CN112980733A - Bacillus cereus N15-2 with inhibition effect only on pathogenic bacteria and no inhibition effect on pathogenic fungi and application thereof - Google Patents
Bacillus cereus N15-2 with inhibition effect only on pathogenic bacteria and no inhibition effect on pathogenic fungi and application thereof Download PDFInfo
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- CN112980733A CN112980733A CN202110293278.2A CN202110293278A CN112980733A CN 112980733 A CN112980733 A CN 112980733A CN 202110293278 A CN202110293278 A CN 202110293278A CN 112980733 A CN112980733 A CN 112980733A
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- bacillus cereus
- bacterial
- rice
- inhibition effect
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a biological control agent, wherein the effective active ingredient of the biological control agent consists of Bacillus cereus N15-2. The method is characterized in that: the biocontrol bacterium only has an inhibition effect on pathogenic bacteria and has no inhibition effect on pathogenic fungi; the biocontrol microbial inoculum is an aqueous agent and comprises the following components in percentage by weight: bacillus cereus N15-2: 89.5 to 99.8 percent; sodium dodecyl sulfate: 0.1-10%; potassium sorbate: 0.1 to 0.5 percent. When the water aqua is applied, the water aqua is irrigated to roots to prevent and control soft rot of Chinese cabbages and bacterial wilt of hot peppers, and is uniformly sprayed on rice leaf surfaces to prevent and control bacterial leaf blight of rice and bacterial leaf streak of rice. The advantages are that: the aim of preventing and treating plant bacterial diseases by using a biocontrol microbial inoculum is achieved by introducing bacillus cereus N15-2 which is safe to human and livestock; the dosage of chemical agents is reduced; meanwhile, the ecological environment is improved, and the residual quantity of chemical agents in agricultural products is reduced.
Description
Technical Field
The invention relates to a biocontrol microbial inoculum and application thereof, in particular to a bacillus cereus N15-2 and application thereof in preventing and treating plant bacterial diseases.
Background
Bacterial diseases of plants are important diseases in agricultural production, the damage degree of the bacterial diseases exceeds that of viruses, and the bacterial diseases become second to fungi. The prevention and treatment difficulty is increased due to the complex prevention and treatment measures, various propagation modes, and the lack of prevention and treatment agents and the long-term continuous cropping planting mode. According to incomplete data statistics, the occurrence area of bacterial diseases of plants in China is 1.2 hundred million acres per year at present. Bacterial diseases are diseases caused by pathogenic bacterial infection, such as soft rot, bacterial wilt, bacterial leaf blight and the like. Bacteria that attack plants can invade through natural orifices (stomata, skin pores, water pores, etc.) and wounds, spread by running water, rain water, insects, etc., and overwinter in disease residues, seeds, soil. The main symptoms of bacterial diseases are decay, necrosis, wilting and perforation, etc. Bacterial mucus overflows from diseased parts in humid weather at the later stage of the bacterial disease, and the bacterial disease is obviously malodorous and is the main characteristic of the disease.
In production, the prevention and the treatment of plant bacterial diseases mainly depend on various copper preparations (such as thiabendazole copper, copper oxychloride, copper hydroxide, cuaminosulfate, copper citrate, thiacetone and the like) and a few antibiotics (such as agricultural streptomycin sulfate, kasugamycin, zhongshengmycin and the like). Agricultural streptomycin has good control effect on most bacterial diseases and has been widely applied in the past. In 2016, 6, 14 days, the last registration certificate of agricultural streptomycin sulfate in China is due and does not continue to be developed, which means that agricultural streptomycin sulfate formally exits the stage of bacterial disease control, and bacterial disease control is replaced by other medicaments. Streptomycin sulfate is forbidden in agricultural crops, and the main reason is that streptomycin sulfate is an antibiotic shared by human and livestock, and after being applied to crops, the streptomycin sulfate can enter human and livestock bodies through a food chain, so that resistance risks are generated. And the long-term and large-scale use of chemical pesticides such as copper preparations and the like not only causes serious pollution to the ecological environment, but also directly increases the residues of toxic chemical substances in agricultural products and brings serious harm to human health. In order to achieve the purposes of effectively controlling bacterial diseases and reducing the use amount of chemical pesticides, methods for controlling plant diseases by using biological control technologies are receiving increasing attention from governments and people. Biological control technology mainly utilizes biological diversity, and can continuously control plant diseases by releasing or introducing beneficial microorganisms in the nature. Researches for preventing and treating plant bacterial diseases by using bio-control bacteria have been carried out at home and abroad since 1950, and the bio-control bacteria mainly comprise Bacillus (Bacillus) and pseudomonas (Pseudomonas).
A novel biocontrol strain N15-2 is obtained by separating and purifying soil samples collected by agricultural academy of sciences of Jiangsu province from Liuhe pea field of Nanjing city of Jiangsu province. The results of the previous studies show that: the biocontrol strain N15-2 has good control effect on various bacterial diseases (soft rot, bacterial wilt, bacterial leaf blight, bacterial streak, black rot and the like) in production. The strain N15-2 is identified as Bacillus cereus by the detection and identification of the institute of microbiology of Chinese academy of sciences and the comprehensive analysis of experimental data such as cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and the like. Bacillus cereus N15-2 has been registered in China general microbiological culture Collection center at 6.1.1.2021, the address is No. 3 of Xilu 1 of Beijing university facing Yang district, and the preservation number is CGMCC No. 21599.
Bacillus cereus (also sometimes called waxy) has been reported in recent years as a Plant growth-promoting rhizobacterium (PGPR) (Gao Tantan et al, 2020, Applied Microbiology and Biotechnology, E1-Sayed M. Desoky et al, 2020, Biocatalysis and Agricutural Biotechnology, Wang Chuanjuan et al, 2012, Agricultural Biotechnology Proc.), for the major control of fungal diseases, including wheat rust (Asghari Bano et al, 2020, Jouranl of Plant Pathology), Phoma nodosum (Pankaj Kumar et al, 20135, Scientific report), tomato wilt and early blight (Kahika S et al, Thienene et al, 2019, Yongsu Mitsugaku et al, cucumber Yokoku et al, Yokogaku et al, Yokokura, Yokogaku et al, Yokoku koku et al, tomato wilt, tomato yellow spot, tomato yellow mold, tomato, the 2016 doctoral paper; liuyuan soldiers, etc., 2015, "agricultural science and management"), rice false smut (Rong of Wang, 2018, "agricultural development and Equipment"), poplar rot (Tianxiaxia, 2017, "Zhejiang agriculture and forestry university Proc.), wheat powdery mildew (Gong Shuang Jun et al, 2016, Chinese Biocontrol Proc.), cereal cyst nematode (Zhang Jie et al, 2016, Biological Control), Aspergillus arachidis (Navya HM et al, 2015," Annals of Applied Biology "), sheath blight of rice (Chengjun et al, 2014, Chinese Biological Control), southern leaf blotch (Chien-Jui Huangang et al, 2010," Annals of Applied Biology), and so on. The Bacillus cereus AR156 has a certain control effect on the bacterial wilt of hot pepper (Wang Yong et al, 2014, Proc. Biometrics) in addition to the prevention effect on the above-mentioned rice sheath blight (Cheng Liu et al, Ming Liang Xuan, Proc. Bioprevention and treatment of China) and root knot nematode (Liao Jie et al, 2019, Notification of microbiology; Jiang Chun No. 2016, Proc. Phr. thesis), and also has a certain control effect on the bacterial wilt of hot pepper (Wang Yong et al, 2014, Proc. Phytopathy). The main control objects of the existing bacillus cereus application and granted patents are also fungal diseases including rice blast, rice sheath blight, rice bakanae disease, rice leaf spot, rice false smut, strawberry powdery mildew, root-knot nematode and the like (patent application numbers: CN 201610799877.0; CN 201611027048.7; CN 201410510935.4; CN201810472191. X; CN 201710837023.1). And some combined fertilizers containing bacillus cereus have good growth promoting and disease preventing effects, and have good control effects on root-knot nematodes, sheath blight and blight in soil (patent application No.: CN 201610694583.1; CN 201410012023.4). In addition, Bacillus cereus has SOD enzyme to raise the oxygen eliminating capacity of crop to produce oxygen in vivo, regulate crop's cell microbial environment, maintain normal cell metabolism and biochemical reaction, raise stress resistance, speed growth and raise yield and quality (patent application No. CN 201910264931.5).
At present, bacillus cereus (waxy) is used for preventing and treating eggplant bacterial wilt and tomato bacterial wilt, 2 application patents (patent application numbers: CN 201810928087.7; CN201811123658.6) and 1 literature report (Qiaokang and the like, 2012, pesticide science and management) exist in China, and the application patents and the literature report directly use a single dose or a compound product of the bacillus cereus (waxy) in field prevention and treatment tests without indoor bioassay tests. The action mechanism of the bacillus cereus N15-2 obtained by the applicant is completely different from that reported by the prior people, and the action mechanism is as follows: 1. unlike other bacillus cereus (waxy) which is reported in the past and has better inhibition effect on a plurality of pathogenic fungi and pathogenic bacteria, the bacillus cereus N15-2 only has inhibition effect on pathogenic bacteria and has no inhibition effect on pathogenic fungi. 2. Some biocontrol bacteria have obvious indoor bacteriostasis, but the field control effect is unstable. The bacillus cereus N15-2 has better indoor and outdoor inhibition effects on several tested bacterial diseases, and the trend of the indoor inhibition test result is consistent with that of the field control test result. 3. As is well known, the bacterial wilt of pepper, the bacterial wilt of tomato and the bacterial wilt of eggplant are Lauerella, but the infection of different hosts can generate different specialities such as speciality of speciality, pathogenicity and the like. The bacillus cereus N15-2 separated and obtained by the applicant has a control effect on bacterial diseases such as pepper bacterial wilt and the like, and other bacillus cereus (waxy) does not necessarily have a control effect on pepper bacterial wilt. In other words, the reported bacillus cereus (waxy) for controlling tomato bacterial wilt and eggplant bacterial wilt can not necessarily control pepper bacterial wilt.
The Bacillus cereus N15-2 is a new strain obtained by sampling and separating by the applicant, and has an inhibition effect only on pathogenic bacteria and no inhibition effect on pathogenic fungi. The bacillus cereus N15-2 can effectively prevent and treat a plurality of bacterial diseases such as Chinese cabbage soft rot, pepper bacterial wilt, rice bacterial leaf blight, rice bacterial streak and the like, has no relevant reports at home and abroad, and has creativity and novelty.
Disclosure of Invention
The invention aims to provide a biocontrol strain-Bacillus cereus (Bacillus cereus) N15-2 with better application prospect, which has been registered in China general microbiological culture Collection center at 1 month and 6 days of 2021, the address is No. 3 of West Lu No. 1 of the sunward area in Beijing, and the preservation number is CGMCC No. 21599.
The invention provides a biocontrol microbial inoculum, wherein the effective active ingredient of the biocontrol microbial inoculum consists of bacillus cereus N15-2.
The biocontrol microbial inoculum disclosed by the invention has the advantages that the bacillus cereus N15-2 only has an inhibition effect on pathogenic bacteria and has no inhibition effect on pathogenic fungi.
The biocontrol microbial inoculum disclosed by the invention comprises only one antibacterial related substance of protease in a metabolite secreted by the bacillus cereus N15-2.
The biocontrol microbial inoculum is an aqueous agent.
The water aqua of the invention is characterized in that: the components and the weight percentage content are as follows: bacillus cereus N15-2: 89.5 to 99.8 percent; sodium dodecyl sulfate: 0.1-10%; potassium sorbate: 0.1 to 0.5 percent; the pH value is 6.5-7.0.
The aqueous agent disclosed by the invention is used for preventing and treating soft rot of Chinese cabbage and pepper bacterial wilt by root irrigation, and is used for preventing and treating bacterial leaf blight of rice and bacterial leaf streak of rice by uniformly spraying on rice leaf surfaces.
The invention has the advantages that: the bacillus cereus N15-2 which is safe to human and livestock and has better application prospect is developed, and the biocontrol resource is enriched; the biocontrol microbial inoculum can be used for preventing and treating various plant bacterial diseases, reducing the using amount of chemical agents in production, improving the ecological environment and reducing the residual amount of the chemical agents in agricultural products.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 shows the inhibitory effect of Bacillus cereus N15-2 on pathogenic bacteria
FIG. 2 shows the production of protease by Bacillus cereus N15-2.
In the figure, A: bacillus cereus N15-2; b: chinese cabbage soft rot; c: bacterial wilt of capsicum; d: rice bacterial blight; e: bacterial leaf streak disease of rice.
The specific implementation mode is as follows:
example 1
Inhibition of pathogenic bacteria by Bacillus cereus N15-2
A. Pathogenic bacteria to be tested
The tested bacteria comprise cabbage soft rot, pepper ralstonia solanacearum, rice leaf blight and rice bacterial streak germ, which are provided by the plant protection research institute of agricultural academy of sciences of Jiangsu province. Culturing with LB culture solution (peptone 10.0g, yeast powder 5.0g, NaCl 10g, distilled water 1000mL) at 28 deg.C under 150rpm shaking for 2d, diluting with sterile water to 10%8cfu/mL for use.
B. The test method comprises the following steps:
activating Bacillus cereus N15-2 screened by agricultural academy of sciences of Jiangsu province on LB culture medium, transferring into 50mL LB culture solution, culturing at 28 deg.C under shaking at 150rpm for 2d, diluting with sterile water to 10%8cfu/mL for use.
Sucking 5 mu L of bacillus cereus N15-2 bacterial liquid, dotting the liquid at the center of an LB flat plate, culturing at 28 ℃ for 48h, spraying pathogenic bacteria diluent, culturing at 28 ℃ overnight, and investigating the diameter of a bacteriostatic circle. Each treatment was repeated 3 times and averaged. And setting clear water as a reference.
C. And (3) test results:
the test results are shown in FIG. 1. The bacillus cereus N15-2 bacterial liquid has certain inhibition effect on 4 kinds of bacteria to be tested, and the diameter of an inhibition zone for cabbage soft rot germs is 40.3 mm; the diameter of a bacteriostasis zone for the pseudomonas solanacearum is 25.9 mm; the diameter of the inhibition zone for rice bacterial blight is 36.0 mm; the diameter of the inhibition zone for rice bacterial streak germ is 39.8 mm.
As shown in fig. 1.
Example 2
Inhibition of various plant pathogenic fungi by bacillus cereus N15-2
A. Test pathogenic fungi:
the 17 pathogenic fungi comprise rice blast, rice sheath blight, rice false smut, rice bakanae, rice leaf spot, wheat gibberellic disease, wheat root rot, wheat sheath blight, wheat take-all, rape sclerotinia, tomato gray mold, tomato late blight, tomato early blight, watermelon wilt, eggplant verticillium wilt, pepper blight and apple rot, and are provided by plant protection research institute of agricultural academy of sciences of Jiangsu province. Culturing in PDA culture medium (200g potato, 20g glucose, 20g agar and 1000mL distilled water, pH 6.5-7.0) at 28 deg.C for 5 d.
B. The test method comprises the following steps:
the bacterial liquid of Bacillus cereus N15-2 was prepared in the same manner as in example 1.
The pathogenic fungus cake (diameter 6mm) was placed on one side of a blank PDA plate 30mm from the edge of the plate. And 5 mu L of bacillus cereus N15-2 bacterial liquid is spotted on the other side of the plate 30mm away from the edge of the plate. Culturing at 28 deg.C, setting clear water as control, investigating the growth diameter of pathogenic bacteria when the control plate pathogenic bacteria colony is full, and calculating the inhibition rate. Each treatment was repeated 3 times and averaged.
The ratio of pathogenic fungi inhibition is [ (control pathogen growth diameter-treated pathogen growth diameter)/control pathogen growth diameter ] × 100%.
C. And (3) test results:
the test result shows that: the bacillus cereus N15-2 has no inhibitory effect on the tested 17 pathogenic fungi, unlike other bacillus cereus reported at present.
Example 3
Production of Bacillus cereus N15-2 metabolic secretion-antibiotic related substance
Test materials: protease detection medium: 100g of skimmed milk powder and 20g of agar, and the volume is up to 1000 mL. And (3) detecting a siderophore culture medium: CAS 60.5mg, 10mL ferric iron solution (1 mmol. L)-1FeCl3·6H2O), HDTMA 72.9mg, agar 20g, constant volume to 1000mL, pH 7.0. Chitinase assay medium: chitin 15g, MgSO4·7H2O 0.5g，FeSO4·7H2O 0.01g，K2HPO40.7g，KH2PO40.3g of agar and 20g of agar, and the volume is up to 1000mL, and the pH value is 7.0-7.2. Cellulase detection culture medium: 10g of peptone, 10g of yeast powder, 10g of sodium carboxymethylcellulose, 5g of NaCl and KH2PO41g of agar and 20g of agar, and the volume is up to 1000mL, and the pH value is 7.0.
The bacterial liquid of Bacillus cereus N15-2 was prepared in the same manner as in example 1.5 mu L of Bacillus cereus N15-2 bacterial liquid is inoculated to the center of each detection medium plate, and each treatment is repeated for 3 times. Culturing at 28 deg.C for 3-7 days, and observing.
And (3) protease detection: the presence or absence of the transparent ring was observed. If a transparent ring is generated, the antagonistic bacteria can secrete the protease.
Detecting siderophore: after culturing at 28 ℃ for 7-10 days, observing whether yellow halo is generated. If a yellow halo is produced, the antagonistic bacteria can secrete the siderophore.
Chitinase detection: the presence or absence of the transparent ring was observed. If a transparent ring is generated, the antagonistic bacteria can secrete chitinase and degrade chitin.
And (3) cellulase detection: the colony was observed for the presence of a transparent ring around it. If a transparent circle is generated, the antagonistic bacteria can secrete the cellulase.
The test result shows that: bacillus cereus N15-2 only secreted protease, and utilized the protein in the assay plate to form a clear ring around the colony (FIG. 2); cellulase, siderophore and chitinase cannot be secreted.
As shown in fig. 2.
Example 4
Obtaining of Bacillus cereus N15-2 aqueous solution
A. Preparing and activating strains.
LB medium (same as example 1) was sterilized by adding agar and slant-slant culture. Inoculating Bacillus cereus N15-2, and culturing at 28 deg.C for 2 days.
B. And (5) liquid fermentation.
Primary fermentation: transferring the cultured test tube slant with an inoculating loop to 300mL LB culture solution. Shaking and culturing at 28 deg.C and 150rpm for 2 d.
Secondary fermentation:the formula of the culture medium is as follows: 1% of soybean cake powder, 0.5% of soybean meal, 0.15% of yeast extract, 1% of corn flour, 0.5% of corn steep liquor, 0.5% of cane sugar, 0.05% of NaCl, CaCO30.18 percent. pH 7.0 before sterilization. 1 ton of material was charged into a 2 ton fermentor. Sterilizing at 121 deg.C for 30 min. And (3) cooling to about 30 ℃, and inoculating 300mL of the primary fermentation strain liquid. The liquid fermentation temperature is 32 ℃, and the stirring speed is 300 rpm. The ventilation rate is 1: 1.5. The fermentation period was 18 hours.
And (3) three-stage fermentation: the culture medium formula is the same as the secondary fermentation. 10 tons of feed are fed into a 15 ton fermenter. Sterilizing at 121 deg.C for 30 min. And (3) inoculating 1 ton of the secondary fermentation strain liquid when cooling to about 30 ℃. The liquid fermentation temperature is 32 ℃, and the stirring speed is 300 rpm. The ventilation rate is 1: 1.5. The fermentation period is about 24 hours, and the mixture is put into a tank.
C. And (4) preparing an aqueous agent.
Putting the fermentation liquor into a storage tank, adding 0.1-10% of sodium dodecyl sulfate and 0.1-0.5% of potassium sorbate, and uniformly stirring. The pH value of the fermentation liquor is 6.5-7.0, and the number of viable bacteria is 109cfu/mL. Filling into a bacillus cereus N15-2 water agent.
Example 5
Test for preventing and treating soft rot of Chinese cabbage by using bacillus cereus N15-2 aqueous solution
Test site: a dry-land crop test base of the institute of plant protection, academy of agricultural sciences, Jiangsu province.
The test varieties are as follows: chinese cabbage of Zhongshi head is provided by Hongkong agricultural department in Xuhuai region of Jiangsu province.
Test and control agents: aqueous bacillus cereus N15-2 solution is provided by example 4. The control agent is 2% kasugamycin wettable powder, purchased from Chunlei biological pharmaceutical industry Co., Ltd, Yanbian, Jilin province, and used according to the recommended dosage in the field.
The test method comprises the following steps: irrigating roots of the Chinese cabbage for 10 days, 17 days and 25 days respectively for 1 time, and irrigating 250ml of the pesticide liquid for each plant. The experiment was set to 5 treatments: : 100 times of diluent of bacillus cereus N15-2 water agent, 200 times of diluent of bacillus cereus N15-2 water agent, 750 times of diluent of 2% kasugamycin wettable powder, 1500 times of diluent of 2% kasugamycin wettable powder and clear water for comparison. 4 times of repetition, 20 cells in total, each cell surfaceProduct of 20m2And are randomly arranged. Randomly sampling in each cell at 35d and 55d after the administration, investigating 50 Chinese cabbages, investigating the disease incidence, and calculating the disease incidence index and the prevention effect. Disease grading criteria are as follows:
level 0: no symptoms;
level 1: the leaves begin to appear water stain-like brown scabs;
and 3, level: the base of the leaf at the bottom 1-2 has obvious scab;
and 5, stage: the outer leaves are 1/3-1/2 rotten;
and 7, stage: the outer leaves are all rotten;
and 9, stage: the whole plant is rotten.
Disease index ═ number of onset of disease at each stage × representative value at each stage)/(total number of investigated plants × highest representative value) ] × 100
Control effect (%) < control disease index > < 100%
The test results are shown in Table 3. After the bacillus cereus N15-2 aqueous solution is applied for 35 days, the control effect of the 100-time diluent is the highest and is 72.06%, the control effect of the 2% kasugamycin wettable powder serving as a contrast agent is the lowest and is 65.20%, and the control effects of the bacillus cereus N15-2 aqueous solution and the kasugamycin wettable powder are not obviously different. The control effect of the bacillus cereus N15-2 aqueous solution 200 times of diluent is equivalent to that of 2% kasugamycin wettable powder 1500 times of diluent. 55 days after the application, the disease index of the clear water contrast is obviously increased, the control effect of the bacillus cereus N15-2 aqueous solution 100 times of diluent is 75.38%, the control effect of the 2% kasugamycin wettable powder 750 times of diluent is 68.68%, and the control effects of the bacillus cereus N15-2 aqueous solution and the kasugamycin wettable powder are obviously different. The results show that: the bacillus cereus N15-2 has obvious effect of preventing and treating Chinese cabbage soft rot.
TABLE 3 field control effect of Bacillus cereus N15-2 aqua on cabbage soft rot
Note: different lower case letters in the same column indicate significant differences.
Example 6
Bacillus cereus N15-2 aqueous solution for field plot experiment for preventing and treating pepper bacterial wilt
Test site: the pepper greenhouse is a facility of the Liuhe test base of the agricultural academy of sciences of Jiangsu province.
The test varieties are as follows: su jiao No. 17, provided by vegetable research institute of academy of agricultural sciences in Jiangsu province.
Test agents: the bacillus cereus N15-2 aqueous solution is provided by example 4, and the reference medicament is 3% Zhongshengmycin wettable powder purchased from Shenzhen Nuopuxin agrichemical Co., Ltd and used according to the recommended dosage in the field.
The test method comprises the following steps: selecting 1 greenhouse with severe natural disease of pepper bacterial wilt as a test greenhouse. The test seedlings are subjected to two stages of channel plug seedling and field planting according to conventional operation. When the hole tray is used for sowing, diluting the bacillus cereus N15-2 aqueous solution according to the proportion of 1: 50, and irrigating 50 milliliters of the aqueous solution in each hole; during field planting and transplanting, root irrigation is carried out on the bacillus cereus N15-2 aqueous solution according to the proportion of 1: 100, and 200 milliliters of root irrigation is carried out on each seedling; and (3) irrigating roots of the bacillus cereus N15-2 aqueous solution according to the proportion of 1: 100 every 15 days after field planting, wherein 500 ml of roots are irrigated to each seedling. Test agents: bacillus cereus N15-2 water agent, 3% zhongshengmycin wettable powder and clear water as a control. Each treatment cell is about 500 seedlings, the treatment cell is repeated for 2 times, and the treatment cells are randomly distributed. And (5) investigating the disease condition when the tomatoes and the peppers are attacked in the control plot, and calculating the control effect.
Incidence rate ═ number of diseased strains in survey cell/number of total strains investigated × 100
Control efficacy (incidence of treatment cells-incidence of control cells)/incidence of control cells
The test results are shown in Table 3. The incidence rate of the pepper bacterial wilt in a control disease field is 45.72 percent, the field control effect of the bacillus cereus N15-2 aqueous solution on the pepper bacterial wilt is 66.91 percent, the control effect of the 3 percent zhongshengmycin wettable powder is 44.31 percent, and the two are obviously different. The test result shows that: the bacillus cereus N15-2 aqueous solution can effectively prevent and treat the pepper bacterial wilt.
TABLE 3 field control of bacterial wilt of capsicum by Bacillus cereus N15-2 aqua
Note: different lower case letters in the same column indicate significant differences.
Example 7
Bacillus cereus N15-2 aqueous solution for field plot experiment for preventing and treating bacterial leaf streak and bacterial blight of rice
Test site: the plant protection research institute of Jiangsu province academy of agricultural sciences rice test field.
Test agents: bacillus cereus N15-2 aqueous solution is provided in example 4, and the control agent is 20% thifluzazole wettable powder, available from longwan chemical company, zhejiang), used according to the instructions of the agent.
Spraying the pesticide on the leaves in the early stage of bacterial leaf blight and bacterial streak disease (sporadic disease in the field), and performing secondary control at the interval of about 10 days. The investigation method comprises the following steps: at the initial stage of disease onset, 4 points were randomly selected per cell, 5 points per point, and 3 leaves at the uppermost part of each tillering were investigated. The disease index was investigated again after the initial administration of 21 days.
Grading standard: grade 0, no scab; grade 1, the scab occupies less than 5 percent of the leaf area; grade 3, the lesion area accounts for 6 to 15 percent of the leaf area; grade 5, the lesion area accounts for 16 to 30 percent of the leaf area; 7, the lesion area accounts for 31 to 50 percent of the leaf area; grade 9, the lesion area accounts for more than 50% of the leaf area.
The disease index ═ Σ (number of onset at each stage × representative value at each stage)/(total number of examined leaves × highest representative value) × 100;
the preventing and treating effect is [1- (after-drug disease index in the treatment area x before-drug disease index in the control area)/(before-drug disease index in the treatment area x after-drug disease index in the control area) ] × 100%.
The test results are shown in Table 4. The control medicament, namely 20 percent of the thiazozole wettable powder has the control effects on the bacterial leaf streak and the bacterial blight of rice of 42.89 percent and 45.44 percent respectively according to the using dose of 50 g/mu, and has the control effects on the bacterial leaf streak and the bacterial blight of rice of 80.15 percent and 76.28 percent respectively according to the using dose of 100 g/mu. The control effect of the bacillus cereus N15-2 aqueous solution on the bacterial leaf streak and the bacterial blight of rice is respectively 38.17-44.22% and 35.32-42.73% according to the use dose of 33.3-50 ml/mu, and the control effect is equivalent to the use dose of 50 g/mu of a contrast medicament; the control effect on the bacterial leaf streak and bacterial blight of rice is 78.64% and 73.56% respectively according to the using dose of 100 ml/mu, and the control effect is equivalent to the using dose of 100 g/mu of the contrast medicament.
TABLE 4 field control of bacterial leaf streak and bacterial blight of rice with aqueous solution of Bacillus cereus N15-2
Claims (2)
1. A biological control agent and application thereof are characterized in that: the effective active ingredient in the biological control agent consists of Bacillus cereus N15-2; the bacillus cereus N15-2 only has an inhibiting effect on pathogenic bacteria and has no inhibiting effect on pathogenic fungi; the metabolite secreted by the bacillus cereus N15-2 only comprises one antibacterial related substance of protease; the biocontrol microbial inoculum is an aqueous agent; the biocontrol microbial inoculum is irrigated to roots and used for preventing and controlling soft rot of Chinese cabbages and bacterial wilt of hot peppers, and is uniformly sprayed on rice leaf surfaces and used for preventing and controlling bacterial leaf blight of rice and bacterial leaf streak of rice.
2. The biocontrol microbial inoculum of claim 1 wherein: the components and the weight percentage content are as follows: bacillus cereus: 89.5 to 99.8 percent; sodium dodecyl sulfate: 0.1-10%; potassium sorbate: 0.1 to 0.5 percent.
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