CN112779183A

CN112779183A - Tomato hemifacial paralysis prevention and control bio-organic fertilizer, preparation method and application thereof

Info

Publication number: CN112779183A
Application number: CN202011633332.5A
Authority: CN
Inventors: 姚燕来; 朱为静; 洪春来; 何科伟; 朱凤香; 王卫平; 洪磊东; 陈杨清
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-11

Abstract

The invention relates to the technical field of biological control, and particularly discloses a tomato hemifacial paralysis disease control bio-organic fertilizer, a preparation method and application thereof, wherein the bio-organic fertilizer contains Bacillus belgii with the preservation number of CGMCC NO. 19860; the preservation unit of the Bacillus belgii is the China general microbiological culture Collection center; the collection name of the Bacillus belgii is Bacillus belgii velezensis WSW 2; the preservation time of the Bacillus belgii is 5 months and 11 days in 2020. The invention has the characteristics of good prevention and treatment effect and low morbidity.

Description

Tomato hemifacial paralysis prevention and control bio-organic fertilizer, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological control, and particularly relates to a tomato hemifacial paralysis disease control bio-organic fertilizer, a preparation method and application thereof.

Background

The tomatoes are also called tomatoes, the fruits of the tomatoes are rich in nutrition and have special flavor, the tomatoes are one of important vegetable crops all over the world, the tomatoes are widely planted in various regions of China, the planting area of the tomatoes in China is about 1600 ten thousand mu, the annual output is about 5500 ten thousand tons, and the tomatoes account for about 7% of the total amount of the vegetables. However, with the increasing of the tomato planting area, the tomato continuous cropping phenomenon is very common, common soil-borne diseases such as blight and bacterial wilt of tomato planting bases are in a high-emergence state, meanwhile, due to the fact that seeds and seedlings are transferred across the country, a plurality of new tomato soil-borne diseases are spread across the country, if a tomato 'hemifacial paralysis' disease which is newly appeared in the south county of Wenzhou province in Zhejiang province, also called a tomato hemifacial paralysis disease, is caused by the tomato disease, the conventional disease incidence reaches 50% -60%, the disease incidence in a high-emergence area can reach more than 80%, the yield reduction is less by 30% -40%, the disease is 60% -80%, even the loss is caused, the normal production of the tomato is seriously affected, and at present, the disease is still lack of a control medicine, so that the development of a novel green control technology becomes an urgent need in tomato cultivation.

Biological control is becoming more and more important because it is environmentally friendly and pathogenic bacteria are not easily resistant. The biological organic fertilizer is a novel fertilizer with organic fertilizer and microbial functions, and compared with a single microbial agent, the biological organic fertilizer is rich in organic matters and organic nutrients, can improve the content of the organic matters in soil, increases the foundation soil fertility of the soil, and plays roles in loosening the soil, improving the granular structure of the soil and rooting and promoting the roots; compared with a single organic fertilizer, the organic fertilizer is rich in functional microorganisms, can increase the number of useful microorganisms at the rhizosphere, inhibit the propagation of harmful pathogenic bacteria, enhance the disease resistance of crops, and has the effects of improving the nutritional conditions and the growth environment of the crops, stimulating the growth and development of the crops and resisting the damage of diseases and insects by the life activities of the microorganisms, thereby achieving the effects of digging the potential fertility of soil and improving the yield and quality of agricultural products. The existing multiple researches show that the biological organic fertilizer not only has an activating effect on nitrogen, phosphorus and potassium in soil, but also can stabilize the supply of nitrogen, phosphorus and potassium nutrients, regulate and control the microbial community structure of the soil, inhibit the occurrence of soil-borne diseases, and improve the yield and the quality of various vegetables, fruit and vegetable crops such as tomatoes, eggplants, Chinese cabbages, cucumbers and the like. Therefore, the biological organic fertilizer is an important direction for developing green agriculture as a novel fertilizer.

Therefore, the existing prevention and treatment for the tomato hemifacial paralysis have the problems of lack of prevention and treatment medicines and high morbidity.

Disclosure of Invention

The invention provides a tomato hemifacial paralysis prevention and control bio-organic fertilizer with good prevention and control effect and low morbidity, a preparation method and application thereof, aiming at solving the technical problems existing in the existing tomato hemifacial paralysis prevention and control.

The first technical scheme of the invention is as follows: the tomato semi-frontier wind disease prevention and control bio-organic fertilizer contains Bacillus belgii with the preservation number of CGMCC NO. 19860; the preservation unit of the Bacillus belgii is the China general microbiological culture Collection center; the collection name of the Bacillus belgii is Bacillus belgii velezensis WSW 2; the preservation time of the Bacillus belgii is 5 months and 21 days in 2020; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The screened Bacillus belgii has strong plate inhibition effect, greenhouse potting prevention and control effect and field prevention and control effect on the tomato hemifacial paralysis, enriches the existing tomato hemifacial paralysis biocontrol bacteria resource library, has the Bacillus belgii with antagonistic effect, and has outstanding technical effect on solving the tomato hemifacial paralysis; the invention selects a healthy tomato plant sample planted in a disease area in a Wenzhou region, slightly takes soil to pull out the plant, slightly shakes off loose attached soil on the plant root system, cuts off the soil from the root stem, weighs the plant root system and the rhizosphere soil thereof, obtains an antagonistic bacterial strain with strong nutrition competition capability to the rhizosphere by adopting a gradient dilution separation method, screens antagonistic bacteria again by adopting a plate opposition method after scribing and purification to obtain an antagonistic bacterial strain with high-efficiency disease resistance, observes the size of an inhibition zone, selects a bacterial strain with a larger inhibition zone by comparing the size of the inhibition zone, shows the bacterial colony to be round, opaque, light soil yellow, has unsmooth edge and folds in the middle of the bacterial colony, observes by adopting a microscope to find that the cell is in a short rod shape to form spores which are positioned in the middle of the cell, and determines the classification position of the antagonistic bacteria by physiological and biochemical characteristics and 16S rRNA gene identification, the bacillus is Bacillus belgii, wherein the maximum similarity rate of 16S rRNA genes with the existing strains is 99 percent through comparison; the optimal growth conditions of the strain are as follows: the temperature is 30 ℃, the culture medium (LB) is peptone 1% (W/V), yeast extract 0.5% (W/V), sodium chloride 2% (W/V) and agar 2% (W/V), the pH value is 5.0-8.0, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; culturing on an LB flat plate at a constant temperature of 30 ℃ for 60-84 h; the Bacillus velezensis WSW2 provided by the invention is a Bacillus, has higher application value in the aspect of preventing and controlling tomato hemifacial paralysis, and especially can be separated aiming at the local environment and is suitable for the local climate and the growth environment, so that the Bacillus velezensis WSW2 has positive effects on improving the crop yield, reducing the pesticide consumption and constructing a good ecological environment; as a green prevention and control microbial agent vigorously advocated by China at present, the tomato semisideline air antagonistic effect is good, so that the tomato semisideline air morbidity is reduced by 60-80%, the occurrence of diseases is reduced, the yield and quality of agricultural products are greatly improved, and the normal production of tomatoes is ensured; the hidden danger of pollution to agricultural products does not exist in the using process, pathogenic bacteria are not easy to generate drug resistance, and the requirements of weight reduction, drug reduction and green production development mode advocated by the state are met; the tomato hemifacial wind disease prevention and control bio-organic fertilizer has the characteristics of both an organic fertilizer and a novel microbial fertilizer, is rich in organic matters and organic nutrients, can improve the content of organic matters in soil, increase the soil foundation fertility, play a role in loosening the soil, improving the soil aggregate structure and rooting and promoting the root, is rich in functional microbes, can increase the number of useful microbes at the rhizosphere, inhibit the propagation of harmful pathogenic bacteria and enhance the disease resistance of crops, has the functions of improving the nutritional condition and the growth environment of the crops by the life activities of the microbes, stimulating the growth and development of the crops and resisting the harm of diseases and insects, and further achieves the effects of excavating the potential fertility of the soil and improving the yield and quality of agricultural products; the tomato hemifacial paralysis prevention and control bio-organic fertilizer has an activating effect on soil nitrogen, phosphorus and potassium, can also stabilize the supply of nitrogen, phosphorus and potassium nutrients, regulate and control the microbial community structure of soil, inhibit the occurrence of soil-borne diseases and improve the yield and quality of tomatoes.

Preferably, in the dry sample of the bioorganic fertilizer for preventing and controlling the tomato hemifacial paralysis per gram, the viable count of the Bacillus belgii is 1.0 multiplied by 10⁸CFU/g～1.0×10¹⁰CFU/g dry sample. Namely, in the dry sample of the tomato hemifacial paralysis prevention and control bio-organic fertilizer per gram, the concentration of the antagonistic bacteria is not less than 1.0 multiplied by 10⁸CFU/g。

The second technical scheme of the invention is as follows: the preparation method of the tomato semi-frontier wind disease prevention and control bio-organic fertilizer comprises the steps of mixing a Bacillus belief fungicide and a decomposed organic material, and carrying out aerobic composting treatment to obtain the tomato semi-frontier wind disease prevention and control bio-organic fertilizer. According to the invention, the Bacillus belgii is combined with a decomposed organic material carrier, and aerobic composting fermentation is carried out to obtain the tomato hemifacial paralysis prevention and control bio-organic fertilizer, so that nutrients are provided for the growth of the Bacillus belgii, the physical, chemical and biological properties of soil can be improved, the growth of plants is promoted, and the occurrence of diseases is reduced.

Preferably, the bacillus beilesensis microbial inoculum is a liquid microbial inoculum; the viable count of the Bacillus belgii in the liquid microbial inoculum is 1.0 multiplied by 10⁸CFU/ml～1.0×10¹⁰CFU/ml. Preferably, the preparation of the Bacillus belgii agent comprises the steps of,

(A) inoculating Bacillus belgii to an LB slant culture medium for activation;

(B) inoculating the activated Bacillus belgii to an LB liquid culture medium to obtain a culture solution;

(C) the culture solution is placed in a shaking table and cultured for 60 to 84 hours under the conditions of the rotating speed of 120 to 180rpm/min and the temperature of 10 to 50 ℃, thus obtaining the liquid microbial inoculum.

Preferably, in the step (C), the culture temperature of the Bacillus belgii is 25-35 ℃; the concentration of NaCl in the culture solution is 0.5-4%; the pH of the culture solution is 4.0-8.0. More preferably, the pH of the culture solution is 5.0 to 7.0.

Preferably, the decomposed organic material is decomposed tea leaves.

Preferably, the preparation of the decomposed tea leaves comprises the steps of,

(a) collecting tea branches and leaves;

(b) crushing the collected tea branches and leaves to below 2cm by using a crusher;

(c) adjusting the water content of the broken tea branches and leaves to 55-65% by using wood chips, mushroom dregs and the like;

(d) piling the broken tea branches and leaves meeting the water content requirement into a strip pile shape with the height of 0.5-1.5 m and the width of 1-2 m, and starting to perform primary aerobic composting fermentation;

(e) when the fermentation temperature of the first aerobic composting fermentation is increased to more than 50 ℃, the tea leaves are turned and thrown once a day until the fermentation temperature begins to decrease, and then the thoroughly decomposed tea leaves are prepared.

Preferably, when the fermentation temperature in the step (e) is reduced to 37-41 ℃, the microbial agent is sprayed on the decomposed tea leaves after the first aerobic composting fermentation; wherein the spraying amount of the microbial agent is calculated according to the standard that 10L of the microbial agent is added to each ton of the decomposed tea leaves;

and continuously carrying out secondary aerobic composting fermentation on the decomposed tea leaves sprayed with the microbial agent, turning and throwing once a day, continuously reducing the fermentation temperature to the ambient temperature, and finishing the fermentation until the water content in the dry stack is 32-38% to obtain the tomato hemifacial paralysis disease control bio-organic fertilizer.

According to the invention, the Bacillus belgii is combined with a decomposed organic material carrier, and then secondary fermentation is carried out to obtain the tomato hemifacial paralysis prevention and control bio-organic fertilizer, so that nutrients are provided for the growth of the Bacillus belgii, the physical, chemical and biological properties of soil can be improved, the growth of plants is promoted, and the occurrence of diseases is reduced; the Bacillus belgii fungicide is combined with an organic carrier for secondary fermentation to obtain a tomato hemifacial paralysis disease prevention and control bio-organic fertilizer product, so that the effective colonization of the Bacillus belgii in soil is ensured, the application effect of the Bacillus belgii in prevention and control is ensured, and the bio-control efficiency can be obviously improved; aerobic composting fermentation can be carried out in an intelligent composting reactor, and the method is simple and easy to operate.

The third technical scheme of the invention is as follows: application of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer in the prevention and control of the tomato hemifacial paralysis disease. The quantity of antagonistic bacteria in the rhizosphere soil of the tomato in the seedling stage and after transplanting is guaranteed, invasion of pathogenic bacteria of the tomato hemifacial paralysis can be resisted, and finally diseases can be prevented and controlled; the method is characterized in that a tomato semi-frontier wind disease prevention and control bio-organic fertilizer containing Bacillus belgii is added into a matrix in the tomato seedling and/or tomato planting process, and the tomato semi-frontier wind disease prevention and control bio-organic fertilizer is added in the tomato seedling and transplanting periods, so that an antagonistic bacteria community system is constructed in the tomato rhizosphere, the number of antagonistic bacteria in the tomato rhizosphere is increased, the resistance of the tomato rhizosphere to pathogenic bacteria is enhanced, and the occurrence of diseases is reduced; specifically, the application method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer comprises the following aspects: adding the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer into a tomato cup seedling culture medium according to the proportion of 2% for each cup of seedlings, and fully and uniformly mixing; when transplanting the cup seedlings, the cup seedlings are moved into potting soil on the premise of not damaging the shape of a matrix; before the potting soil is transplanted, the tomato hemifacial wind disease prevention and control bio-organic fertilizer is applied in advance, and the application proportion is 1%.

The invention has the following beneficial effects:

(1) the screened Bacillus belgii has strong plate inhibition effect, greenhouse potting prevention and control effect and field prevention and control effect on the tomato hemifacial paralysis, enriches the existing tomato hemifacial paralysis biocontrol bacteria resource library, has the Bacillus belgii with antagonistic effect, and has outstanding technical effect on solving the tomato hemifacial paralysis;

(2) the Bacillus velezensis WSW2 is a Bacillus, has high application value in the aspect of preventing and controlling tomato hemifacial paralysis, and particularly can be separated from the local environment to adapt to the local climate and growth environment, so that the Bacillus velezensis WSW2 has positive effects on improving crop yield, reducing pesticide consumption and constructing a good ecological environment;

(3) as a green prevention and control microbial agent vigorously advocated by China at present, the antagonistic effect on the tomato semisideline is good, so that the morbidity of the tomato semisideline is reduced by 60-80%, the occurrence of diseases is reduced, the yield and the quality of agricultural products are greatly improved, and the normal production of tomatoes is ensured; the hidden danger of pollution to the quality safety of agricultural products does not exist in the using process, pathogenic bacteria are not easy to generate drug resistance, and the requirements of weight reduction, drug reduction and green production development mode advocated by the state are met;

(4) the tomato hemifacial paralysis prevention and control bio-organic fertilizer has the characteristics of an organic fertilizer and a novel microbial function fertilizer, is rich in organic matters and organic nutrients, can improve the content of organic matters in soil, increase the soil foundation fertility, play a role in loosening the soil, improving the soil granular structure and rooting and promoting the root, is rich in functional microbes, can increase the number of useful microbes in the rhizosphere, inhibit the propagation of harmful pathogenic bacteria and enhance the disease resistance of crops, has the functions of improving the nutritional condition and the growth environment of the crops by the life activities of the microbes, stimulating the growth and development of the crops and resisting the harm of diseases and insects, and further achieves the effects of excavating the potential fertility of the soil and improving the yield and quality of agricultural products; the tomato hemifacial paralysis prevention and control bio-organic fertilizer has an activating effect on soil nitrogen, phosphorus and potassium, can also stabilize the supply of nitrogen, phosphorus and potassium nutrients, regulate and control the microbial community structure of soil, inhibit the occurrence of soil-borne diseases and improve the yield and quality of tomatoes;

(5) after the bacillus beilis is combined with a decomposed organic material carrier, aerobic composting fermentation is carried out to obtain the tomato hemifacial paralysis prevention and control bio-organic fertilizer, so that nutrients are provided for the growth of the bacillus beilis, the physical, chemical and biological properties of soil can be improved, the growth of plants is promoted, and the occurrence of pests is reduced;

(6) the method is favorable for ensuring the quantity of antagonistic bacteria in the rhizosphere soil of the tomatoes in the seedling stage and after transplanting, resisting the invasion of pathogenic bacteria of the tomato hemifacial paralysis, and finally preventing and controlling the diseases.

Drawings

FIG. 1 is a colony morphology map of number KS1 with a larger inhibition zone in the isolated WSW2 strain of the present invention;

FIG. 2 is a morphological diagram showing the round, opaque and light earthy yellow colonies formed by the application of the strain WSW2 to LB plates according to the present invention;

FIG. 3 is a morphology of colonies showing non-smooth edges and wrinkled middles when strain WSW2 was spread on LB plates according to the present invention;

FIG. 4 is a micrograph of a colony in which cells are short rod-shaped and form spores, which are located in the middle of the cells, observed by a microscope when the strain WSW2 is spread on an LB plate in the present invention;

FIG. 5 is a bar graph showing the growth of strain WSW2 in the present invention at different temperatures;

FIG. 6 is a bar graph showing the growth of strain WSW2 in the present invention at different NaCl concentrations;

FIG. 7 is a bar graph of the growth of strain WSW2 in the present invention at various pH;

FIG. 8 is a bar graph showing the utilization of various monosaccharides by strain WSW2 according to the present invention;

FIG. 9 is a bar graph showing the utilization of different disaccharides by strain WSW2 according to the invention;

FIG. 10 is a bar graph showing the utilization of various polysaccharides by strain WSW2 of the present invention;

FIG. 11 is a bar graph showing the utilization of different esters by strain WSW2 in accordance with the present invention;

FIG. 12 is a bar graph showing the utilization of various alcohols by strain WSW2 in accordance with the present invention;

FIG. 13 is a bar graph showing the utilization of various amino acids by strain WSW2 in accordance with the present invention;

FIG. 14 is a bar graph showing the utilization of various amines by strain WSW2 in accordance with the present invention;

FIG. 15 is a bar graph showing the utilization of various organic acids by strain WSW2 in accordance with the present invention;

FIG. 16 is a diagram showing the growth state of untreated tomato plants in the continuous cropping plot of the invention, wherein the continuous cropping plot is selected from the Ma Zhen Town tomato in the Xanthina county and has a serious incidence of hemifacial paralysis;

FIG. 17 is a diagram showing the growth state of tomato plants after being treated with organic fertilizer in a continuous cropping plot selected from Siberian Town tomato in the city of Hannan county and in the town of Ma, and having severe incidence of hemifacial paralysis;

FIG. 18 is a comparison of the blank control, Fusarium solani pathogen control and Bacillus belgii disease resistant treated potted plants of example 5 of this invention;

FIG. 19 is a bar graph of the incidence of potting disease for the blank control, the Fusarium solani pathogen control and the Bacillus beijerinckii disease resistant treatment of example 5 of the present invention.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.

The main raw and auxiliary materials, reagents and instrument equipment involved in the invention are as follows: separating pathogenic bacteria to be tested from tomato hemifacial paralysis plants planted in the endemic area of the Wenzhou region; separating the antagonistic bacteria from rhizosphere soil samples in healthy plants in the disease area, sampling, storing at low temperature, and immediately separating in a laboratory; the culture medium adopts potato glucose culture medium-PDA and LB culture medium; MSSPX-250 type biochemical incubator; MLS-3020 autoclave; SW-CJ-1FB type single double-face purification workbench; E360K centrifuge; constant temperature shaking table HWY-100; PCR Instrument Eppendorf No: 5345, mixing the raw materials together; PCR premix solution, and the rest reagents are analytically pure.

The bacillus beleisi, the preservation number of the bacillus beleisi is CGMCC NO. 19860; the preservation unit of the Bacillus belgii is the China general microbiological culture Collection center; the collection name of the Bacillus belgii is Bacillus belgii velezensis WSW 2; the preservation time of the Bacillus belgii is 5-month-21-day 2020.

The application of Bacillus belgii as an antagonist of tomato hemifacial paralysis.

A microbial agent comprising the Bacillus belgii of claim 1. The microbial agent is a liquid microbial agent; the viable count of the Bacillus belgii in the liquid microbial inoculum is 1.0 multiplied by 10⁸CFU/ml～1.0×10¹⁰CFU/ml。

The preparation method of the microbial agent comprises the following steps,

(A) inoculating Bacillus belgii to an LB slant culture medium for activation;

(C) the culture solution is placed in a shaking table and cultured for 60 to 84 hours under the conditions of the rotating speed of 120 to 180rpm/min and the temperature of 10 to 50 ℃, thus obtaining the liquid microbial inoculum. More preferably, the culture temperature of Bacillus belgii is 25 to 35 ℃.

The concentration of NaCl in the culture solution is 0.5-4%; the pH of the culture solution is 5.0-8.0.

Growth of the Bacillus belgii in a culture medium using one or more monosaccharide compounds selected from the group consisting of beta-formyl-D-glucoside, alpha-D-glucose, D-mannose, D-fructose, and D-galactose as a carbon source; the Bacillus belgii grows by taking one or more disaccharide compounds of D-maltose, D-trehalose, alpha-D-lactose or melibiose as a carbon source; the growth of the Bacillus belgii in the culture solution takes one or more than one type of disaccharide compounds in raffinose, dextrin or pectin as a carbon source; the growth of the Bacillus belgii in the culture solution takes one or more esters of Tween 40, methyl pyruvate or D-methyl lactate as a carbon source; the growth of the Bacillus beleisi in the culture solution takes one or more alcohol substances of D-sorbitol, D-mannitol, D-arabitol or inositol as a carbon source; the growth of the Bacillus beleisi in the culture solution takes one or more amino acids of aminoacetyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid or L-serine as a carbon source; the growth of the Bacillus beleisi in the culture solution takes one or more organic acids of D-galacturonic acid, D-gluconic acid, p-hydroxy-phenylacetic acid, L-lactic acid, L-malic acid, bromo-succinic acid, r-amino-butyric acid, beta-hydroxy-D, L-butyric acid or formic acid as a carbon source.

The Bordetella sessilifolia disease control bio-organic fertilizer is characterized in that the viable count of the Bacillus belgii is 1.0 multiplied by 10 in each gram of dry tomato Bordetella sessilifolia disease control bio-organic fertilizer sample⁸CFU/g～1.0×10¹⁰CFU/g dry sample.

The preparation method of the tomato hemifacial wind disease prevention and control bio-organic fertilizer comprises the steps of mixing a microbial agent with a decomposed organic material, and carrying out aerobic composting treatment to obtain the tomato hemifacial wind disease prevention and control bio-organic fertilizer. (ii) a The decomposed organic material is decomposed tea leaves.

The preparation of the decomposed tea leaves comprises the following steps,

(a) collecting tea branches and leaves;

(b) crushing the collected tea branches and leaves by using a crusher until the width of the tea branches and leaves is less than 2 cm;

(c) adjusting the water content of the broken tea branches and leaves to 55-65% by using wood chips and/or mushroom residues;

(e) when the fermentation temperature of the first aerobic composting fermentation is increased to more than 50 ℃, turning and throwing once every day until the fermentation temperature begins to decrease, thus obtaining the decomposed tea leaves; when the fermentation temperature is reduced to 37-41 ℃, the microbial inoculum is sprayed on the thoroughly decomposed tea leaves after the first aerobic composting fermentation; wherein the spraying amount of the microbial agent is calculated according to the standard that 10L of the microbial agent is added to each ton of the decomposed tea leaves;

Example 1: separation, screening and identification of Bacillus velezensis CGMCC NO.19860

(1) Isolation and screening of antagonistic bacteria

Gradient dilution separation method: selecting a healthy tomato plant sample planted in a disease area in the Wenzhou region, slightly pulling out the plant with soil, slightly shaking off loose attached soil on a plant root system, cutting off the plant root system from a root stem, weighing 10g of a plant root system and rhizosphere soil thereof, placing the plant root system and the rhizosphere soil in a conical flask containing sterilized glass beads and 90mL of sterile water, and carrying out treatment at the temperature of 28 ℃ and the temperature of 150 r.min^-1Is oscillated for 30min under the condition of stirring speed to obtain the concentration of 10^-1The rhizosphere soil suspension is respectively diluted into 10 by a gradient concentration dilution method^-4、10^-5And 10^-6Diluting the solution; 0.1ml 10 of each^-4、10^-5And 10^-6And respectively coating the three gradients of diluent on an LB culture medium, repeating each gradient for 3 times, carrying out inverted culture for 18h at the temperature of 28 ℃, selecting a single typical bacterial colony which grows rapidly and has different forms, namely obtaining rhizosphere microbial strains with strong rhizosphere nutrition competition capability, carrying out streak purification on the selected strains on an LB flat plate for 3-4 times, respectively numbering, adding the purified strains into sterilized glycerol with the volume fraction of 15%, and preserving at the temperature of-80 ℃ for later use.

Screening antagonistic bacteria by a plate confronting method: in order to screen and obtain functional antagonistic bacterial strains with high-efficiency antagonistic action, a plate confronting method is adopted, 1 tomato hemifacial pathogenic bacterium separated from a plant is inoculated into a PDA plate, meanwhile, separated rhizosphere microbial strains are inoculated on the periphery of the plate, a confronting test is carried out, the plate is cultured in a culture box at 28 ℃, the size of an inhibition zone is observed, and the antagonistic bacterial strains with larger inhibition zone, namely antagonistic bacterial strains with high-efficiency disease resistance, are selected by comparing the size of the inhibition zone and are used for further verification and purification. Experiments show that the KS1 strain shown in figure 1 in the separated strains has a larger inhibition zone, which shows that the separated strains have stronger antibacterial activity and are used for further identification and functional verification, and the separated strains are named as WSW2 according to the preservation requirements of laboratory strains.

(2) Identification of antagonistic bacteria

And (3) carrying out molecular identification on the obtained antagonistic bacterial strain by adopting a molecular biology means and utilizing 16S rDNA sequencing.

The primers for PCR amplification of the 16S rDNA fragment are a pair of universal primers;

forward primer BSF8/20SEQ ID N0: 1: 5'-AGAGT TTGAT CCTGG CTCAG-3', respectively;

reverse primer BSR1541/20SEQ ID N0: 2: 5'-AAGGA GGTGA TCCAG CCGCA-3', respectively;

the PCR reaction system was 50. mu.l: 10 XPCR buffer 5.0. mu.l, MgCl₂(25mM) 3.0. mu.l, dNTP 4.0. mu.l, each 1.0. mu.l of the primer BSF8/20 and the primer BSR1541/20, template DNA 1.0. mu.l, Taq enzyme (10000U/mL, Takara Bio Inc.) 0.25. mu.l, and redistilled water 36.0. mu.l.

The PCR amplification procedure was: (1) 5min at 94 ℃; (2) 1min at 94 ℃, 1min at 55 ℃, 1.5min at 72 ℃, and circulating for 35 times in the step (2); (3) 10min at 72 ℃; (4) 10min at 4 ℃. The amplified product is separated and identified by 1 percent agarose gel electrophoresis, and the PCR amplified product is subjected to bidirectional sequencing in Biotechnology engineering (Shanghai) GmbH.

The 16S rDNA sequence SEQ ID N0:3 is:

TTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCG GGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAAC AGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTT GCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCC TATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCC CCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCACGCTTTCGCTCCTCAGCGTCAGTTAC AGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACT CAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAA TAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCC GCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACT TTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTC GGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTT TATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGT TACTCACCCGTCCGCCGCTAACATCAGGAGAAGTCCCATCTGTCCGCTCGACTTGATGTATTAGGCACGCCGCCAGCGTTCGCCTGAGCCA GATCAAAACTCTAAA

the 16S rDNA sequence obtained by sequencing is compared on an NCBI website or an RDP website, and the similarity with Bacillus velezensis (T) is the highest and reaches 99 percent after comparison.

The antagonistic strain was spread on an LB plate, and as shown in FIGS. 2 and 3, the colony was found to be round, opaque, light yellow, unsmooth at the edge, and wrinkled in the middle; further, as shown in FIG. 4, the cells were observed under a microscope, and were found to be short rods, to form spores, which were located in the middle of the cells.

The obtained antagonistic bacteria is determined to be Bacillus belgii by combining the colony morphology and the microscopic observation morphology of the strain with high similarity, and is named as Bacillus belgii WSW 2.

Example 2: growth condition of Bacillus velezensis CGMCC NO.19860

(1) Effect of temperature on growth of Strain WSW2

The culture medium adopts LB culture medium, the initial pH is 7.0, the liquid loading of the shake flask is 50ml/250ml, the inoculation amount is 0.5%, the culture temperature is respectively set to 10 ℃, 20 ℃, 30 ℃, 40 ℃ and 50 ℃, the rotation speed of the shaking table is 150rpm, the culture time is 24h, and the OD of the bacterial liquid OD at different temperatures is measured after the culture is finished₆₀₀Values, each temperature was repeated 3 times.

As a result: bacterial liquid OD of bacterial strain WSW2 cultured for 24h at different temperatures₆₀₀As shown in FIG. 5, it can be seen from FIG. 5 that the bacterial cell concentration of the strain WSW2 was low and the average OD was low at a culture temperature of 10 ℃₆₀₀At 0.085, strain WSW2 was essentially non-growing; the highest growth of the strain at 30 ℃ and OD₆₀₀2.504 is achieved; the strain WSW2 can tolerate higher culture temperature, and the growth amount OD of the strain is 50 DEG C₆₀₀Still reaching 1.19.

(2) Effect of different NaCl concentrations (w/v) on the growth of the Strain WSW2

The culture medium adopts LB culture medium, the initial pH is 7.0, the NaCl concentration is set to 4 gradients which are 0.5%, 1%, 2% and 4%, the growth curve of the strain under different NaCl concentrations is measured by adopting a microorganism growth curve analyzer, the rotating speed is medium speed, the culture temperature is 30 ℃, the culture time is 48h, and the bacterial liquid absorbance values (OD) under different temperatures are measured after the culture is finished₆₀₀Values), each inoculum size was repeated 4 times.

As a result: the test results of different NaCl concentrations are shown in FIG. 6, and it can be seen from FIG. 6 that the strain has strong osmotic pressure resistance and can grow at NaCl concentrations of 0.5% -4.0%, but different NaCl concentrations have certain influence on the growth of the strain, when the NaCl concentration is 0.5% -2.0%, the inoculated strain has no lag phase, and has certain promotion effect on the growth of the strain at 2.0%, and has the maximum OD at 2.0% NaCl concentration₆₀₀A value; the 4% NaCl had an effect on the initial growth of the strain, with a growth lag phase longer than other concentrations, up to about 5h, and a growth rate lower than other concentrations.

(3) Effect of initial pH on growth of Strain WSW2

The culture medium adopts LB culture medium, the initial pH is set with 5 gradients of 4.0, 5.0, 6.0, 7.0 and 8.0, the inoculation amount is 0.5%, the growth curve of the strain under different initial pH is measured by a microorganism growth curve analyzer, the rotating speed is medium speed, the culture temperature is 30 ℃, the culture time is 48h, and the OD of the bacterial liquid OD under different temperatures is measured after the culture is finished₆₀₀Values, repeated 4 times per pH.

As a result: measuring the influence of different pH values on the growth of the strain WSW2, and determining the OD of the strain liquid₆₀₀The change with time is shown in FIG. 7, and it can be seen from FIG. 7 that the growth retardation of strain WSW2 is significantly prolonged when the initial medium has a pH of 4.0, and the growth phase starts to enter the logarithmic growth phase after 38 h; when the pH is 5.0, the strain WSW2 grows at the fastest speed and is grown 2h after inoculationEntering a logarithmic growth phase, entering a stationary phase at 12h, and enabling the OD value to be the maximum and to reach 1.62; when the pH value is between 6.0 and 8.0, the growth rate of the bacterial strain WSW2 is faster and closer, the bacterial strain WSW2 enters a stable period at 14 hours, but the maximum growth amount is less than that at pH 5.0.

(4) Analysis of the ability of strain WSW2 to utilize various carbon sources and the sensitivity to compounds

The carbon source utilization capacity of the strain is identified by using a BIOLOG GENIII microorganism identification plate.

Firstly, inoculating an activated WSW2 strain on a BUG culture medium, culturing at the temperature of 30 ℃ until a single colony is formed, inoculating the single colony to a clean inoculation pipe containing an inoculation liquid, and adjusting the thallus concentration in the inoculation liquid to 90-98% T by using a turbidity meter; secondly, pouring the prepared bacterial suspension into a V-shaped sample adding water tank, sucking the bacterial suspension into a suction head of a pipette by using an 8-channel pipette, sequentially adding the bacterial suspension into all the holes of the microplate according to the amount of 100 mu l per hole, and covering the cover of the microplate; and finally, putting the microplate into an incubator at the temperature of 33 ℃ for incubation, reading by using a BIOLOG identification system after 24 hours, and determining the utilization capacity of the strain WSW2 to different carbon sources and the sensitivity of the strain WSW2 to compounds.

(4.1) utilization of monosaccharides by Strain

As shown in FIG. 8, the strain WSW2 can utilize monosaccharide compounds such as β -formyl-D-glucoside, α -D-glucose, D-mannose, D-fructose and D-galactose as carbon sources, has low creatinine-utilizing activity, and cannot utilize D-salicin, 3-formylglucose, D-fucose, L-fructose, L-rhamnose and 6-PO₄D-glucose-6-phosphate and D-fructose-6-phosphate as carbon sources.

(4.2) utilization of disaccharide by Strain

As shown in FIG. 9, the strain WSW2 can use disaccharide compounds such as D-maltose, D-trehalose, α -D-lactose and melibiose as carbon sources, but cannot use disaccharide compounds such as D-cellobiose, gentiobiose, sucrose and D-turanose as carbon sources.

(4.3) utilization of polysaccharide substance by Strain

As shown in FIG. 10, the strain WSW2 can utilize polysaccharides such as raffinose, dextrin and pectin as carbon sources, but cannot utilize stachyose.

(4.4) utilization of esters by Strain

As shown in FIG. 11, strain WSW2 can utilize esters such as Tween 40, methyl pyruvate and D-methyl lactate as carbon source, but cannot utilize L-galacturonolactone as carbon source.

(4.5) utilization of alcohol substances by Strain

As shown in FIG. 12, the strain WSW2 can utilize D-sorbitol, D-mannitol, D-arabitol and inositol as carbon sources, but cannot utilize glycerol as a carbon source.

(4.6) utilization of amino acids by Strain

As shown in FIG. 13, the strain WSW2 can utilize various amino acids such as aminoacetyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid and L-serine as carbon sources, wherein the utilization rates of L-alanine and L-arginine are highest, but D-aspartic acid, D-serine and L-pyroglutamic acid cannot be utilized as carbon sources.

(4.7) utilization of amine-based substance by Strain

As shown in FIG. 14, none of the strains WSW2 could utilize amine compounds such as N-acetyl-D-glucosamine, N-acetyl- β -D-mannosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, L-histamine and glucuronamide as carbon sources.

(4.8) utilization of organic acid substances by Strain

As shown in fig. 15, the strain WSW2 can use various organic acids such as D-galacturonic acid, D-gluconic acid, p-hydroxy-phenylacetic acid, L-lactic acid, L-malic acid, bromo-succinic acid, r-amino-butyric acid, β -hydroxy-D, L-butyric acid, and formic acid as carbon sources, but cannot use D-glucuronic acid, mucic acid, quinic acid, glyconic acid, citric acid, α -keto-glutaric acid, D-malic acid, α -hydroxy-butyric acid, α -keto-butyric acid, acetoacetic acid, propionic acid, and acetic acid as carbon sources.

Example 3: preparation of bacterial strain WSW2 tomato hemibarbus disease prevention and control bio-organic fertilizer

Antagonistic bacterium agentPreparation: inoculating Bacillus belgii to LB slant for activation, inoculating activated Bacillus belgii to LB liquid culture medium, shake culturing at 30 deg.C in shaker at 150rpm for 3 days to obtain antagonistic bacteria agent, and determining viable count of the antagonistic bacteria agent to be 8.9 × 10¹⁰CFU/ml。

Tomato semi-sideline wind disease prevention and control bio-organic fertilizer: the tomato semi-sideline wind disease prevention and control bio-organic fertilizer usually takes decomposed organic materials as carriers, and through comparison of different materials, specifically, an organic material with pH meeting the most appropriate growth requirement of antagonistic bacteria and the lowest content of infectious microbe rate is selected as a carrier, and finally, decomposed tea leaves are taken as an organic material carrier, wherein the physical and chemical properties of the organic materials of the specifically decomposed tea leaves are shown in table 1;

table 1: physicochemical properties of organic material of decomposed tea leaves

Collecting the trimmed waste tea branches and leaves, crushing the waste tea branches and leaves to be less than 2cm by a crusher, adjusting the water content to be about 60 percent, stacking the waste tea branches and leaves into a stack shape with the height of about 1m and the width of about 1.5m, performing aerobic high-temperature fermentation, and turning and throwing the waste tea branches and leaves once a day when the temperature rises to be more than 50 ℃ until the temperature of the compost is gradually reduced; when the temperature is reduced to about 40 ℃, 10L of the prepared anti-disease microbial inoculum is added according to 1 percent of inoculation amount, namely, 10L of the prepared anti-disease microbial inoculum is added to each ton of decomposed auxiliary materials, so that the number of viable bacteria in the materials reaches 10⁸And (3) uniformly spraying the microbial inoculum to the tea leaf residue organic material fermented and decomposed at high temperature, continuously turning over the material to promote the colonization and proliferation of the antagonistic bacteria on the material, and ending the fermentation when the fermentation temperature is continuously reduced to the environmental temperature for 30 days to obtain the tomato windbreak disease control bio-organic fertilizer.

Determination of effective bacterial count of tomato hemifacial paralysis prevention and control bio-organic fertilizer: after the waste tea branches are fermented and decomposed at high temperature, WSW2 microbial inoculum is added for secondary fermentation to prepare the disease-resistant tomato hemibarbus stroke disease prevention and control bio-organic fertilizer, and after the secondary fermentation is finished, the tomato hemibarbus stroke disease is measuredThe effective viable count of the harmful prevention and control bio-organic fertilizer is measured once every 1 month, the survival rate of the antagonistic bacteria in the tomato hemifacial paralysis prevention and control bio-organic fertilizer is observed, the number of the effective bacteria in the organic material is increased after secondary fermentation for 6 months, the number of the effective bacteria in the organic material is reduced within 2 months of initial storage, but the subsequent storage period is not obviously changed, and the number of the effective bacteria still reaches 7.9 x 10 after 6 months of storage⁸CFU/g, the specific measurement results are shown in Table 2.

Table 2: effective bacterium number in tomato semi-frontier wind disease prevention and control bio-organic fertilizer

Example 4: application of strain WSW2 tomato hemibarbus disease prevention and control bio-organic fertilizer in tomato hemibarbus disease prevention and control

According to the research, a disease-causing plant and a healthy plant are collected from a tomato continuous cropping disease-causing plot, pathogenic microorganisms are separated from the disease-causing plant and identified as Fusarium solani (Fusarium solani), a plurality of rhizosphere microorganisms are separated and screened from rhizosphere soil of the healthy plant, one efficient antagonistic microorganism strain is selected, the disease resistance of the disease-causing plant is verified through plate confrontation, a rotten organic material suitable for preparing a tomato hemifacial disease prevention and control bio-organic fertilizer is further obtained through screening of organic materials, the disease-preventing and control bio-organic fertilizer for preventing the tomato hemifacial wind diseases is prepared, a field test is carried out, good bacteriostasis and disease prevention effects are obtained, and a new green technology is provided for biological prevention and control of the tomato hemifacial wind diseases.

The disease-resistant test point selects a continuous cropping plot with serious disease of the tomato semi-frontier wind in the town of horses in the south of the canes to evaluate the disease-resistant effect of the bio-organic fertilizer for preventing and controlling the tomato semi-frontier wind disease; the experiment sets 1 blank control and 1 tomato hemifacial paralysis disease prevention and control bio-organic fertilizer treatment, 3 repeats are set, each repeats 1 cell, the area of the cell is 20m²。

Blank control: cultivating according to a conventional mode, and applying base fertilizer according to 50 kg/muUsing compound fertilizer, selecting N-P as compound fertilizer₂O₅-K₂O: 15-15-15; the growth state is shown in fig. 16.

Treating the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer: cultivating according to a conventional method, applying a compound fertilizer to the base fertilizer according to 40 kg/mu, wherein the compound fertilizer is N-P₂O₅-K₂O: 15-15-15, and simultaneously applying the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer according to 200 kg/mu; the growth state is shown in fig. 17.

Tomato seedling culture: selecting common tomato seeds, adopting a commercial seedling culture substrate, and sowing the tomato seeds in a plug tray with 75 holes, wherein 1 seed is planted in each hole; after 25 days, transplanting when the tomato seedlings grow to be three leaves and one heart.

Transplanting the tomato seedlings: after the tomatoes grow to three leaves and one heart, transplanting the tomato seedlings into a blank control cell and a disease-resistant tomato hemifacial paralysis prevention and control bio-organic fertilizer treatment cell, and planting 50 plants in each cell.

Disease incidence survey: the number of tomato attacks was regularly observed and recorded, and the incidence was counted as shown in table 3.

Table 3: control and treatment cell morbidity statistics

And (3) analyzing test effects: as can be seen from Table 3, the incidence of the tomatoes in the test is observed after the tomatoes are planted in a fixed manner, wherein the disease is earlier compared with three repeat regions of a plot, the disease symptoms already appear in part of plants before the tomatoes bloom, and the average incidence reaches 70% by statistics after the tomatoes are in a fruiting period; the treatment adopts the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer to partially replace three repeat areas of a fertilizer, the morbidity is obviously reduced, the morbidity in the flowering and fruiting period is only 24.67 percent, the morbidity is reduced by 65.09 percent compared with a control, and the morbidity of tomato soil-borne diseases caused by tomato hemifacial paralysis is obviously reduced.

Example 5: application of strain WSW2 in tomato hemifacial paralysis prevention and control

According to the research, a disease-causing plant and a healthy plant are collected from a disease-causing plot of a tomato continuous cropping tomato, pathogenic microorganisms are separated from the disease-causing plant and identified as Fusarium solani, a plurality of bacillus beijerinckii antagonistic microorganisms are separated and screened from rhizosphere soil of the healthy plant, a high-efficiency bacillus beijerinckii antagonistic microorganism is preferably selected, plate confrontation and pot experiment are carried out, good bacteriostasis and convenience effects are obtained, and a new prevention and control means is provided for biological prevention and control of the tomato 'semifrontier' disease.

(1) Activating strains: the preserved fusarium solani is inoculated to a large PDA slant culture medium for activation, and the Bacillus belgii is inoculated to an LB slant culture medium for activation.

(2) Preparing pathogenic bacteria and preparing antagonistic bacteria: inoculating activated Fusarium solani to PDA plate, culturing at 30 deg.C in incubator, washing Fusarium solani spore with sterile water after 7 days of culture, and adjusting spore concentration to 10⁶CFU/ml as a pathogen inoculation solution; inoculating activated Bacillus belgii into LB liquid medium, shake culturing at 30 deg.C in shaker at 150rpm for 3 days, and determining the amount of antagonistic antibacterial agent to be 10¹⁰CFU/ml, diluted to 10⁸CFU/ml is used as antagonist bacteria inoculum.

(3) Tomato seedling culture: selecting common tomato seeds, adopting a commercial seedling culture substrate, and sowing the tomato seeds in a plug tray with 75 holes, wherein 1 seed is planted in each hole; after 25 days, transplanting when the tomato seedlings grow to be three leaves and one heart.

(4) Preparation of cultivation soil: collecting greenhouse soil in a test area of agricultural academy of sciences of Zhejiang province in dry areas of Hangzhou, Zhejiang province, performing pot culture test, loading 10kg of soil in each pot, and applying 4g of compound fertilizer in each pot according to normal fertilizer application amount, wherein N-P is selected as the compound fertilizer₂O₅-K₂O：15-15-15。

(5) And (3) test treatment: the experiment was set with 2 controls and 1 treatment, which were blank control, fusarium solani pathogen inoculation control and bacillus beilesiensis disease-resistant treatment as shown in fig. 18, respectively; blank control is NoConventional cultivation without inoculation of pathogenic bacteria of fusarium solani and disease-resistant microorganisms of bacillus belgii is carried out by any treatment; inoculating the prepared fusarium solani pathogen spore suspension into soil, and adding the pathogenic pathogen spore suspension according to 100 ml/pot to ensure that the concentration of the fusarium solani pathogen in the soil reaches 10⁴CFU/g; the disease-resistant treatment of the Bacillus beilis comprises inoculating 100ml of Bacillus beilis anti-pathogenic bacteria agent on the basis of inoculating fusarium solani pathogenic bacteria contrast to ensure that the concentration of the Bacillus beilis anti-pathogenic bacteria in soil is 10⁶CFU/g。

(6) Transplanting the tomato seedlings: after the tomatoes grew to three leaves and one heart, as shown in fig. 18, tomato seedlings were transplanted into the corresponding control and treated pots for pot experiments, 1 plant per pot.

(7) Disease incidence survey: the number of tomato diseases was regularly observed and recorded, and the disease incidence was counted, and the statistical result is shown in fig. 19.

And (3) test results: the incidence of the plants in the test is regularly observed, as can be seen from fig. 19, the conventional cultivation blank control potted plant CK without any treatment does not find the occurrence of the tomato hemifacial paralysis in the tomato cultivation; the control CK-F inoculated with fusarium solani pathogenic bacteria starts to attack at the flowering and fruiting period of the tomatoes, and the incidence rate reaches 100 percent; the disease-resistant treatment of the Bacillus belgii has low morbidity which is only 20 percent and is reduced by 80 percent compared with the control CK-F of the fusarium solani.

The screened Bacillus belgii has strong plate inhibition effect, greenhouse potting prevention and control effect and field prevention and control effect on the tomato hemifacial paralysis, enriches the existing tomato hemifacial paralysis biocontrol bacteria resource library, has the Bacillus belgii with antagonistic effect, and has outstanding technical effect on solving the tomato hemifacial paralysis; the invention selects a healthy tomato plant sample planted in a disease area in a Wenzhou region, slightly takes soil to pull out the plant, slightly shakes off loose attached soil on the plant root system, cuts off the soil from a root stem, weighs a plant root system and rhizosphere soil thereof, obtains a rhizosphere microorganism strain with strong nutrition competition capability to the rhizosphere by adopting a gradient dilution separation method, screens antagonistic bacteria again by adopting a plate opposition method after scribing and purification to obtain an antagonistic strain with high-efficiency disease resistance, selects the antagonistic strain with a larger inhibition zone by comparing the size of the inhibition zone, has circular, opaque, light yellow soil and unsmooth edges and folds in the middle, observes by adopting a microscope to find that the cell is in a short rod shape, forms spores which are positioned in the middle of the cell, and determines the classification status of the antagonistic bacteria as Belisbacillus through physiological and biochemical characteristics and 16S gene identification, the similarity of the 16S rRNA gene sequences with Bacillus belgii is 99% at most by comparison in a database, the screened Bacillus belgii is different from other various bacilli, and the application effect of the screened Bacillus belgii on biological prevention and control of soil-borne diseases is rarely reported, particularly on tomato hemifacial paralysis caused by Fusarium solani; the optimal growth conditions of the strain are as follows: the temperature is 30 ℃, the culture medium (LB) is peptone 1% (W/V), yeast extract 0.5% (W/V), sodium chloride 2% (W/V) and agar 2% (W/V), the pH value is 5.0-8.0, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; culturing on an LB flat plate at a constant temperature of 30 ℃ for 60-84 h; the Bacillus velezensis WSW2 provided by the invention is a Bacillus, has higher application value in the aspect of preventing and controlling tomato hemifacial paralysis, and especially can be separated aiming at the local environment and is suitable for the local climate and the growth environment, so that the Bacillus velezensis WSW2 has positive effects on improving the crop yield, reducing the pesticide consumption and constructing a good ecological environment; as a green prevention and control microbial agent vigorously advocated by China at present, the tomato semisideline air antagonistic effect is good, so that the tomato semisideline air morbidity is reduced by 60-80%, the occurrence of diseases is reduced, the yield and quality of agricultural products are greatly improved, and the normal production of tomatoes is ensured; the hidden danger of pollution to agricultural products does not exist in the using process, pathogenic bacteria are not easy to generate drug resistance, and the requirements of weight reduction, drug reduction and green production development mode advocated by the state are met; the tomato hemifacial wind disease prevention and control bio-organic fertilizer has the characteristics of both an organic fertilizer and a novel microbial fertilizer, is rich in organic matters and organic nutrients, can improve the content of organic matters in soil, increase the soil foundation fertility, play a role in loosening the soil, improving the soil aggregate structure and rooting and promoting the root, is rich in functional microbes, can increase the number of useful microbes at the rhizosphere, inhibit the propagation of harmful pathogenic bacteria and enhance the disease resistance of crops, has the functions of improving the nutritional condition and the growth environment of the crops by the life activities of the microbes, stimulating the growth and development of the crops and resisting the harm of diseases and insects, and further achieves the effects of excavating the potential fertility of the soil and improving the yield and quality of agricultural products; the tomato hemifacial paralysis prevention and control bio-organic fertilizer has an activating effect on soil nitrogen, phosphorus and potassium, can also stabilize the supply of nitrogen, phosphorus and potassium nutrients, regulate and control the microbial community structure of soil, inhibit the occurrence of soil-borne diseases and improve the yield and quality of tomatoes.

Introduction of tomato hemifacial paralysis conditions:

the cang-nan county in Wenzhou, Zhejiang province is an important base for supplying spring tomatoes in China, and the planting area of the tomatoes reaches more than 4 ten thousand mu. With the extension of planting years and the popularization and application of exogenous tomato seed seedlings, in recent years, besides common tomato bacterial wilt, blight and other soil-borne diseases, a plurality of new tomato soil-borne diseases appear, wherein tomato hemifacial paralysis is a newly discovered tomato soil-borne disease in recent years. The disease starts to be attacked at the beginning of flowering phase after the tomatoes are planted and gradually becomes worse, leaves of the tomato plants at one side first become yellow and withered when the disease attacks, then the leaves of the tomato plants at the other side also become yellow and withered gradually, meanwhile, a plurality of small raised lumps appear at the base parts of the tomato stems, the stems of the plants which are cut off and attacked can be found, the vascular bundle parts at the roots of the plants become grey and gradually become brown and are lignified, and finally the whole plants become yellow, withered and die. Typical symptoms caused by the disease are similar to the diseases of eggplant caused by hemifacial paralysis, and are not common verticillium wilt caused by verticillium dahliae, and the area is called as tomato hemifacial paralysis. The disease causes tomato incidence is high, the common rate is 50-60%, the serious disease land reaches more than 80%, and even the tomato is not harvested. In the investigation, pathogenic plants are collected to separate pathogenic bacteria, the same pathogenic microorganism is found at the root, the middle part and the top of a plant with serious disease, the pathogenic bacteria is identified to be Fusarium solani (Fusarium solani), the bacterial strain grows well on a PDA flat plate, a large amount of white hypha is generated on the surface of a bacterial colony, the back surface of the bacterial colony is purple red, small-sized conidia are grown in a false head-shaped mode under the observation of a microscope, the spores are oval, and the bacterial strain can cause the occurrence of tomato hemidrosis shown by a tieback test.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> tomato hemifacial paralysis prevention and control bio-organic fertilizer, preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Bacillus velezensis

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 20

<212> DNA

<213> Bacillus velezensis

<400> 2

aaggaggtga tccagccgca 20

<210> 3

<211> 1467

<212> DNA

<213> Bacillus velezensis

<400> 3

ttcggcggct ggctccataa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60

tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120

ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180

atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240

gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300

ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360

gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420

acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480

acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540

cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600

tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc atcgtttacg 660

gcgtggacta ccagggtatc taatcctgtt cgctcccacg ctttcgctcc tcagcgtcag 720

ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780

gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt ccaatgaccc 840

tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg agccctttac 900

gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960

tagccgtggc tttctggtta ggtaccgtca aggtgccgcc ctatttgaac ggcacttgtt 1020

cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 1080

gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 1200

tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagccga 1260

agccaccttt tatgtctgaa ccatgcggtt cagacaacca tccggtatta gccccggttt 1320

cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc gtccgccgct 1380

aacatcagga gaagtcccat ctgtccgctc gacttgatgt attaggcacg ccgccagcgt 1440

tcgcctgagc cagatcaaaa ctctaaa 1467

Claims

1. Tomato hemilateral wind disease prevention and control bio-organic fertilizer, characterized by: the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer contains Bacillus belgii, wherein the preservation number of the Bacillus belgii is CGMCC NO. 19860; the preservation unit of the Bacillus belgii is the China general microbiological culture Collection center; the collection name of the Bacillus belgii is Bacillus belgii velezensis WSW 2; the preservation time of the Bacillus belgii is 5 months and 11 days in 2020.

2. The tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 1, which is characterized in that: in each gram of biological organic fertilizer dry sample, the viable count of the bacillus beleisi is 1.0 multiplied by 10⁸CFU/g～1.0×10¹⁰CFU/g dry sample.

3. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer according to claim 1, which is characterized by comprising the following steps: and mixing the Bacillus beilis microbial inoculum with the decomposed organic material, and performing aerobic composting treatment to obtain the bio-organic fertilizer.

4. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 3, which is characterized in that: the Bacillus belgii microbial inoculum is a liquid microbial inoculum; the viable count of the Bacillus belgii in the liquid microbial inoculum is 1.0 multiplied by 10⁸CFU/ml～1.0×10¹⁰CFU/ml。

5. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 3, which is characterized in that: the preparation method of the Bacillus belgii agent comprises the following steps,

(A) inoculating Bacillus belgii to an LB slant culture medium for activation;

6. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 5, which is characterized in that: in the step (C), the culture temperature of the Bacillus belgii is 25-35 ℃; the concentration of NaCl in the culture solution is 0.5-4%; the pH of the culture solution is 4.0-8.0.

7. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 3, which is characterized in that: the decomposed organic material is decomposed tea leaves.

8. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 7, which is characterized in that: the preparation of the decomposed tea leaves comprises the following steps,

(a) collecting tea branches and leaves;

9. The preparation method of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer as claimed in claim 8, which is characterized in that: when the fermentation temperature in the step (e) is reduced to 37-41 ℃, spraying the microbial agent on the decomposed tea leaves after the first aerobic composting fermentation; wherein the spraying amount of the microbial agent is calculated according to the standard that 10L of the microbial agent is added to each ton of the decomposed tea leaves;

10. Application of the tomato hemifacial paralysis disease prevention and control bio-organic fertilizer in the prevention and control of the tomato hemifacial paralysis disease.