Bacillus subtilis, bacterial agent and application thereof for preventing and treating citrus canker

Abstract

The invention provides a Bacillus subtilis for preventing and treating citrus canker, a bacterial agent and an application thereof, belonging to the technical field of microorganisms. The Bacillus subtilis is classified and named as Bacillus subtilis Bacillus subtilis , the deposit number is CGMCC No.24778, and the deposit date is April 26, 2022; compared with other Bacillus subtilis, the Bacillus subtilis of the present application has a stronger preventive and therapeutic effect on citrus canker. The Bacillus subtilis JQ-K98 of the present invention treats the citrus detached leaves inoculated with the canker pathogen, and the incidence of canker is only 6.25%, and the preventive and therapeutic effect of citrus detached leaf canker is 92.86%; the Bacillus subtilis JQ-K98 of the present invention is used for field prevention and control of citrus canker, and the prevention effect is 87.84%.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and specifically provides a Bacillus subtilis, a bacterial agent and application thereof for preventing and treating citrus canker. Background Art

[0002] The main symptoms of citrus canker are yellow, oil-soaked spots the size of a needle tip on the leaves, most of which appear on the back of the leaves and gradually expand into a circle with bulges on all sides and a depression in the middle, resembling a crater. The symptoms on the fruit are similar to those on the leaves and are less severe on the branches, but no matter where they are, they will eventually weaken the tree, and in severe cases will lead to early leaf and flower drop, tree decline, reduced fruit yield, and lower quality, affecting the economic income of growers.

[0003] Citrus canker is a devastating global quarantine disease that has occurred in many countries and regions around the world, causing great economic losses to the citrus industry.

[0004] Citrus canker occurs when lesions on old leaves are the source of infection. When the temperature and air humidity are suitable, the disease overflows and spreads through wind, rain, and insects, infecting young citrus tissues through the host's stomata and wounds. Therefore, conventional measures to prevent and control canker include pest control, timely garden cleaning, and pruning of diseased branches and leaves, which can reduce or prevent the occurrence and spread of canker. However, this method is costly and the effect is not guaranteed.

[0005] Copper preparations are commonly used in the chemical control of citrus canker, which can reduce the accumulation of fungi on new leaves and alleviate subsequent infections. However, excessive use of copper preparations has led to outbreaks of red spider mites in citrus orchards, excessive copper content in fruits, and easy liver damage when consumed. In addition, the control effect is getting worse and worse.

[0006] Microorganisms are an important part of biological control. Some microorganisms can directly inhibit the growth of pathogens by secreting secondary metabolites such as antibiotics; some microorganisms can also compete with pathogens for iron ions by secreting siderophores, thereby indirectly inhibiting the growth of pathogens; some microorganisms can enhance the disease resistance of plants by inducing systemic resistance in plants.

[0007] However, the microbial agents in the prior art are less effective in preventing and treating citrus canker. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a Bacillus subtilis, a bacterial agent and its application for preventing and treating citrus canker, to achieve the following invention objectives:

[0009] A microorganism with good effect in preventing and controlling citrus canker was screened and used for preventing and controlling citrus canker. The prevention efficiency in field experiments reached more than 85%; at the same time, it can also improve the quality of citrus fruits.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A bacillus subtilis for preventing and treating citrus canker, wherein the classification of the bacillus subtilis is named as bacillus subtilis Bacillus subtilis The deposit number is CGMCC No.24778, and the deposit date is April 26, 2022.

[0012] The following are further improvements to the above technical solution:

[0013] A bacterial agent for preventing and treating citrus canker, the bacterial agent is the fermentation liquid of the above-mentioned Bacillus subtilis, and the bacterial content of Bacillus subtilis in the bacterial agent is 112×10 8 cfu / mL.

[0014] The method for preparing the fermentation liquid of Bacillus subtilis comprises strain activation, seed liquid preparation and fermentation; the fermentation comprises primary seed tank culture, secondary seed tank culture and fermentation tank culture.

[0015] The method for activating the strain is to perform three-zone streaking on the Bacillus subtilis on the LB solid culture medium, and culture it at 37° C. for 24 hours to obtain the activated strain.

[0016] The method for preparing the seed solution is to pick a single colony from the activated strain and inoculate it into 150 mL of seed culture medium, and culture it at 37° C. and 200 rpm for 14 hours to obtain the seed solution.

[0017] The method for culturing in the first-level seed tank is as follows: according to an inoculation amount of 10% by volume, the seed liquid is connected to the seed culture medium of the first-level seed tank, the temperature is controlled to be 37°C, the ventilation is 0.8V / Vm, the pH is 7.5, the rotation speed is 200 rpm, and the culturing time is 12h to obtain a first-level seed culture; the method for culturing in the second-level seed tank is as follows: according to an inoculation amount of 10%, the first-level seed culture is inoculated into the seed culture medium of the second-level seed tank, the temperature is controlled to be 37°C, the ventilation is 0.8 V / Vm, the pH is 7.5, the rotation speed is 100 rpm, and the culturing time is 12h, sampling and microscopic examination of the bacteria are in the logarithmic growth phase to obtain a second-level seed culture; for the fermentation tank culture, the second-level seed culture is transferred to the fermentation tank for culturing, the temperature is controlled to be 37°C, the ventilation volume from 0 to 6h is 0.8V / Vm, the ventilation volume after 6h is adjusted to 2V / Vm, the pH is 7.5, the rotation speed is 100 rpm, and the culturing time is 32h.

[0018] The seed culture medium and the fermentation culture medium in the fermenter are composed of the following ingredients, calculated by mass percentage: 3% maltodextrin, 6.5% soybean meal powder, 0.3% glucose, 0.1% magnesium sulfate heptahydrate, 0.2% calcium carbonate, 0.05% disodium hydrogen phosphate, 0.02% manganese sulfate, 0.15% defoaming agent, and deionized water is supplemented to 100%.

[0019] The bacterial agent is used for preventing and controlling citrus canker.

[0020] Dilute the bacterial agent with clean water to 500 million / mL to obtain the bacterial agent dilution solution, and spray 50L of the bacterial agent dilution solution on each acre of leaves.

[0021] The strain JQ-K98 screened by the present invention is classified and named as Bacillus subtilis Bacillus subtilis , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 26, 2022, and the deposit number is CGMCC No.24778.

[0022] Compared with the prior art, the present invention achieves the following beneficial effects:

[0023] 1) The Bacillus subtilis JQ-K98 of the present invention has an inhibition zone diameter of 2.27 cm against the pathogen of citrus canker.

[0024] 2) The Bacillus subtilis JQ-K98 of the present invention was used to treat detached leaves infected with citrus canker. The incidence rate on the 10th day was only 6.25%, while the incidence rate of the control group was 87.5%. The control effect of the Bacillus subtilis JQ-K98 of the present invention on the detached leaves infected with citrus canker was 92.86%.

[0025] 3) The existing Bacillus subtilis has a poor control effect on citrus canker. The Bacillus subtilis JQ-K98 of the present invention has a stronger control effect on citrus canker than the existing Bacillus subtilis.

[0026] The Bacillus subtilis JQ-K98 of the present invention is used for controlling citrus canker in the field, and the prevention effect reaches 87.84%; the existing Bacillus subtilis agent is used for controlling citrus canker in the field, and the prevention effect is only 25.08%, and the prevention effect of copper preparations is 44.22%; it can be seen that the Bacillus subtilis of the present invention has a higher prevention effect on citrus canker than the existing Bacillus subtilis and copper preparations, and achieves unexpected technical effects.

[0027] 4) The microbial agent described in the present application is used to prevent and treat citrus canker, which can solve the problem of excessive heavy metals caused by the large-scale application of existing copper preparations, and can improve the quality and yield of fruits and increase the income of fruit farmers.

[0028] After the citrus is sprayed with the microbial agent of the present invention, the single fruit weight is 170.6g, the soluble solid content is 13.9wt%, the reducing sugar content is 5.2wt%, the solid-acid ratio is 29.3, the sugar-acid ratio is 10.9, and the vitamin C content is 26.6mg / 100g. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the colony morphology of Bacillus subtilis JQ-K98 on a plate;

[0030] Figure 2 This is a diagram showing the antibacterial effect of Bacillus subtilis JQ-K98 on citrus canker bacteria;

[0031] Figure 3 This is a graph showing the protective effect of Bacillus subtilis JQ-K98 of the present invention on citrus detached leaves inoculated with pathogenic bacteria of ulcer disease;

[0032] 3a is the water control, 3b is the leaves treated with Bacillus subtilis JQ-K98;

[0033] Figure 4 This is a picture of the field control of citrus canker in Example 5. DETAILED DESCRIPTION

[0034] Example 1 Acquisition of JQ-K98 strain

[0035] The Bacillus subtilis (JQ-K98 strain) of the present invention is isolated from the citrus root soil of Naxi Village, Xianhu Town, Wuming District, Nanning City, Guangxi.

[0036] The specific separation method is as follows:

[0037] (1) Soil collection: 100 g of soil sample was collected from the soil surface to 10 cm. Five samples were collected in parallel at one location, mixed and passed through a 40-mesh sieve, and 100 g was taken for subsequent separation tests.

[0038] (2) Culture medium preparation: Prepare NA culture medium and PDA culture medium separately and make culture medium plates.

[0039] (3) Separation and activation

[0040] Accurately weigh 10 g of soil sample and place it in a 250 mL conical flask. Add 99 mL of sterile water and place it on a shaker. Shake at 200 rpm and 37 °C for 30 min to make a soil suspension. Dilute it to 10 by 10 times in a gradient. -8 , obtaining a diluted soil suspension.

[0041] The diluted soil suspension was isolated from single colonies in NA medium and PDA medium, respectively.

[0042] NA medium is used to isolate bacteria. The single colony isolated on the NA medium is further activated and cultured in the NA medium at a temperature of 37° C. for 4 days to obtain an activated NA medium plate.

[0043] PDA medium is used to isolate fungi. The single colony isolated on the PDA medium is further activated and cultured in the PDA medium. The activation temperature is 28° C. and the activation time is 8 days to obtain a PDA medium plate after activation and culture.

[0044] (4) Screening

[0045] The isolated and activated single colonies were subjected to a plate confrontation experiment with the purified cultured citrus canker pathogen. The following is the screening process confrontation experiment and its results:

[0046] The citrus canker pathogen was prepared as 10 6 Take 100 μL of the bacterial suspension with a concentration of 100 cfu / mL and spread it evenly on the NA medium plate. Punch holes on the NA medium plate and the PDA medium plate after activation and culture in step (3) to isolate multiple single colonies. Place the single colonies on the plate coated with pathogens, invert and culture at 37°C for 3 days, and measure the diameter of the inhibition zone.

[0047] Table 1 Diameter of inhibition zone of isolated single colonies against ulcer pathogens (cm)

[0048]

[0049] As can be seen from Table 1, the average diameter of the inhibition zone of the JQ-K98 strain reached 2.27 cm, and it had the highest antibacterial activity against citrus canker.

[0050] The JQ-K98 strain was stored on LB slant medium for later use.

[0051] Example 2 Identification of JQ-K98 strain

[0052] The JQ-K98 strain isolated and activated in Example 1 was identified as follows:

[0053] 1. Microbiological characteristics: After JQ-K98 strain was cultured on NA medium at 37℃ for 1 day, single colonies were round, milky white, opaque, with irregular edges, wrinkled and raised surfaces. It was identified as a positive bacterium by Gram staining, rod-shaped, spore-forming, and aerobic growth (see Figure 1 ).

[0054] 2. Molecular biology identification:

[0055] The 16S rDNA sequence of the JQ-K98 strain was sequenced to obtain a 1414 bp long base sequence, as shown in SEQ ID NO.1 in the sequence table. It was compared with the NCBI database. Bacillus subtilis The 16S rDNA sequence homology of Y17B strain (GenBank: MT571500.1) reached 100%. Combined with the morphological characteristics, physiological and biochemical properties of JQ-K98 strain, it was determined that the detected JQ-K98 strain belonged to Bacillus subtilis.

[0056] The strain JQ-K98 screened by the present invention is classified and named as Bacillus subtilis Bacillus subtilis , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 26, 2022, and the deposit number is CGMCC No.24778.

[0057] Example 3 Cultivation of Bacillus subtilis JQ-K98

[0058] (1) Preparation of culture medium

[0059] The composition of LB liquid culture medium: peptone 10g, yeast extract powder 5g, sodium chloride 10g, deionized water to 1000mL, pH 7.0.

[0060] The composition of LB solid medium: peptone 10g, yeast extract powder 5g, sodium chloride 10g, agar 15g, deionized water to 1000mL, pH 7.0.

[0061] The composition of the seed culture medium and fermentation culture medium of Bacillus subtilis JQ-K98, by mass percentage, includes the following components: 3% maltodextrin, 6.5% soybean meal, 0.3% glucose, 0.1% magnesium sulfate heptahydrate, 0.2% calcium carbonate, 0.05% disodium hydrogen phosphate, 0.02% manganese sulfate, 0.15% defoaming agent, and deionized water makes up to 100%.

[0062] (2) Strain activation

[0063] The Bacillus subtilis JQ-K98 preserved on the LB slant medium was picked up, three-zoned on the LB solid medium, and cultured at 37° C. for 24 h to obtain an activated strain.

[0064] (3) Seed solution preparation

[0065] Take a 1000ml Erlenmeyer flask and fill it with 150ml of seed culture medium; pick a single colony from the activated strain and inoculate it into the Erlenmeyer flask, culture it at 37°C and 200 rpm for 14h to obtain seed solution.

[0066] (4) Fermentation

[0067] A. Primary seed tank culture

[0068] The seed tank is filled with seed culture medium, and seed liquid is added according to 10% inoculation amount (volume ratio). The culture conditions are as follows: temperature 37°C, ventilation 0.8V / Vm, pH 7.5, rotation speed 200 rpm, and culture time 12h to obtain a primary seed culture.

[0069] B. Secondary seed tank culture

[0070] The primary seed culture was inoculated into the secondary seed tank at a 10% inoculation rate. The culture conditions were: temperature 37°C, ventilation 0.8 V / Vm, pH 7.5, rotation speed 100 rpm, and culture time 12 h. Samples were taken for microscopic examination and the bacteria were in the logarithmic growth phase to obtain the secondary seed culture.

[0071] C. Fermentation tank culture

[0072] The secondary seed culture was transferred to the fermentation tank for culture. Culture conditions: temperature 37°C, ventilation: 0.8V / Vm for 0-6h, gradually adjusted to 2V / Vm after 6h, pH 7.5, speed 100 rpm, culture time 32h. The sporulation rate of sampling microscopy reached 92%, and the fermentation was completed. The fermentation liquid of Bacillus subtilis JQ-K98 was obtained, and the number of live bacteria in the fermentation liquid was 112×10 8 cfu / mL.

[0073] (5) Antibacterial effect of flat plate

[0074] Pick a single colony of citrus canker bacteria from the plate, inoculate it into a shake flask containing 30 mL of liquid LB medium, and culture it at 37°C and 200 rpm for 12 hours to obtain the canker bacteria culture solution. Take 100 μL of it and spread it evenly on the plate; take 5 μL of Bacillus subtilis JQ-K98 fermentation solution and spot it on the plate coated with canker bacteria, with 3 spots on one plate in a triangular distribution; place the front side in a 37°C incubator and culture it for 48 hours to observe whether there is an inhibition zone, such as Figure 2 As shown. Figure 2 It can be seen that Bacillus subtilis JQ-K98 exhibits a good inhibitory effect on citrus canker bacteria, and the diameters of the inhibition zones at the three points are 2.2 cm, 2.3 cm, and 2.3 cm, respectively.

[0075] Example 4 Experiment on the efficacy of JQ-K98 against citrus detached leaves inoculated with pathogenic bacteria of ulcer disease

[0076] (1) Collect new leaves that have just fully expanded from citrus (the cultivar is Wogan) plants. Each group is treated with 8 leaves. The surface of the leaves is rinsed clean with sterile water, dried at room temperature, and placed in a sterilized plate lined with moisturizing filter paper.

[0077] (2) Take a slant of pure bacteria of ulcer pathogen activated at 28°C for 3 days, wash out the bacterial moss with 5 ml of sterile water and prepare a bacterial suspension stock solution (bacterial concentration is 105×10 8 cfu / mL), and then diluted to 10 by 10-fold dilution method -3 times to obtain a bacterial suspension for inoculation.

[0078] (3) Inoculation method: Use a sterilized inoculation needle to puncture a small hole in the leaf. Then use a sterilized filter paper disc (diameter 5mm) to absorb enough bacterial suspension for inoculation (about 50 uL) and stick it on the puncture point. Every 2 puncture points form a group, 3 groups per leaf, a total of 6 puncture points. The inoculated leaves are placed in a culture dish moisturized with absorbent paper (place 2 layers of ordinary absorbent paper in the culture dish and spray with sterile water), 2 leaves are placed in each dish, 4 dishes are treated, and cultured at 30℃. During the culture period, keep a certain humidity in the dish (wrap the petiole with soaked cotton to keep it moist), and remove the filter paper after 24 hours.

[0079] (4) After the filter disc was removed, 2 mL of Bacillus subtilis JQ-K98 fermentation liquid was sprayed into the culture dish of the treatment group;

[0080] The control group was sprayed with the same amount of sterile water.

[0081] (5) Observe the disease condition of the leaves. At the same time, pay attention to replenishing the water (sterile water) in the culture dish in time to ensure the humidity inside the culture dish. Check the disease condition once a day and count the incidence rate of the acupuncture point. The judgment is based on whether small lesions that can be seen by the naked eye appear.

[0082] (6) The final incidence rate and control effect were calculated after 10 days (see Tables 2 and 3).

[0083] Incidence rate (%) = (number of acupuncture point incidences / total number of acupuncture points) × 100%

[0084] Prevention and treatment effect (%) = (number of acupuncture point cases in the control group - number of acupuncture point cases in the treatment group) / number of acupuncture point cases in the control group × 100%

[0085] Table 2 Number of acupuncture point incidences

[0086]

[0087] From Table 2, it can be seen that the initial onset time of citrus canker on citrus leaves in the experimental group was generally significantly delayed compared with the control group, and the number of cases in the experimental group was significantly reduced compared with the control group on the 10th day. In addition, the number of cases in the control group stabilized on the 5th day, while the number of cases in the experimental group remained stable around the 8th day, so the stabilization period of the number of cases in the experimental group was also delayed compared with the control group.

[0088] Table 3 Morbidity and control effects on the 10th day

[0089]

[0090] From Table 3, it can be seen that the morbidity of the water control group is 87.50%, while the morbidity of the strain JQ-K98 test group is 6.25%, and the prevention effect can reach 92.86%. The morbidity of the test group has been significantly reduced compared with the control group, indicating that the strain JQ-K98 of the present invention has a good prevention and control effect on citrus canker (see Figure 3 ).

[0091] Example 5 Field plot test of microbial inoculants

[0092] (1) Selection of trial sites

[0093] An ulcer disease-affected area was selected in Liangjiang Town, Wuming District, Nanning City, Guangxi Province for the experiment.

[0094] (2) Test time

[0095] August 2019 - October 2020

[0096] (3) Experimental crops

[0097] Citrus, cultivar Wogan

[0098] (4) Experimental design

[0099] The experimental field was set up with 1 control and 3 treatments, each repeated 3 times. The plot area was 150 m2 and they were arranged randomly. One protection row was set up between the plots.

[0100] Control group: spray water on leaves;

[0101] Treatment 1: Foliar spraying of a dilution of Bacillus subtilis inoculant (20 billion / g of Bacillus subtilis inoculant) produced by Shandong Dehe Mingxing Biotechnology Co., Ltd.

[0102] Treatment 2: foliar spray of a diluted copper preparation;

[0103] Treatment 3: Spray the leaves with a diluted solution of Bacillus subtilis JQ-K98 bacterial solution.

[0104] The investigation was conducted on citrus, and the specific contents were as follows: the application of the drug began when the autumn shoots sprouted to 2 cm, and sprayed once every 8 days for 3 times in a row;

[0105] The specific method of processing 1 is:

[0106] The purchased existing Bacillus subtilis inoculant was diluted with clean water to 500 million / mL to obtain the inoculant dilution solution, and 50L of the inoculant dilution solution was sprayed on each acre of leaves.

[0107] The specific method of processing 2 is:

[0108] Use thiamethoxam and crown fungicide alternately, that is, use thiamethoxam the first time, crown fungicide the second time, and thiamethoxam the third time; spray once after each strong wind and rain to reduce the invasion of bacteria through the wound and cause a major outbreak of ulcer disease.

[0109] The dosage of thiamethoxam per mu is 60g, which is diluted to 500 times liquid and sprayed on the leaves;

[0110] The dosage of Guanjunqing preparation per mu is 10g, which is diluted to 1500 times liquid and sprayed on leaves;

[0111] The copper thiamethoxam preparation has an active ingredient content of 20wt% and is in the form of a suspension, and is produced by Zhejiang Dongfeng Chemical Co., Ltd.

[0112] The described crown bacterium preparation has an active ingredient content of 57.6wt% and is in the form of water-dispersible granules, produced by Newfarm Australia Ltd.

[0113] The specific method for processing 3 is:

[0114] The Bacillus subtilis JQ-K98 fermentation liquid prepared by the present invention is diluted with clean water to 500 million / mL to obtain a diluted liquid, and 50 L of the diluted liquid is sprayed on each acre of leaves.

[0115] (5) Disease statistics

[0116] Disease investigation method: For each citrus tree, select branches with relatively consistent growth in the same direction as the investigation object, mark them well, and only investigate the disease situation of new leaves. Select 5 trees in each plot for investigation, take samples at 5 points in the east, south, west, north and center of each tree, investigate all leaves on 2 new shoots at each point, record leaves of different levels, and calculate the disease index and prevention and control effect. The classification standard of damaged leaves is:

[0117] Level 0: no disease;

[0118] Level 1: 1 to 5 lesions per leaf;

[0119] Level 3: 6 to 10 lesions per leaf;

[0120] Level 5: 11 to 15 lesions per leaf;

[0121] Level 7: 16 to 20 lesions per leaf;

[0122] Level 9: There are more than 21 lesions on each leaf.

[0123] Disease index and prevention efficacy calculation formula:

[0124]

[0125] The control effect of each group of citrus is shown in Figure 4 .

[0126] (6) Citrus quality evaluation

[0127] During harvest, the Wogan oranges are sent to the Microbiological Fertilizer and Edible Fungi Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture and Rural Affairs / National Famous, Special and New Agricultural Products Nutritional Quality Evaluation and Identification Agency for quality testing.

[0128] Table 4 Survey on disease grade of citrus canker

[0129]

[0130] As can be seen from Table 4, after using the JQ-K98 fungicide, the disease control efficiency reached 87.84%, and the disease incidence of citrus was significantly reduced compared with the control of clear water, dead grass and the treatment of copper preparation alone, which has great value for industrial promotion.

[0131] The Wogan oranges were sent to the Microbiological Fertilizer and Edible Fungi Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture and Rural Affairs / National Famous, Special and New Agricultural Products Nutritional Quality Evaluation and Identification Agency for citrus quality testing. The weight of a single fruit was 170.6g, the soluble solids content was 13.9wt%, the reducing sugar content was 5.2wt%, the solid-acid ratio was 29.3, the sugar-acid ratio was 10.9, and the vitamin C content was 26.6mg / 100g.

[0132] From the above data, it can be seen that the JQ-K98 bacterial agent of the present invention can effectively prevent and treat citrus canker and at the same time, can also significantly improve the quality of citrus.

[0133] The above disclosure is only the preferred embodiment of the present disclosure, which certainly cannot be used to limit the scope of rights of the present disclosure. Therefore, equivalent changes made according to the scope of the patent application of the present disclosure are still within the scope covered by the present disclosure.

Claims

1. A Bacillus subtilis for preventing and treating citrus canker, characterized in that: The Bacillus subtilis is classified and named as Bacillus subtilis Bacillus subtilis The deposit number is CGMCC No.24778, and the deposit date is April 26, 2022.

2. A bacterial agent for preventing and treating citrus canker, characterized in that: The bacterial agent is the fermentation broth of Bacillus subtilis according to claim 1, and the bacterial content of Bacillus subtilis in the bacterial agent is 112×10 8 cfu / mL.

3. The bacterial agent according to claim 2, characterized in that: The method for preparing the fermentation liquid of Bacillus subtilis comprises strain activation, seed liquid preparation and fermentation; the fermentation comprises primary seed tank culture, secondary seed tank culture and fermentation tank culture.

4. The bacterial agent according to claim 3, characterized in that: The method for activating the strain is to perform three-zone streaking on the Bacillus subtilis on the LB solid culture medium, and culture it at 37° C. for 24 hours to obtain the activated strain.

5. The bacterial agent according to claim 4, characterized in that: The method for preparing the seed solution is to pick a single colony from the activated strain and inoculate it into 150 mL of seed culture medium, and culture it at 37° C. and 200 rpm for 14 hours to obtain the seed solution.

6. The bacterial agent according to claim 5, characterized in that: The method for culturing in the first-level seed tank is as follows: according to an inoculation amount of 10% by volume, the seed liquid is connected to the seed culture medium of the first-level seed tank, the temperature is controlled to be 37°C, the ventilation is 0.8 V / V·m, the pH is 7.5, the rotation speed is 200 rpm, and the culturing time is 12 hours to obtain a first-level seed culture; the method for culturing in the second-level seed tank is as follows: according to an inoculation amount of 10%, the first-level seed culture is inoculated into the seed culture medium of the second-level seed tank, the temperature is controlled to be 37°C, the ventilation is 0.8 V / V·m, the pH is 7.5, the rotation speed is 100 rpm, and the culturing time is 12 hours, sampling and microscopically examining the bacteria in the logarithmic growth phase to obtain a second-level seed culture; for the fermentation tank culture, the second-level seed culture is transferred to the fermentation tank for culturing, the temperature is controlled to be 37°C, the ventilation volume from 0 to 6 hours is 0.8 V / V·m, the ventilation volume after 6 hours is adjusted to 2 V / V·m, the pH is 7.5, the rotation speed is 100 rpm, and the culturing time is 32 hours.

7. The bacterial agent according to claim 6, characterized in that: The seed culture medium and the fermentation culture medium in the fermenter are composed of the following ingredients, calculated by mass percentage: 3% maltodextrin, 6.5% soybean meal powder, 0.3% glucose, 0.1% magnesium sulfate heptahydrate, 0.2% calcium carbonate, 0.05% disodium hydrogen phosphate, 0.02% manganese sulfate, 0.15% defoaming agent, and deionized water is supplemented to 100%.

8. The use of the bacterial agent according to claim 2, characterized in that: The bacterial agent is used for preventing and treating citrus canker.

9. The use according to claim 8, characterized in that: Dilute the bacterial agent with clean water to 500 million / mL to obtain the bacterial agent dilution solution, and spray 50L of the bacterial agent dilution solution on each acre of leaves.

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