Bacillus subtilis, bacterial agent and plant disease control agent containing the same and their applications

Abstract

The present invention relates to the field of plant control, and discloses a Bacillus subtilis, a bacterial agent containing the bacterium, a plant disease control agent, and their applications. Specifically, the present invention provides a strain of Bacillus subtilis, and the preservation number of the Bacillus subtilis is CGMCC NO.17161. The bacterial agent provided by the present invention contains the above-mentioned Bacillus subtilis. The plant disease control agent provided by the present invention contains the above-mentioned Bacillus subtilis and / or the above-mentioned bacterial agent. The Bacillus subtilis of the present invention has a significant inhibitory effect on bitter melon virus disease, has a good antiviral effect, and has a significant field control effect, and can effectively reduce the occurrence of bitter melon virus disease.

Description

Technical Field

[0001] The invention relates to the field of plant control, in particular to a bacillus subtilis, a bacterial agent containing the bacillus, a plant disease control agent and applications thereof. Background Art

[0002] Bitter melon virus disease is a viral disease caused by single or combined infection of cucumber mosaic virus (CMV) and watermelon mosaic virus (WMV). Bitter melon virus disease is the main disease of bitter melon, which is widely distributed. The disease is more serious in open field planting in summer and autumn. The disease rate is generally 5-10%, which will affect the yield and quality of bitter melon to a certain extent. When the disease is serious, the incidence rate can reach more than 15%, which seriously affects the production of bitter melon. When bitter melon virus disease occurs, the whole plant is generally affected, mainly invading the leaves and growth points of the plant, and the symptoms are most obvious on the top young stems and vines; the leaves of the early infected plants become smaller and wrinkled, the internodes are shortened, the whole plant is obviously dwarfed, and no melons or few melons are produced; the disease occurs in the middle to late stages, the leaves in the middle and upper parts are wrinkled, the leaf color is uneven, the young vines are deformed, the growth is blocked, and the melons are small or twisted; the fields with a high incidence of infected plants have a sharp drop in yield or even a loss of harvest.

[0003] In recent years, due to changes in crop layout and the development of economic crops such as vegetables, the number of virus-source crops and transmission vectors has increased, increasing the infection and harm of viruses, causing their harm to increase year by year, and causing huge losses to bitter melon production. Viral diseases have become an important limiting factor in bitter melon production.

[0004] For a long time, the prevention and control of viral diseases has mainly relied on chemical control and the cultivation of virus-resistant varieties. There are many problems in chemical control. The large-scale application of chemical pesticides has caused pesticide residues, food safety, rampant pests and serious ecological environmental pollution. At present, there are not many satisfactory practical varieties of anti-plant virus agents, especially special therapeutic agents. Breeding disease-resistant varieties is considered to be an economical and effective way to prevent and control bitter melon virus diseases, but there are currently no high-quality and highly resistant varieties that can be promoted on a large scale. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a Bacillus subtilis, a bacterial agent containing the bacterium and a plant disease control agent and their applications. The sterile fermentation liquid and crude protein liquid of the strain have a highly effective inhibitory effect on bitter melon virus disease.

[0006] In order to achieve the above object, the present invention provides a Bacillus subtilis in a first aspect. The deposit number of the Bacillus subtilis is CGMCC NO.17161.

[0007] The second aspect of the present invention provides a bacterial agent, which contains the above-mentioned Bacillus subtilis.

[0008] Preferably, the bacterial agent contains at least one of live bacteria, dead bacteria and fermentation products of the Bacillus subtilis; preferably live bacteria and / or fermentation products.

[0009] Preferably, the bacterial agent is a liquid bacterial agent and / or a solid bacterial agent, preferably a liquid bacterial agent.

[0010] The third aspect of the present invention provides a plant disease control agent, which contains the above-mentioned Bacillus subtilis and / or the above-mentioned bacterial agent.

[0011] Preferably, the method for preparing the plant disease control agent comprises: fermenting the Bacillus subtilis to obtain a fermentation liquid, and subjecting the fermentation liquid to solid-liquid separation I to obtain a sterile fermentation liquid.

[0012] Preferably, the method for preparing the plant disease control agent further comprises: subjecting the sterile fermentation liquid to precipitation and solid-liquid separation II to obtain crude protein, and subjecting the crude protein to dialysis and desalination to obtain a protein solution.

[0013] Preferably, the fermentation culture conditions at least meet the following conditions: inoculation amount of 3-6 volume %, temperature of 28-37° C., rotation speed of 150-250 rpm, and time of 48-90 h;

[0014] The solid-liquid separation process I comprises: centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain the sterile fermentation liquid;

[0015] The centrifugal conditions at least meet the following conditions: temperature of 0-8°C, rotation speed of 8000-12000rpm, and time of 10-20min;

[0016] The pore size of the filter is 0.1-0.45 μm.

[0017] Preferably, the precipitation process comprises: mixing the sterile fermentation liquid with a precipitant and stirring the mixture, and then letting the mixture stand; the precipitant is preferably ammonium sulfate;

[0018] The static conditions at least meet the following requirements: temperature of 0-8°C and time of 10-15h;

[0019] The conditions of the solid-liquid separation II at least meet the following requirements: temperature of 0-8°C, rotation speed of 6000-10000rpm, and time of 10-20min;

[0020] The molecular weight cut-off of the dialysis desalination is 8000-14000.

[0021] The fourth aspect of the present invention provides the use of the above-mentioned Bacillus subtilis, the above-mentioned bacterial agent, and the above-mentioned plant disease control agent in preventing and controlling bitter melon virus disease.

[0022] Through the above technical scheme, the Bacillus subtilis provided by the present invention has a significant inhibitory effect on bitter melon virus disease, and is a green, safe, and environmentally friendly biological control medium. The inhibition rate of the sterile fermentation liquid of Bacillus subtilis on bitter melon virus in vitro measured by the system mosaic method and the local dead spot method is more than 93%; the crude protein liquid prepared by the precipitation of the sterile fermentation liquid of Bacillus subtilis also has an inhibitory effect on bitter melon virus disease, has a good antiviral effect, and has a significant field prevention effect, and can effectively reduce the occurrence of bitter melon virus disease.

[0023] Biological Deposit

[0024] The strain provided by the present invention is Bacillus subtilis, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on January 11, 2019 (the abbreviation of the depository is CGMCC), and the deposit number is CGMCC No.17161. DETAILED DESCRIPTION

[0025] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0026] In the first aspect, the present invention provides a Bacillus subtilis, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on January 11, 2019 (the abbreviation of the depository is CGMCC), and the deposit number is CGMCC No.17161.

[0027] The Bacillus subtilis provided by the present invention is isolated from a healthy bitter melon strain from a bitter melon virus disease-infected field in Changsha City, Hunan Province. The Bacillus subtilis can be isolated by a conventional method for isolating new strains in the art, such as tissue separation.

[0028] The tissue separation method may specifically include: taking the rhizosphere soil of healthy bitter melon plants from the field where bitter melon virus disease occurs, dissolving it with sterile water and stirring it, and then standing it until the soil particles are fully sunk; taking the supernatant and washing it with sterile water at 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 After gradient dilution, spread them in NA culture medium respectively, culture them upside down in a constant temperature incubator, observe the growth of colonies, and promptly pick different single colonies for streaking and purification 2-3 times.

[0029] The Bacillus subtilis provided by the present invention can produce a large amount of live cells and / or fermentation products of Bacillus subtilis after fermentation culture. The present invention has no particular restrictions on the fermentation culture method, as long as the Bacillus subtilis can be proliferated in large quantities by the fermentation culture method. For example, Bacillus subtilis can be inoculated into a liquid culture medium with an inoculation amount of 3-6 volume%, and the fermentation liquid is obtained after shaking culture for 48-90 hours at a temperature of 28-37°C and a rotation speed of 150-250rpm. Wherein, the culture medium can be a culture medium conventionally used in the art, for example, the liquid culture medium can be NB culture medium, the concentration of peptone is 8-12g / L, the concentration of beef extract powder is 2-4g / L, the concentration of sodium chloride is 4-6g / L, and the pH is 7-7.4.

[0030] In a second aspect, the present invention provides a bacterial agent, which contains the above-mentioned Bacillus subtilis.

[0031] In the present invention, there is no particular limitation on the concentration of Bacillus subtilis in the bacterial agent, and the concentration can be selected according to specific circumstances.

[0032] According to the present invention, the bacterial agent preferably contains at least one of live cells, dead cells and fermentation products of the Bacillus subtilis, more preferably live cells and / or fermentation products. In the present invention, the term "fermentation product" refers to metabolites (including intracellular metabolites and / or extracellular metabolites) produced by Bacillus subtilis during fermentation or cultivation. Preferably, the bacterial agent contains extracellular metabolites of Bacillus subtilis.

[0033] According to the present invention, there is no particular limitation on the dosage form of the microbial agent. It can be prepared into different dosage forms according to different intended uses, and corresponding auxiliary materials (excipients) and other ingredients can be added. For example, the microbial agent can be a liquid microbial agent and / or a solid microbial agent, preferably a liquid microbial agent, for example, a sterile fermentation liquid obtained by removing the bacterial cells from the fermentation liquid of Bacillus subtilis, or a sterile fermentation liquid obtained by removing the bacterial cells from the fermentation liquid of Bacillus subtilis, and then the sterile fermentation liquid is precipitated and separated to obtain a crude protein liquid. Among them, what kind of auxiliary materials are added to what dosage form of the microbial agent is well known to those skilled in the art, and will not be described in detail here.

[0034] In a third aspect, the present invention provides a plant disease control agent, which contains the above-mentioned Bacillus subtilis and / or the above-mentioned bacterial agent.

[0035] According to the present invention, the plant disease control agent contains at least one of the live bacteria, dead bacteria and fermentation products of the Bacillus subtilis, and the plant disease control agent may also include other herbicidal active ingredients, which include chemical substances and / or living organisms.

[0036] According to the present invention, the plant disease control agent can be a sterile fermentation liquid obtained by removing the bacterial cells from the fermentation liquid of Bacillus subtilis. The specific preparation process includes: fermenting the Bacillus subtilis to obtain a fermentation liquid, and subjecting the fermentation liquid to solid-liquid separation I to obtain a sterile fermentation liquid.

[0037] Preferably, the fermentation culture conditions at least meet: the inoculation amount is 3-6 volume%, specifically 3 volume%, 4 volume%, 5 volume%, 6 volume%, or any value between the above two values; the temperature is 28-37°C, specifically 28°C, 30°C, 32°C, 34°C, 36°C, 37°C, or any value between the above two values; the rotation speed is 150-250rpm, specifically 150rpm, 170rpm, 190rpm, 210rpm, 230rpm, 250rpm, or any value between the above two values; the time is 48-90h, specifically 48h, 58h, 68h, 78h, 88h, 90h, or any value between the above two values.

[0038] According to the present invention, solid-liquid separation I can adopt any conventional separation method, such as filtration and centrifugation. Preferably, the process of solid-liquid separation I includes: centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain the sterile fermentation liquid. The centrifugation conditions at least meet the following conditions: the temperature is 0-8°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, or any value between the above two values; the speed is 8000-12000rpm, specifically 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, or any value between the above two values; the time is 10-20min, specifically 10min, 12min, 14min, 16min, 18min, 20min, or any value between the above two values; the pore size of the filtration is 0.1-0.45μm, specifically 0.22μm, 0.45μm, or other pore size specifications within this range.

[0039] According to the present invention, the plant disease control agent can be a protein extract produced by fermentation of Bacillus subtilis. The specific preparation process can include: subjecting the above-mentioned sterile fermentation liquid to precipitation and solid-liquid separation II to obtain crude protein, and subjecting the crude protein to dialysis and desalination to obtain protein solution, that is, using the protein solution as the plant disease control agent.

[0040] According to the present invention, the precipitation process comprises: mixing the sterile fermentation liquid with the precipitant and stirring the mixture and then letting it stand.

[0041] Preferably, the precipitant is ammonium sulfate; the amount of the precipitant is preferably 50-60:100 in a mass volume ratio (w / v) to the sterile fermentation broth. Exemplarily, the precipitant is slowly added to the sterile fermentation broth in an ice water bath while stirring to precipitate the protein in the sterile fermentation broth.

[0042] According to the present invention, the standing conditions at least meet: the temperature is 0-8°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, or any value between the above two values; the time is 10-15h, specifically 10h, 11h, 12h, 13h, 14h, 15h, or any value between the above two values.

[0043] According to the present invention, according to the present invention, solid-liquid separation II can adopt any conventional separation method, such as filtration, centrifugation. Preferably, the conditions of the solid-liquid separation II at least meet the following conditions: the temperature is 0-8°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, or any value between the above two values; the rotation speed is 6000-10000rpm, specifically 6000rpm, 7000rpm, 8000rpm, 9000rpm, 10000rpm, or any value between the above two values; the time is 10-20min, specifically 10min, 12min, 14min, 16min, 18min, 20min, or any value between the above two values. The molecular weight cutoff of the dialysis desalination is 8000-14000.

[0044] In a fourth aspect, the present invention provides the use of the above-mentioned Bacillus subtilis, the above-mentioned bacterial agent, and the above-mentioned plant disease control agent in preventing and controlling bitter melon virus disease.

[0045] The Bacillus subtilis provided by the invention has a significant inhibitory effect on bitter melon virus disease. The inhibition rate of the aseptic fermentation liquid of the Bacillus subtilis on bitter melon virus in vitro measured by the system mosaic method and the local dead spot method reaches more than 93%. The crude protein liquid prepared by precipitation of the aseptic fermentation liquid of the Bacillus subtilis also has an inhibitory effect on bitter melon virus disease, has a good antiviral effect, and has a significant field prevention effect, and can effectively reduce the occurrence of bitter melon virus disease.

[0046] The present invention will be described in detail below through examples.

[0047] In the following examples, the components of the NA medium are: peptone concentration of 10 g / L, beef extract concentration of 3 g / L, sodium chloride concentration of 5 g / L, agar 15 g / L, pH of 7.3, and high pressure steam sterilization at 121°C for 30 min;

[0048] The components of NB medium are: peptone concentration is 10g / L, beef extract concentration is 3g / L, sodium chloride concentration is 5g / L, pH is 7.2, and high-pressure steam sterilization is performed at 121°C for 30 minutes.

[0049] In the following examples, healthy bitter melon and bitter melon virus disease susceptible plants were collected from a bitter melon virus disease field in Changsha City, Hunan Province, and other raw materials and reagents were commercially available.

[0050] Example 1

[0051] Several healthy bitter melon plants were found from a bitter melon virus disease-infected field in Changsha City, Hunan Province. 5 g of rhizosphere soil was taken and diluted to 50 mL with sterile water. After magnetic stirring for 30 min, the soil was allowed to stand until the soil particles were fully settled. The supernatant was taken and diluted with sterile water to 10 -2, 10 -3 , 10 -4 , 10 -5 , 10 -6 After gradient dilution, the strains were spread on NA medium and incubated in a 28°C constant temperature incubator for 1-3 days. The growth of the colonies was observed and different single colonies were streaked and purified 2-3 times to obtain pure culture strain me-1.

[0052] The isolated pure culture strain me-1 was grown on NA medium plates at 28°C. The colonies were white and opaque, dull, with irregular edges, water-soaked and sticky in the early stage, and wrinkled in the later stage. They were Gram-positive, endogenous spores, with capsules and flagella, and were short rod-shaped bacteria with blunt ends.

[0053] The genomic DNA of the isolated pure culture strain me-1 was extracted, and the universal primers 27F / 1492R were used to perform PCR amplification of the 16SrDNA fragment. The amplified fragment was sequenced by Shanghai Bioengineering Co., Ltd., and the measured sequence was compared for homology in the NCBI database. The results showed that the homology of its 16S rDNA sequence with Bacillus subtilis was the highest, reaching 99.3%. Through morphological observation and 16S rDNA sequence molecular identification, it was determined that the strain of the present invention was Bacillus subtilis (Bacillus subtilis), and was named Bacillus subtilis me-1.

[0054] According to the results of morphological identification and molecular biological identification, the strain was identified as Bacillus subtilis, and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 11, 2019, with the deposit number CGMCC No.17161.

[0055] Example 2

[0056] (1) After activating the Bacillus subtilis me-1 isolated in Example 1, the inoculum was inoculated into NB medium at an inoculum amount of 3%, and fermented at a temperature of 37° C. and a rotation speed of 150 rpm for 90 h to obtain a fermentation liquid;

[0057] (2) The fermentation liquid obtained in step (1) was centrifuged at 0°C and 8000 rpm for 20 min to remove the bacteria to obtain a supernatant, and the supernatant was filtered through a bacterial filter (pore size 0.45 μm) to obtain a sterile fermentation liquid.

[0058] Example 3

[0059] (1) Activating the Bacillus subtilis me-1 isolated in Example 1, inoculating it into NB medium at an inoculum amount of 4 volume %, and fermenting it for 64 hours at a temperature of 32° C. and a rotation speed of 180 rpm to obtain a fermentation liquid;

[0060] (2) centrifuging the fermentation liquid obtained in step (1) at a temperature of 4° C. and a rotation speed of 10,000 rpm for 15 min to remove the bacteria to obtain a supernatant, and filtering the supernatant through a bacterial filter (pore size of 0.45 μm) to obtain a sterile fermentation liquid;

[0061] (3) placing the sterile fermentation broth obtained in step (2) in an ice water bath, slowly adding ammonium sulfate (the mass volume ratio of ammonium sulfate to the sterile fermentation broth is 56.1:100), stirring while adding, and then standing at 4° C. for 12 hours to precipitate the protein in the sterile fermentation broth, and centrifuging at 4° C. and 8000 rpm for 15 minutes to obtain a crude protein solid and a deproteinized liquid;

[0062] (4) The crude protein solid obtained in step (3) is dissolved in PBS, placed in a dialysis bag (molecular weight cut-off: 8000-14000), and dialyzed to remove salt to obtain a protein solution.

[0063] Example 4

[0064] (1) After activating the Bacillus subtilis me-1 isolated in Example 1, the inoculum was inoculated into NB medium at an inoculum amount of 6 volume %, and fermented at a temperature of 28° C. and a rotation speed of 250 rpm for 48 hours to obtain a fermentation liquid;

[0065] (2) The fermentation liquid obtained in step (1) was centrifuged at 8°C and 12000 rpm for 10 min to remove the bacteria to obtain a supernatant, and the supernatant was filtered through a bacterial filter (pore size 0.22 μm) to obtain a sterile fermentation liquid.

[0066] Test Example 1

[0067] 0.1 g of dry leaves infected with bitter melon virus were ground into a homogenate, and PBS buffer was added at a mass volume ratio of 1:20 (W / V) to prepare a 20-fold bitter melon virus solution; healthy bitter melon plants with uniform growth and at the 6-leaf stage were selected and divided into a treatment group and a control group (5 replicates were set for each group), and the whole plant was sprayed and inoculated, spraying water 2-3 times a day, and the number of diseased leaves was counted after 3 days, and the inhibition rate was calculated. The results are shown in Table 1, and the prevention effect was calculated by the number of diseased leaves; wherein the spraying liquid of the treatment group was equal volumes of the sterile fermentation liquid obtained in step (2) of Example 3 and the bitter melon virus solution, and the spraying liquid of the control group was prepared by equal volumes of NB culture medium and virus mixed solution, and the mixing time was 10 minutes.

[0068] The modes of action on the pathogen are divided into three types: spraying the sterile fermentation liquid obtained in step (2) of Example 3 with the bitter melon virus liquid after mixing for 10 minutes, spraying the sterile fermentation liquid obtained in step (2) of Example 3 6 hours and 24 hours later and then spraying an equal volume of the bitter melon virus liquid, and spraying the bitter melon virus liquid for 6 hours and 24 hours respectively and then spraying the sterile fermentation liquid obtained in step (2) of Example 3, which are recorded as the three treatments of passivation effect, preventive effect, and therapeutic effect, respectively.

[0069] Inhibition rate = (number of control diseased leaves - number of treated diseased leaves) / number of control diseased leaves × 100%.

[0070] Table 1 Inhibitory effect of sterile fermentation liquid on bitter melon virus disease

[0071]

[0072] As shown in Table 1, the sterile fermentation liquid obtained in step (2) of Example 3 exhibited an obvious passivation effect when acting on bitter melon virus disease. The inhibition rate of both was 71.43% when they were passivated in vitro for 10 minutes. The preventive effect was 66.67% and 50% when the bitter melon virus liquid was inoculated 6 hours and 24 hours after spraying the sterile fermentation liquid, respectively. The therapeutic effect was 40% and 50% when the sterile fermentation liquid was sprayed 6 hours and 24 hours after inoculation of the virus, respectively.

[0073] Test Example 2

[0074] The sterile fermentation liquid obtained in step (2) of Example 3, the crude protein liquid and the deproteinized liquid obtained in step (3) were respectively sprayed on the bitter melon virus disease susceptible plants to observe the inhibition rate of bitter melon virus, and the results are shown in Table 2. As shown in Table 2, compared with the sterile fermentation liquid, the antiviral effect of the crude protein liquid is reduced, but much higher than that of the deproteinized liquid; based on this, it can be preliminarily determined that the effective component of the Bacillus subtilis provided by the present invention against bitter melon virus disease is protein.

[0075] Table 2

[0076] Treatment fluid Sterile fermentation broth Crude protein liquid Protein removal solution Inhibition rate 93.59％ 85.32％ 27.43％

[0077] Test Example 3

[0078] A bitter melon virus-infected field in Ningxiang, Changsha City, Hunan Province was selected, and 12 rows of healthy bitter melon plants with equal growth and soil fertility were selected, with 40 plants in each row. The disease was naturally caused, and rows 1-6 were marked as blank groups (sprayed with clean water), and rows 7-12 were marked as treatment groups (sprayed with the sterile fermentation liquid prepared in Example 2). The drugs were used three times on March 27, April 1, and April 5, 2019, respectively. After 30 days, the incidence of bitter melon virus disease was investigated. The results are shown in Table 3.

[0079] Investigate the bitter melon plants in each group, classify them according to the disease classification standard, and calculate the disease index of each treatment. The disease classification standard and formula are as follows:

[0080] Level 0: New and old leaves have no chlorosis, mosaic, bright veins, yellowing and other virus symptoms;

[0081] Level 1: New leaves show chlorosis, mosaic, bright veins, and yellowing;

[0082] Level 3: A large number of new leaves are chlorotic, mosaic, yellow, and wrinkled;

[0083] Level 5: More than half of the leaves of the whole plant are chlorotic, yellow, mosaic, and wrinkled;

[0084] Level 7: The leaves of the entire plant become chlorotic, yellow, mosaic, and wrinkled, accompanied by dwarfing and even death.

[0085] Incidence rate (%) = number of diseased plants / total number of plants investigated × 100,

[0086] Disease index = ∑ (number of diseased plants at each level × relative level value) / (total number of plants investigated × 7) × 100

[0087] Relative control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0088] Table 3

[0089]

[0090] After three times of drug treatment, a survey on the incidence of bitter melon was conducted on May 5. As shown in Table 3, the aseptic fermentation liquid of the Bacillus subtilis strain obtained by the present invention has a reduced incidence rate and disease index compared with clean water, and the prevention effect is more significant. The aseptic fermentation liquid of the Bacillus subtilis obtained by the present invention has a field prevention effect of 59.12% on bitter melon virus disease. Therefore, the Bacillus subtilis of the present invention has an effective component for resisting bitter melon virus disease, and has a certain reference value for the prevention and treatment of this disease.

[0091] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A plant disease control agent for preventing and controlling bitter melon virus disease, characterized in that: The plant disease control agent contains a sterile fermentation liquid of Bacillus subtilis, wherein the Bacillus subtilis ( Bacillus subtilis )’s deposit number is CGMCC NO.17161.

2. The plant disease control agent according to claim 1, characterized in that The preparation method of the plant disease control agent comprises: fermenting the Bacillus subtilis to obtain a fermentation liquid, and separating the fermentation liquid into a solid-liquid to obtain a sterile fermentation liquid.

3. The plant disease control agent according to claim 2, characterized in that: The fermentation culture conditions at least meet the following requirements: inoculation volume of 3-6% by volume, temperature of 28-37° C., rotation speed of 150-250 rpm, and time of 48-90 h; The solid-liquid separation process includes: centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain the sterile fermentation liquid; The centrifugal conditions at least meet the following conditions: temperature of 0-8°C, rotation speed of 8000-12000rpm, and time of 10-20min; The pore size of the filter is 0.1-0.45 μm.

4. Use of the plant disease control agent according to any one of claims 1 to 3 in controlling bitter melon virus disease.

Images