CN109593682B

CN109593682B - Bacillus subtilis GUMT332 and application thereof

Info

Publication number: CN109593682B
Application number: CN201811639287.7A
Authority: CN
Inventors: 彭丽娟; 丁海霞; 余水
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2022-09-13
Anticipated expiration: 2038-12-29
Also published as: CN109593682A

Abstract

The invention discloses a bacillus subtilis GUMT332 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2018874. The invention also discloses an application of the bacillus subtilis GUMT332 in preventing and treating tobacco black shank. The bacillus subtilis GUMT332 has obvious capacity of antagonizing tobacco phytophthora parasitica and capacity of antagonizing pepper anthracnose.

Description

Bacillus subtilis GUMT332 and application thereof

Technical Field

The invention relates to bacillus subtilis GUMT332 and application thereof, and belongs to the technical field of biological control.

Background

Tobacco black shank (Phytophthora nicotiana) is one of the most destructive soil-borne diseases in Guizhou tobacco production, and the economic loss caused by the disease is nearly ten thousand yuan every year, thus seriously affecting the economic income of tobacco growers. At present, medicaments are mostly adopted for prevention and treatment in production, but the main medicaments have long service life, the drug resistance of pathogenic bacteria is increased year by year, and the negative effects of pesticide residue, environmental pollution and the like are gradually increased. Therefore, the biological control is a hot field of research at present.

Bacillus spp is the most common microorganism species in the current biological control research, has the advantages of industrial prospect, various action mechanisms, low production cost, easy storage and transportation, difficult generation of drug resistance, safety to people and livestock and the like, and is an important direction for developing biological control preparations. At present, various natural bacillus isolates with excellent properties are used as living microbial preparations at home and abroad to be successfully applied in a commercialized way, and the development potential is huge. BioYield developed by Gustafson corporation, a fungicide made by blending Bacillus amyloliquefaciens (B.amyloliquefaciens) FZB42 and Bacillus subtilis GB122, has been used for the control of various plant diseases. The yield increasing series products prepared from bacillus cereus, bacillus subtilis and the like, which are researched by Chinese agriculture university at the earliest time in China, have good yield increasing and disease preventing effects on rice, wheat, vegetables and more economic crops.

At present, the development of a biocontrol preparation suitable for preventing and controlling black shank diseases of Guizhou tobacco is realized, good preparations can be used relatively rarely, and a plurality of reports prove that the drug resistance of black shank pathogenic bacteria in tobacco producing areas of Guizhou province is increased year by year, so that better biological strains are needed to be developed to control the generation of the black shank diseases of the tobacco.

In addition, the pepper anthracnose is one of the main diseases in pepper production, and can be harmful in the whole growth period of the pepper, thereby causing great loss. Guizhou province is one of the main cultivation places of pepper in China, the disease area of anthracnose caused by long-term continuous cropping of pepper is continuously enlarged, and the income of farmers is seriously influenced. At present, chemical pesticides are mainly adopted to prevent and treat diseases in production, the drug resistance of pathogenic bacteria is increased year by year, the field dosage is correspondingly increased, and pesticide residues and environmental pollution are easily caused. Therefore, the biological control is a hot field of research at present.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a bacillus subtilis GUMT332 which has better control effect on tobacco black shank and pepper anthracnose so as to overcome the defects of the prior art.

The technical scheme of the invention is as follows: the bacillus subtilis GUMT332 is preserved in China center for type culture Collection (address: Wuhan university in Wuhan, China) in 2018 at 12 and 22 months, and the preservation number is CCTCC NO: m2018874, name: bacillus subtilis GUMT332(Bacillus subtilis GUMT 332).

The invention also provides application of the bacillus subtilis GUMT332 in preventing and treating tobacco black shank.

The invention also provides application of the bacillus subtilis GUMT332 in preventing and treating tobacco bacterial wilt.

The invention also provides application of the bacillus subtilis GUMT332 in preventing and treating pepper anthracnose.

The invention has the beneficial effects that: the method collects rhizosphere soil from flue-cured tobacco planting plots in Zunyi City of Guizhou province, screens and separates the rhizosphere soil to obtain the Bacillus subtilis GUMT332 which has obvious capability of antagonizing tobacco black shank bacteria (Phytophthora nicotianae) and capability of antagonizing pepper anthracnose. GUMT332 is identified as bacillus subtilis (B.subtilis) through morphological, physiological and biochemical characteristics and 16S rDNA gene.

Drawings

FIG. 1 shows the antagonistic effect of Bacillus on tobacco black shank screening, CK: tobacco black shank (Phytophthora nicotianae); GUMT332 represents a screened Bacillus strain with antagonistic effect;

FIG. 2 strain GUMT 33216S rDNA gene evolutionary tree;

FIG. 3 identification of the GUMT332 strain antibiogram;

FIG. 4 strains GUMT332, GUMT332 and GUMT332 biofilms, protease, cellulase, siderophore and phosphate assays.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments:

separation and screening of Bacillus subtilis GUMT332

1. Materials and methods

1.1 materials

In 2017 and 2018, a plot with serious tobacco black shank attack is selected from Meitan county in Zunyi city in Guizhou province, the rhizosphere soil of healthy tobacco plants is collected, and 1 part of soil sample is mixed in the same plot by a 5-point sampling method. The aseptic valve bag is used for collecting soil samples, and the soil collecting tool is disinfected by 75% alcohol, so that soil microorganisms among different fields are prevented from being mixed.

1.2 culture Medium

The LB culture medium is used for separating and storing bacillus; the PDA culture medium is used for a plate confrontation culture experiment; the protease detection culture medium, the cellulase detection culture medium, the siderophin detection culture medium and the phosphatase detection culture medium are used for detecting whether the bacillus produces protease, cellulase, siderophin, phosphatase and the like.

1.3 test strains

The test strain, Nicotiana tabacum (Phytophthora nicotianae), was supplied by the Phytopathology laboratory of the college of agriculture of Guizhou university.

2. Isolation and preservation of rhizosphere bacillus

The soil dilution is subjected to water bath in a water bath kettle at 85 ℃ for 30min by adopting a dilution plate method to kill most of non-spore bacteria. Different single colonies were picked, the strains were purified and stored in a-80 ℃ freezer.

3. Tobacco black shank bacterium and ralstonia solanacearum antagonistic bacillus screening method

The method comprises the steps of taking tobacco black shank bacteria as test plant pathogenic bacteria, screening biological control strains with obvious tobacco black shank prevention and control effects by a plate confronting method, observing whether an inhibition zone is generated or not by primary screening and secondary screening, and measuring the inhibition diameter by a cross method. And detecting whether a biological membrane, protease and cellulase are produced or not by a specific plate, and selecting 3 strains with the best effect for a subsequent field test, wherein the number of the strains is GUMT332 (shown in figure 1).

Identification of bacillus subtilis GUMT332, where the bacteriostatic rate (%) is [ (control group colony diameter-treatment group colony diameter)/treatment group colony diameter ] × 100 ″, and

1. biocontrol strain identification

The selected strains are identified by morphological, physiological and biochemical characteristics and 16S rDNA genes.

1.1 colony morphology Observation

Inoculating the strain into a test tube containing 5mL of LB culture solution, shaking overnight at 37 deg.C, centrifuging to collect thallus, washing thallus with sterile water for 3 times, and resuspending with sterile water and diluting to 1 × 10 ⁸ CFU/mL, 10. mu.L of the bacterial suspension was dropped on an LB plate, and the colony morphology was observed after overnight culture at 37 ℃.

1.2 physiological and Biochemical characteristics

The physiological and biochemical properties of the strains were determined according to Bergey's Manual of systematic bacteriology.

1.3 molecular biological identification

50mg/mL lysozyme was treated with water bath at 37 ℃ for 1h, and genomic DNA was extracted using a Biomiga bacterial genomic DNA extraction kit. And (3) taking the genome DNA as a template, carrying out PCR amplification by adopting a 16S rDNA universal primer 27F/1492R, and sending the PCR product to Shanghai workers for PCR product sequencing after the PCR product is verified to be correct. And performing BLAST alignment analysis on the sequencing result in an NCBI database, and constructing a phylogenetic tree by utilizing CLUSTAL X software to perform multiple sequence alignment and a Neighbor-Joining algorithm in a phylogenetic analysis software MEGA 6.

After analysis: GUMT332 can form a relatively complex colony structure on LB medium. The physiological and biochemical characteristics of the strain are shown in table 1, the strain is G +, glucose, rhamnose and mannitol can be decomposed to generate catalase and nitrate reductase, methyl red is positive in staining, gelatin can be liquefied, and the like. Through 16S rDNA gene sequence analysis, a 16S rDNA gene evolutionary tree of the strain GUMT332 is constructed, and the result shows that the strain GUMT332 and bacillus subtilis are gathered into a branch (figure 2). GUMT332 is identified as bacillus subtilis (B.subtilis) through morphological, physiological and biochemical characteristics and 16S rDNA gene.

TABLE 1 physiological and biochemical characteristics of the Strain GUMT332

+: positive reaction; -: negative reaction

2. Identification of antibiogram of biocontrol strain

The strain GUMT332 has spectrum antagonistic activity, and has good antibacterial effect on multiple pathogenic fungi such as tobacco phytophthora parasitica, tobacco brown spot, pepper colletotrichum and sorghum spot (see figure 3). The strain has potential biological control effect on the diseases.

3. Bacterial biofilm, protease and cellulase assays

The GUMT332 bacillus can generate a more complex biomembrane structure and can generate protease, cellulase, phosphatase and siderophore (see figure 4), and is related to the promotion of plant growth by the bacillus. The bacillus colonizes the root system of the plant and generates a complex biofilm structure, can effectively occupy favorable sites on the root surface, limits the invasion of pathogenic bacteria hyphae, can induce the plant to generate resistance to the pathogenic bacteria, and achieves the effects of preventing diseases and increasing yield.

Application of bacillus subtilis GUMT332 in preventing and treating plant diseases

1. Tobacco black shank prevention and control field test

The test varieties are: and (4) cloud smoke 87. Test site: meitan county copy le town. Test site: the fertility is uniform, and the cured tobacco is planted in 2017 and the black shank disease is in the field.

And (3) experimental design: treating with Bacillus subtilis GUMT332, using a 30% agent methyloxazilin water aqua (Zhongnong Hua (Tianjin) agricultural chemicals limited) as a positive control, using greenish kang (the greenish kang is a microecological preparation developed by Zhongnong greenkang (Beijing) limited responsibility company and used for improving the effects of soil microecological disease control and growth promotion and yield increase) as a positive control, using a blank control without any agent treatment, totally 3 treatments, 3 times of repetition, 30 cigarettes in each cell, and arranging all the treatment random blocks. The treatment concentration and the dosage of the medicament refer to the description of the medicament, and the medicament is applied by root irrigation for 2 times when the tobacco seedlings are transplanted; the Bacillus concentration is 10 ⁸ And (3) irrigating the roots with the CFU/mL and 50mL of bacterial liquid for 3 times, irrigating the roots 1 time when the tobacco seedlings are transplanted, and irrigating the roots 1 time every 7-10 days. Transplanting in 2018, 4 and 23 days, wherein the field cultivation management method is consistent with that of the local area, and no other bactericide is applied.

And (3) field investigation: in the field investigation time, blank control is selected, the disease is started after the disease is collected, the blank control is carried out for 3 times before the disease is collected, the incidence rate of the black shank of tobacco plants in all treated cells is investigated, and the severity of the disease is investigated in a grading manner. The grading standard of the tobacco black shank disease is carried out according to GB/T23222-2008, the morbidity and disease index of the disease are calculated, and the data are subjected to variance analysis by adopting a Duncan new complex polarization method of a DPS data processing system. The results are shown in Table 2.

TABLE 2 Metan area tobacco black shank's field district is prevented imitating

As can be seen from Table 2, the bacillus has a certain control effect on the tobacco black shank, but the control effect is not as good as that of the first frost hymexazol. The metalaxyl-hymexazol is a main medicament for preventing and treating the tobacco black shank in the current production, and the effect of preventing and treating the tobacco black shank in the test is good. Reports show that the tobacco phytophthora parasitica has drug resistance to metalaxyl-manganese zinc in production. Therefore, by combining the advantages and the disadvantages of chemical agents and biological bactericides, the biocontrol strain and chemical pesticide can be tried to be applied in a compounding way, and the purpose of reducing the dosage of the chemical pesticide is achieved.

2. Tobacco bacterial wilt prevention and control field test

The test varieties are: and (4) cloud smoke 87. Test site: meitan county copy le town. Test site: the fertility is uniform, and the cured tobacco is planted in 2017 and the plots are planted in the disease of bacterial wilt.

And (3) experimental design: the bacillus subtilis GUMT332 is used for treating, zhongshengmycin (zhongshengmycin is a novel agricultural antibiotic successfully developed by biocontrol of Chinese academy of agricultural sciences, is an antibiotic produced by Hainan variety of Streptomyces lavendulae and belongs to an N-glycoside alkaline water-soluble substance, the processing dosage form of the bacillus subtilis is a protective bactericide with a wider bactericidal spectrum, has contact killing and osmosis effects, has very high activity on bacterial diseases and partial fungal diseases of crops, and also has a certain yield increasing effect, is a common biological bactericide in the current production, has an obvious effect on preventing and treating solanaceae bacterial wilt) as a positive control, greenish as a positive control, is not subjected to any medicament treatment as a blank control, is subjected to 3 treatments for 3 times, is subjected to 30 tobacco blocks in each cell, and is randomly arranged. The treatment concentration and the dosage of the medicament refer to the description of the medicament, and the medicament is applied by root irrigation for 2 times when the tobacco seedlings are transplanted; the Bacillus concentration is 10 ⁸ And (3) irrigating the roots with CFU/mL and 50mL of bacterial liquid for 3 times, irrigating the roots 1 time when the tobacco seedlings are transplanted, and irrigating the roots 1 time every 7-10 days. Transplanting in 2018, 4 and 23 days, wherein the field cultivation management method is consistent with that of the local area, and no other bactericide is applied.

And (3) field investigation: and selecting blank control in field investigation time, starting diseases, then performing 3 times in total before harvesting, investigating the incidence of bacterial wilt of tobacco plants in all treated cells, and investigating the severity of diseases in a grading manner. The tobacco bacterial wilt disease grading standard is carried out according to GB/T23222-2008, the disease incidence and disease index are calculated, and the data is subjected to variance analysis by adopting a Duncan new complex range method of a DPS data processing system. The results are shown in Table 3.

Table 3 Meitan area tobacco bacterial wilt field plot control effect

Experimental results show that the bacillus has good control effect on tobacco bacterial wilt, and is lower than zhongshengmycin but better than greenish green.

3. Field test for controlling pepper anthracnose

The test varieties are: an Party and Wu. Test site: guiyang city Huaxi district.

And (3) experimental design: treating with Bacillus subtilis GUMT332, using commercial biocontrol agent Lukangwei (Zhongnong Lukang (Beijing) biotechnology limited) as positive control, and blank control without any agent treatment, and repeating for 3 times, wherein each cell contains 30 pepper strains, and all treatments are arranged in random blocks. The treatment concentration and the dosage of the medicament refer to the description of the medicament, and the medicament is applied by root irrigation for 3 times when the pepper seedlings are transplanted; the Bacillus concentration is 10 ⁸ And (3) irrigating the roots with the bacterial liquid of CFU/mL and 50mL for 3 times, irrigating the roots for 1 time during transplanting of the pepper seedlings, and irrigating the roots for 1 time at intervals of 7-10 days. The field cultivation management method is consistent with that of the local area, and other bactericides are not applied.

Blank controls are selected in field investigation time to start diseases and then the blank controls are carried out for 2 times, the incidence of the pepper anthracnose in all treatment districts is investigated, and the severity of the diseases is investigated in a grading manner. The grading standard of the pepper anthracnose disease is carried out according to GB/T17980.33-2000, the disease incidence and the disease index of the disease are calculated, and the data is subjected to variance analysis by adopting a Duncan new complex range method of a DPS data processing system.

Disease investigation began when the empty control plot pepper anthracnose began to occur. The survey was conducted 1 st time in 2018, 5 months, 22 days, 1 time every 7-10 days, and 3 times in total. The results are shown in Table 4.

Field plot control effect of bacillus 4 on pepper anthracnose

Experimental results show that the bacillus has obvious prevention effect on pepper anthracnose and has better effect than commercial biocontrol agents.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. Bacillus subtilis capable of preventing and treating tobacco black shank, tobacco bacterial wilt and pepper anthracnoseBacillus subtilis ) GUMT332, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018874.