttttt atcacaaagt ggtaagcgcc ctcccggagg ttaagctacc tacttctttt 60

gcaacccact cccatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgt 120

agcattctga tctacgatta ctagcgattc cgacttcatg gagtcgagtt gcagactcca 180

atccggacta cgacgtactt tatgagttcc gcttgccctc gcggggtcgc ttccctttgt 240

atacgccatt gtagcacgtg tgtagcccta ctcgtaaggg ccatgatgac ttgacgtcat 300

ccccaccttc ctccggttta tcaccggcag tctcctttga gttcccgcca tcacgcgctg 360

gcaacaaagg ataagggttg cgctcgttgc gggacttaac ccaacatttc acaacacgag 420

ctgacgacag ccatgcagca cctgtctcag agttcccgaa ggcaccaaag catctctgct 480

aagttctctg gatgtcaaga gtaggtaagg ttcttcgcgg tgcatcgaat taaaccacaa 540

gctccaccgc ttgtgcgggc ccccgtcaat tcatttgagt tttaaccttg cggccgtact 600

ccccaggcgg tcgacttaac gcgttagctc ccggaagcca cgcctcaagg gcacaacctc 660

caagtcgaca tcgtttacag cgtggactac cagggtatct aatcctgttt gctccccacg 720

ctttcgcacc tgagcgtcag tctttgtcca gggggccgcc ttcgccaccg gtattcctcc 780

acatctctac gcatttcacc gctacacatg gaattctacc cccctctaca agactctagc 840

tgaccagtat cagatgcaat tcccgggtta agcccgggga tttcacatct gactcaatca 900

accgcctgcg tgcgctttac gcccagtaat tcccgattaa cgcttgcaac ctccgtatta 960

ccgcggctgc tggcacggag ttagccggtg cttcttctgt cggtaacgtc aattgccaag 1020

gttattaacc ttgacacctt cctcccgact gaaagtactt tacaacccga aggccttctt 1080

catacacgcg gcatggctgc atcaggcttg cgcccattgt gcaatattcc ccactgctgc 1140

ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg ctgatcatcc tctcagacca 1200

gctagggatc gtcgcctagg taagccgtta cctcacctac tagctaatcc catatgggtt 1260

catctgatgg cgcgaggccc ggaggtcccc cgctttggtc cgaagacatt atgcggtatt 1320

agctaccgtt tccagtagtt atcccccgcc atcaggcaga tccccataca ttactcaccc 1380

gtccgccgct cgtcagcaga gaagcaagct tctccctgtt accgcccgac ttgcatgtgt 1440

tagctgccgc aagttcc 1457

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence, artificial sequence

<400> 4

gcggatccgc ggccgctgca gagtttgatc ctggctcag 39

<210> 5

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence, artificial sequence

<400> 5

ggctcgagcg gccgcccggg ttaccttgtt acgactt 37

Claims

1. A Serratia marcescens, whose deposit number is CGMCC No.24567.

2. A microbial agent, comprising Serratia marcescens, wherein the deposit number of Serratia marcescens is CGMCC No. 24567.

3. A pesticide or a plant growth promoter, comprising the microbial agent according to claim 2.

4. The pesticide or plant growth promoter according to claim 3, wherein:

The pesticide or plant growth promoter also includes adjuvants.

5. The pesticide or plant growth promoter according to claim 4, wherein:

The auxiliary agent is selected from one or more of methyl methacrylate and methacrylic acid copolymer nanoparticles, nanogold particles, urea, wettable powder cuprous oxide, ferrous sulfate, sodium alkyl sulfonate, and soapberry extract.

6. The pesticide or plant growth promoter according to claim 5, wherein:

The auxiliary agent is nanoparticles of a copolymer of methyl methacrylate and methacrylic acid.

7. The pesticide or plant growth promoter according to claim 6, wherein:

Calculated by weight percentage of the total weight of the pesticide or plant growth promoter, the adjuvant is 0.1-10wt%.

8. A method for promoting plant growth or controlling fall armyworm, characterized in that it comprises applying the microbial agent according to claim 2 or the pesticide or plant growth promoter according to any one of claims 3 to 7 on the plant, wherein the plant is corn.

9. A microbial agent, comprising Serratia marcescens, Enterococcus and Morganella;

The deposit number of the Serratia marcescens is CGMCC No.24567;

The deposit number of the enterococcus is CGMCC No.24566, and the deposit number of the Morganella is CGMCC No.24593.

10. The microbial agent according to claim 9, wherein

The bacterial agent consists of Serratia marcescens, Enterococcus and Morganella.

11. The microbial agent according to claim 9, wherein

In parts by weight, the mass ratio of the Serratia marcescens, the Enterococcus and the Morganella is 10:1:10.

12. A pesticide or a plant growth promoter, comprising the microbial agent according to any one of claims 9 to 11.

13. The pesticide or plant growth promoter according to claim 12, wherein:

The pesticide or plant growth promoter also includes adjuvants.

14. The pesticide or plant growth promoter according to claim 13, wherein:

15. The pesticide or plant growth promoter according to claim 14, wherein:

16. The pesticide or plant growth promoter according to claim 13, wherein:

17. A method for promoting plant growth or controlling fall armyworm or promoting cannibalism among fall armyworm larvae, characterized in that it comprises applying the microbial agent according to any one of claims 9 to 11 or the pesticide or plant growth promoter according to any one of claims 12 to 16 on the plant, wherein the plant is corn.

18. The method according to claim 17, wherein:

The application method is root dredging and/or spraying.