CN110079483B - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN110079483B CN110079483B CN201910451292.3A CN201910451292A CN110079483B CN 110079483 B CN110079483 B CN 110079483B CN 201910451292 A CN201910451292 A CN 201910451292A CN 110079483 B CN110079483 B CN 110079483B
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- fusarium oxysporum
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- 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
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- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention relates to the technical field of microorganisms, and discloses bacillus subtilis and application thereof. The preservation number of the bacillus subtilis is CGMCC No. 17357. The bacillus subtilis strain has a broad antibacterial spectrum, has an obvious inhibition effect on various crop blight pathogens of over 75 percent, also has obvious inhibition effects on other 26 fungal pathogens and 2 bacterial pathogens, is easy to fix in soil, is not easy to generate drug resistance, has good drug effect durability, can effectively reduce the usage amount of pesticides, is beneficial to pollution-free production of crops, and reduces the expenditure of farmers.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis and application thereof.
Background
Crop fusarium wilt is a soil-borne disease caused by fusarium oxysporum and can damage a plurality of economic crops such as melons, beans, solanaceous fruits and the like, as fusarium oxysporum continuously evolves new physiological races and chlamydospores of the fusarium oxysporum can survive in soil for a long time, the control of the fusarium wilt is very difficult, the crop production is seriously influenced, for example, the melon fusarium wilt is generally reduced by 20-30% in a diseased field, and the yield of the diseased field can reach 50-60% even in a dead field. The crop blight has wide spread range, deep damage degree and great control difficulty, and the crop production in China is seriously influenced, so the effective control of the blight becomes the urgent priority for improving the crop yield in China.
The existing control technology of the blight mainly comprises agricultural control, chemical control and biological control. Although the agricultural control such as crop rotation and stubble changing can reduce the number of pathogenic bacteria in soil, but the method cannot be widely used due to the shortage of agricultural cultivated land in China, the breeding of disease-resistant varieties and the enhancement of field management measures have certain effects on disease control, but the breeding period of the disease-resistant varieties is long, the pathogenic variation of blight germs is large, and the resistance easily disappears. Although the chemical prevention and treatment is quick and effective, pathogenic bacteria are easy to generate drug resistance, environmental pollution and pesticide residue are caused, the harm to human bodies is large, and the chemical prevention and treatment does not meet the policy and policy of national green and agriculture, so the use is limited. In this case, much of the eye's sight begins to shift to biocontrol measures.
The biological control is to inhibit the growth and reproduction of pathogenic microorganisms by utilizing the secondary metabolites of the biocontrol bacteria or in the growth process through the functions of antagonism, parasitism, predation and the like and the ways of competing with pathogenic bacteria for living space, nutrient substances and the like to control plant diseases. The biological control of the blight has small interference to the environment, and accords with the sustainable development concept and the national policy and policy guidelines of green and agriculture, so in recent years, the biological control of the blight is rapidly developed as a safe, environment-friendly, green and effective control means. At present, the biocontrol microbial inoculum for the blight mainly comprises bacillus, bacillus subtilis, pseudomonas fluorescens, trichoderma and the like, and the bacillus subtilis has the advantages of quick growth and propagation, easy field planting, disease treatment, growth promotion, soil improvement and the like, and is particularly popular.
Disclosure of Invention
In view of the above, the present invention aims to provide a bacillus subtilis capable of controlling blight of various crops;
another object of the present invention is to provide a bacillus subtilis with broad-spectrum bacteriostatic effect, which can effectively prevent and treat diseases caused by various fungal and bacterial pathogenic bacteria;
the invention also aims to provide application of the bacillus subtilis in controlling related diseases and inhibiting pathogenic bacteria and application in preparing related microbial preparations.
In order to achieve the above purpose, the invention provides the following technical scheme:
the Bacillus subtilis with the preservation number of CGMCC No.17357 has been preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 2019, 3 and 19, and is named as Bacillus subtilis by classification. The bacillus subtilis is obtained by taking bacillus subtilis screened from soil as a starting bacterium and performing ARTP mutagenesis, and is numbered YJY 19-01.
The results of plate confronting experiments show that the bacillus subtilis strain has a wide antibacterial spectrum, has an inhibiting effect on 8 crop blight pathogens, and also has a good inhibiting effect on 26 crop fungal pathogens such as phytophthora capsici, phytophthora nicotianae, strawberry root rot, cucumber target spot, potato blight, tomato bacterial wilt, cucumber angular leaf spot, tomato gray mold, colletotrichum gloeosporioides, cowpea black spot, apple rot and the like, and 2 crop bacterial pathogens. Based on the technical effect, the invention provides the application of the bacillus subtilis with the preservation number of CGMCC No.17357 in preventing and treating fungal pathogenic bacteria and/or bacterial pathogenic bacteria or in preparing a microbial bacteriostatic agent for the fungal pathogenic bacteria and/or the bacterial pathogenic bacteria.
Preferably, the fungal pathogen is selected from one or more of the following:
tomato fusarium oxysporum, cucumber fusarium oxysporum, melon fusarium oxysporum, watermelon fusarium oxysporum, cotton fusarium oxysporum, banana fusarium oxysporum, stevia rebaudiana, cauliflower fusarium oxysporum, phytophthora capsici, potato rhizoctonia solani, cotton red rot, rice sheath blight, cowpea black rot, cucumber red rot, maize microsporum, strawberry brown rot, strawberry root rot, wheat scab, colletotrichum gloeosporum, soybean alternaria, sclerotinia sclerotiorum, apple rot, Chinese cabbage anthracnose, Hunan phytophthora blight, cucumber scab, fusarium laminarum, cucumber gray mold, tomato gray mold, cauliflower black rot, wheat root rot, cucumber leaf mold, tomato leaf spot and tomato leaf mold.
Preferably, the bacterial pathogen is ralstonia solanacearum and/or alternaria cucumerinum.
The plate confrontation experiment shows that the bacillus subtilis has obvious inhibition effect on 8 crops such as banana, cauliflower, cotton, tomato, watermelon, melon, cucumber and stevia rebaudiana, and the inhibition rate is over 75 percent. Pot experiments show that the bacillus subtilis can effectively control the tomato infection caused by the blight, so that the disease occurrence control effect is achieved, and the disease prevention effect is up to 75.76%. The field experiment shows that the bacillus subtilis microbial inoculum can control the tomato withering disease, and the control effect is kept above 75%. Based on the experimental results, the invention also provides the application of the bacillus subtilis with the preservation number of CGMCC No.17357 in preventing and treating crop blight and/or in preparing microbial agents for preventing and treating crop blight.
Preferably, the crop is selected from one or more of tomato, cucumber, melon, watermelon, cotton, banana, stevia and cauliflower.
According to the application of the bacillus subtilis, the invention also provides a microbial agent which comprises the bacillus subtilis with the preservation number of CGMCC No. 17357. Preferably, the bacillus subtilis has a bacterial concentration of 1 × 109～1010CFU/mL. In a specific embodiment of the invention, the microbial agent is prepared by preparing seed liquid from the bacillus subtilis through an LB culture medium and then fermenting through a fermentation culture medium.
Meanwhile, the invention also provides a preparation method of the microbial agent, which comprises the steps of preparing the bacillus subtilis with the preservation number of CGMCC No.17357 into seed liquid, then inoculating the seed liquid into a fermentation culture medium for expanded culture, and adjusting the concentration of the bacillus subtilis in the fermentation liquid to obtain the microbial agent.
In a specific embodiment of the present invention, the preparation method is as follows:
inoculating the bacillus subtilis strain onto an LB (Langerhans) flat plate, streaking, culturing for 14-16h in a biochemical incubator at 37 ℃, picking out a single colony to be inoculated into an LB liquid culture medium containing 100mL after the single colony grows out, and culturing for 14-16h in a shaking table at 37 ℃ and 170rpm to prepare a seed solution; inoculating the seed liquid into a fermentation culture medium with the inoculation amount of 1 percent for expanding culture for 24 hours, and adjusting the concentration of the bacillus subtilis in the fermentation liquid to 1 multiplied by 109～1010CFU/mL, and obtaining the microbial agent.
Wherein the LB culture medium solid culture medium comprises the following formula: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar powder and 1000mL of water, wherein the pH value is 7.2; the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 1000mL of water and pH 7.2; the fermentation medium is a corn flour and soybean meal culture medium and comprises the following components in percentage by weight: corn flour 2.0%, soybean flour 2.0%, NaCl 0.5%, MnSO4·H20.8 percent of O and the balance of water, and the pH value is 7.2.
On the basis of the microbial agent provided by the invention, any appropriate component can be added to prepare a biological agent containing various antibacterial effects related to the invention, and the microbial agent can be applied in any one or more than two aspects as follows:
preventing and treating crop blight, preparing biological preparation for preventing and treating crop blight, preventing and treating fungal pathogenic bacteria, preparing biological inhibitor for preventing and treating fungal pathogenic bacteria, preventing and treating bacterial pathogenic bacteria and preparing biological inhibitor for preventing and treating bacterial pathogenic bacteria.
According to the technical scheme, the bacillus subtilis strain has a wide antibacterial spectrum, has an inhibition effect of more than 75% on various crop blight pathogens, also has an obvious inhibition effect on other 26 fungal pathogens and 2 bacterial pathogens, is easy to fix in soil, is not easy to generate drug resistance, has good drug effect durability, can effectively reduce the usage amount of pesticides, is beneficial to pollution-free production of crops, and reduces the expenditure of farmers.
Biological preservation Instructions
YJY19-01, classification name: bacillus subtilis is preserved in China general microbiological culture Collection center (CGMCC) on 19.3.2019, with the address of No. 3 of Xilu No.1 of Kyoho, Beijing, and the preservation number of CGMCC No. 17357.
Drawings
FIG. 1 shows the plate confrontation results of the Bacillus subtilis of the present invention against 8 crop blight pathogens;
FIGS. 2 to 6 show the results of plate confrontation of the Bacillus subtilis according to the present invention against various pathogenic bacteria.
Detailed Description
The invention discloses bacillus subtilis and application thereof, and can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the bacillus subtilis and applications of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately modifying and combining the bacillus subtilis and applications described herein without departing from the spirit, scope, and content of the invention.
The invention is further illustrated by the following examples.
Example 1: the bacillus subtilis has the inhibiting effect on 8 crop blight pathogenic fungi
A. Test pathogenic fungi:
pathogenic fungi including tomato wilt pathogen, cucumber wilt pathogen, melon wilt pathogen, watermelon wilt pathogen, cotton wilt pathogen, banana wilt pathogen, stevia rebaudiana wilt pathogen, and cauliflower wilt pathogen are all separated and stored by Jingbo chemical research institute of Huanghe delta.
B. The test method comprises the following steps:
the experiment adopts a plate confronting method, 8 crop blight germs are respectively inoculated on a PDA culture medium (200g of potatoes, 20g of glucose, 15g of agar and 1000mL of distilled water, pH6.5-7.0) plate for activation, and after the plate is fully grown by fungi, the fungi are uniformly beaten into circular fungus blocks with the diameter of 8mm from the outer edge of a bacterial colony by a sterilized puncher. Inoculating hypha blocks in the center of a new PDA plate, inoculating the bacillus subtilis at the positions with the same distance of 25mm from the center of the periphery of the new PDA plate, and setting a blank control. Culturing at 28 deg.C, examining 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 result shows that the bacillus subtilis has obvious inhibition effect on 8 crops of wilting (bananas, cauliflower, cotton, tomatoes, watermelons, melons, cucumbers and stevia rebaudiana), the inhibition rate is more than 75%, and the details are shown in table 1 and figure 1.
TABLE 1 inhibition of crop blight by Bacillus subtilis according to the invention
Note: the inhibition rate is [ (control pathogen diameter-treated pathogen diameter)/control pathogen diameter ] × 100%.
Example 2: determination of the bacteriostatic spectrum of the Bacillus subtilis
A. Test pathogenic fungi:
including 26 fungal pathogens and 3 bacterial pathogens except blight pathogen, all of which were isolated and stored by the institute of chemical engineering, Jingbo, Huanghe Delta, as detailed in Table 2.
B. The test method comprises the following steps:
the method comprises the following steps: the pathogenic bacteria of fungi adopt a plate confronting method. The crop pathogenic bacteria are respectively inoculated on a PDA culture medium (200g of potato, 20g of glucose, 15g of agar and 1000mL of distilled water, pH6.5-7.0) plate for activation, and after the fungi grow on the plate, the fungi are uniformly punched into round fungus blocks with the diameter of 8mm from the outer edge of a colony by using a sterilized puncher. Inoculating hypha blocks in the center of a new PDA plate, inoculating the bacillus subtilis at the positions with the same distance of 25mm from the center of the periphery of the new PDA plate, observing the growth characteristics of pathogenic mould colonies, and judging whether the antagonistic effect is achieved. If the mould colony is round or can cover the screened antagonistic bacteria, no antagonism is shown; if the mold colony is deformed, such as square, diamond, petal shape, etc., it is considered as antagonistic.
The second method comprises the following steps: the bacteria pathogenic bacteria adopt the bacteriostasis ring method. Inoculating bacterial pathogenic bacteria on an LB culture medium for activation, then selecting a single colony to be transferred into 50mL of LB culture solution, carrying out shaking culture at 37 ℃ and 170rpm for 2d, diluting the single colony to 108cfu/mL by using sterile water, inoculating the bacillus subtilis at the central point of an LB plate, spraying the bacillus subtilis by using pathogenic bacteria diluent, carrying out overnight culture at 37 ℃, and observing whether a bacteriostatic circle exists. If the inhibition zone appears, the antagonism is considered to be possessed, and if the inhibition zone does not appear, the antagonism is not expressed.
The results of the bacteriostasis spectra show that the bacillus subtilis has obvious inhibiting effects on other 26 crop fungal pathogenic bacteria and 2 bacterial pathogenic bacteria besides inhibiting effects on 8 crop blight pathogenic bacteria in example 1, and the details are shown in a table 2 and figures 2-6.
TABLE 2 bacterial inhibition spectra of the Bacillus subtilis of the present invention against various crop pathogens
Note: and (3) determination of antagonistic effect: 1. fungi, wherein the bacterial colony of the fungi is round or can cover the screened antagonistic bacteria, which shows no antagonistic effect and is shown by "-"; if the mold colony is deformed, such as square, diamond, petal shape, etc., the mold colony is regarded as having antagonistic effect and is indicated by "+"; 2. the bacteria are regarded as having antagonistic action when the zone of inhibition is present, and are indicated by "+", and the bacteria are regarded as having no antagonistic action when the zone of inhibition is not present, and are indicated by "-".
Example 3: indoor potted planting control effect experiment for tomato wilt
Damaging the tomato seedling in the 2-4 true leaf stage with root hair, and adding into pathogenic spore liquid (1 × 10)6spore/mL) for 20min, transplanting into pots filled with vermiculite, 5 plants per pot, and irrigating root with 20mL of pathogenic spore solution (1 × 10)6spore/mL), and after 24h, root irrigation is carried out on the experimental group by 20mL of antagonistic bacteria liquid (the bacteria content is 10)8cfu/mL); taking 50% carbendazim diluted by 500 times as a medicament control, namely, the medicament control treatment is that the 50% carbendazim diluted by 500 times is irrigated to the roots of tomato seedlings inoculated with the blight germina of tomato, and each plant is 20 mL; using clear water treated tomato seedlings inoculated with the tomato fusarium oxysporum as blank control. After 10 strains are treated, the conventional management is carried out, the disease condition is investigated after 35 days of growth, and the control effect is calculated.
The classification standard of tomato blight disease is as follows:
level 0: no symptoms;
level 1: 1-2 true leaves become yellow or withered and drooping;
and 2, stage: 3-4 true leaves become yellow or withered and droop;
and 3, level: 5 to 6 true leaves become yellow or withered and drooping;
4, level: so that the whole plant is withered and withered.
After the tomato fusarium oxysporum spore fluid is artificially infected, the morbidity of a blank control, a medicament control and a microbial inoculum group is 100.00%, 80.00% and 60.00%, the disease index is 62.50%, 18.97% and 15.15%, and the control effect of the medicament control and the microbial inoculum group is 69.65% and 75.76%, which are detailed in table 3. The result shows that the control effect of the bacillus subtilis on tomato wilt is 500 times higher than that of a conventional medicament, namely 50 percent carbendazim diluent.
TABLE 3 potted plant disease prevention of tomato by the Bacillus subtilis of the present invention (transplantation 35d)
Note: the incidence rate is the number of treated plants/total treated plants multiplied by 100 percent;
disease index ∑ (number of diseased plants at each stage × representative grade value)/(total number of investigated plants × highest representative grade value) × 100%;
the prevention and treatment effect is (1-disease index of test area/disease index of control area) × 100%.
Example 4: field control effect experiment for tomato wilt
Disease prevention experiments in the field are carried out in a shop experimental greenhouse. The test method comprises the following steps: there are 3 treatments in total: the bacillus subtilis preparation comprises a bacillus subtilis preparation group, a 50% carbendazim 500-time diluent preparation group and a clear water control group. Repeating for 3 times, and having 9 cells each with an area of 20m2And are randomly arranged. The test is carried out for the first time of root irrigation in 2018, 8, month and 30 days, and each seedling is 300 mL. Irrigating the roots again for 1 time at an interval of 15 days, and controlling for 2 times totally. And (5) investigating the disease occurrence condition of tomato plant blight in the cell at the end of 9 months, and calculating the control effect.
The field control effect result shows that the field control effect of the bacillus subtilis on tomatoes reaches over 75 percent, and is detailed in table 4.
TABLE 4 disease prevention of tomato by the Bacillus subtilis preparation of the invention in the field (transplantation 35d)
Note: the incidence rate is the number of treated plants/total treated plants multiplied by 100 percent;
disease index ∑ (number of diseased plants at each stage × representative grade value)/(total number of investigated plants × highest representative grade value) × 100%;
the prevention and treatment effect is (1-disease index of test area/disease index of control area) × 100%.
Example 5: the microbial agent of the invention
Inoculating the bacillus subtilis strain onto an LB (Langerhans) flat plate, streaking, culturing for 14-16h in a biochemical incubator at 37 ℃, picking out a single colony to be inoculated into an LB liquid culture medium containing 100mL after the single colony grows out, and culturing for 14-16h in a shaking table at 37 ℃ and 170rpm to prepare a seed solution; inoculating the seed liquid into a fermentation culture medium with the inoculation amount of 1 percent for expanding culture for 24 hours, and adjusting the concentration of the bacillus subtilis in the fermentation liquid to 1 multiplied by 109～1010cfu/mL, obtaining the microbial agent.
Wherein the LB solid medium formula is as follows: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar powder and 1000mL of water, wherein the pH value is 7.2; the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 1000mL of water and pH 7.2; the fermentation medium is a corn flour and soybean meal culture medium and comprises the following components in percentage by weight: corn flour 2.0%, soybean flour 2.0%, NaCl 0.5%, MnSO4H20.8 percent of O and the balance of water, and the pH value is 7.2.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. The bacillus subtilis is characterized in that the preservation number is CGMCC No. 17357.
2. The application of the bacillus subtilis with the preservation number of CGMCC No.17357 in preventing and treating fungal pathogenic bacteria and/or bacterial pathogenic bacteria or in preparing a microbial bacteriostatic agent for the fungal pathogenic bacteria and/or the bacterial pathogenic bacteria; the fungal pathogenic bacteria are selected from one or more than two of the following pathogenic bacteria:
tomato fusarium oxysporum, cucumber fusarium oxysporum, melon fusarium oxysporum, watermelon fusarium oxysporum, cotton fusarium oxysporum, banana fusarium oxysporum, stevia rebaudiana fusarium oxysporum, cauliflower fusarium oxysporum, phytophthora capsici, potato rhizoctonia solani, cotton red rot, rice sheath blight, cowpea black rot, cucumber red rot, corn leaf blight, strawberry brown rot, strawberry root rot, wheat scab, colletotrichum gloeosporum, soybean alternaria, sclerotinia, apple rot, Chinese cabbage anthracnose, Hunan phytophthora blight, cucumber scab, fusarium laminarum, cucumber gray mold, tomato gray mold, cauliflower black rot, botrytis cinerea, wheat root rot, cucumber target spot, tomato leaf mold and tomato leaf mold;
the bacterial pathogenic bacteria are ralstonia solanacearum and/or cucumber angular leaf spot bacteria.
3. A microbial agent is characterized by comprising bacillus subtilis with the preservation number of CGMCC No. 17357.
4. The microbial agent according to claim 3, wherein the Bacillus subtilis has a bacterial concentration of 1X 109～1010CFU/mL。
5. A preparation method of a microbial agent is characterized in that Bacillus subtilis with the preservation number of CGMCC No.17357 is prepared into seed liquid, then the seed liquid is inoculated into a fermentation culture medium for expanded culture, and the concentration of the Bacillus subtilis in the fermentation liquid is adjusted to obtain the microbial agent.
6. The use of the microbial agent of claim 3 or 4 or the microbial agent prepared by the preparation method of claim 5 in any one or more than two of the following aspects:
preventing and treating fungal pathogenic bacteria, preparing biological inhibitor of fungal pathogenic bacteria, preventing and treating bacterial pathogenic bacteria and preparing biological inhibitor of bacterial pathogenic bacteria;
the fungal pathogenic bacteria are selected from one or more than two of the following pathogenic bacteria:
tomato fusarium oxysporum, cucumber fusarium oxysporum, melon fusarium oxysporum, watermelon fusarium oxysporum, cotton fusarium oxysporum, banana fusarium oxysporum, stevia rebaudiana fusarium oxysporum, cauliflower fusarium oxysporum, phytophthora capsici, potato rhizoctonia solani, cotton red rot, rice sheath blight, cowpea black rot, cucumber red rot, corn leaf blight, strawberry brown rot, strawberry root rot, wheat scab, colletotrichum gloeosporum, soybean alternaria, sclerotinia, apple rot, Chinese cabbage anthracnose, Hunan phytophthora blight, cucumber scab, fusarium laminarum, cucumber gray mold, tomato gray mold, cauliflower black rot, botrytis cinerea, wheat root rot, cucumber target spot, tomato leaf mold and tomato leaf mold;
the bacterial pathogenic bacteria are ralstonia solanacearum and/or cucumber angular leaf spot bacteria.
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