CN109504638B - Bacillus belgii GUAL210 and application thereof - Google Patents
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Abstract
The invention discloses Bacillus belgii GUAL210, which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2018871. The invention also discloses application of the Bacillus belgii GUAL210 in simultaneous control of tobacco black shank and tobacco bacterial wilt. The Bacillus belgii GUAL210 has obvious capacity of antagonizing tobacco black shank bacteria and ralstonia solanacearum at the same time, and capacity of antagonizing pepper anthracnose.
Description
Technical Field
The invention relates to Bacillus belgii GUAL210 and application thereof, and belongs to the technical field of biological control.
Background
Tobacco black shank and tobacco bacterial wilt are the most destructive soil-borne diseases in tobacco production in China, huge economic loss is caused each year, Guizhou province is one of main production areas of tobacco production in China, tobacco soil-borne diseases are serious, economic loss caused by diseases is close to hundred million yuan each year, and economic income of tobacco growers is seriously influenced. At present, the disease control in production mainly adopts planting disease-resistant varieties and comprehensive control measures of mainly planting disease-resistant varieties and mainly controlling cultivation and secondarily controlling medicaments, but no proper good varieties for resisting (resisting) soil-borne diseases exist in the production practice. Therefore, chemical control methods are mostly adopted to control diseases at present. At present, the pesticide control depends on metalaxyl, agricultural streptomycin and other chemical pesticides, and because of few varieties of alternative pesticides, the service life of the main pesticide is long, 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, biological control has developed into a hot field of research on control of tobacco diseases in recent years.
The use of bacillus for plant disease control is one of the hot fields of biological control research at present, and the bacillus has the precondition of industrialization and wide adaptability; the action mechanism is various, and the drug resistance is not easy to generate; the fermentation period is short, and the production cost is low; spore can be formed, and the storage and the transportation are easy; the bacillus is environment-friendly and safe to human and livestock, so that the development of a biocontrol agent by using the bacillus becomes an important direction for the development of the biocontrol agent. Some researchers have tried to screen biocontrol bacillus for tobacco soil-borne diseases (tobacco black shank and tobacco bacterial wilt) at home and abroad. In addition, tobacco black shank and tobacco bacterial wilt in Guizhou areas are frequently mixed, and no article reports that bacillus capable of preventing and controlling the two diseases is screened at present.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the Bacillus belgii GUAL210 can simultaneously prevent and control tobacco black shank and tobacco bacterial wilt and pepper anthracnose so as to overcome the defects of the prior art.
The technical scheme of the invention is as follows: the Bacillus belgii GUAL210 is preserved in the China center for type culture Collection (address: Wuhan university in Wuhan, China) in 2018, 12 and 22 months, and the preservation number is CCTCC NO: m2018871, name: bacillus belgii GUAL210(Bacillus velezensis GUAL 210).
The invention also provides application of the Bacillus belgii GUAL210 in simultaneous control of tobacco black shank and tobacco bacterial wilt.
The invention also provides application of Bacillus belgii GUAL210 in preventing and treating tobacco black shank.
The invention also provides application of the Bacillus belgii GUAL210 in preventing and treating tobacco bacterial wilt.
The invention also provides application of the Bacillus belgii GUAL210 in preventing and treating pepper anthracnose.
The invention has the beneficial effects that: the method collects the rhizosphere soil of healthy tobacco strains from areas with serious tobacco black shank and bacterial wilt disease in Anlong county of Guizhou province, and obtains the bacillus GUAL210 by screening and separating the rhizosphere soil, and the bacillus GUAL has obvious capacity of antagonizing tobacco black shank bacteria (Phytophthora nicotianae) and ralstonia solanacearum and capacity of antagonizing pepper anthracnose. GUAL210 was identified as bacillus belgii (b.velezensis) by morphological, physiobiochemical characteristics and 16S rDNA genes.
Drawings
FIG. 1 shows the antagonistic effect of Bacillus on tobacco black shank screening, CK: phytophthora nicotianae (Phytophthora nicotianae); GUAL210 represents a screened bacillus strain having an antagonistic effect;
FIG. 2 shows the antagonistic effect of Bacillus on Ralstonia solanacearum, CK: ralstonia solanacearum (Ralatonia solanacearum); GUAL210 represents a screened bacillus strain having an antagonistic effect;
FIG. 3 strain GUAL 21016S rDNA gene clade;
FIG. 4 bacterial colony morphology and biofilm (biofilm) morphology of strain GUAL 210;
FIG. 5 identification of the strain GUAL210 antibiogram;
FIG. 6 identification of Strain GUAL210 antibacterial substances.
Detailed Description
The invention will be further described with reference to the following drawings and specific embodiments:
separation and screening of Bacillus belgii GUAL210
1. Materials and methods
1.1 materials
Soil samples were collected in 2017 and 2018, healthy tobacco plant rhizosphere soil samples were collected from tobacco black shank and bacterial wilt disease-causing plots in Anlong county, Guizhou, and West, Guizhou province, and 1 part of soil sample was mixed in the same plot by using a 5-point sampling method. The paper bag is used for collecting the soil sample, and the soil collecting tool is disinfected in advance, so that soil microorganism mixing is avoided.
1.2 culture Medium
The LB culture medium is used for separation and preservation of bacillus and a plate confrontation experiment; the PDA culture medium is mainly used in the plate confrontation experiment.
1.3 tobacco disease Strain
The test strains, Phytophthora nicotianae (Phytophthora nicotiana) and ralstonia solanacearum (Ralatonia solanacearum), were provided by the phytopathology laboratory of the college of agriculture of the university of Guizhou.
2. Isolation and preservation of rhizosphere bacillus
The soil dilution is subjected to water bath in a water bath kettle at 85 deg.C for 30min by dilution plate method to kill most of non-spore bacteria. 100. mu.L of the dilution was applied to LB plates, 3 plates per concentration, and cultured at 37 ℃. After 1 day of culture, different single colonies were picked, transferred to tubes 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 and ralstonia solanacearum as test plant pathogenic bacteria, screening bacillus capable of antagonizing the two pathogenic bacteria simultaneously by a plate confronting method, observing whether an inhibition zone is generated or not by primary screening and secondary screening, measuring the inhibition diameter by a cross method, detecting whether a biological membrane, protease, cellulase, siderophin, phosphatase and the like are generated or not by a specific plate, and selecting bacillus 1 strain with better antagonistic effect, wherein the number of the bacillus 1 strain is GUAL210 (see fig. 1 and fig. 2).
The bacteriostatic ratio (%) [ (diameter of control colony-diameter of treatment colony)/diameter of treatment colony ] × 100
Identification of Bacillus belgii GUAL210
1. Biocontrol strain identification
The screened 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 ℃, centrifuging to collect thallus, washing the thallus with sterile water for 3 times, and finally suspending and diluting with sterile water to a concentration of 1 × 108CFU/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 a Neighbor-Joining algorithm in CLUSTALX software for multi-sequence alignment and phylogenetic analysis software MEGA 6.
After analysis: the GUAL210 can form a relatively complex colony structure on LB medium (FIG. 4). The physiological and biochemical characteristics of the strain are shown in Table 1, and the strain G+Can decompose glucose, rhamnose and mannitol to produce catalase and nitrate reductase, and has the characteristics of positive methyl red staining, liquefaction of gelatin and the like. Through 16S rDNA gene sequence analysis, a 16S rDNA gene evolutionary tree of the strain GUAL210 is constructed, and the result shows that the strain GUAL210 and Bacillus velezensis (Bacillus velezensis) are gathered into a branch (figure 3). GUAL210 is identified as Bacillus velezensis (Bacillus velezensis) through morphological, physiological and biochemical characteristics and 16S rDNA gene.
TABLE 1 physiological and biochemical characteristics of the strain GUAL210
+: positive reaction; -: negative reaction
2. Identification of antibiogram of biocontrol strain
The strain GUAL210 has spectrum antagonistic activity, and has good antibacterial effect on multiple pathogenic fungi such as tobacco black shank bacteria, tobacco ralstonia solanacearum, tobacco brown spot bacteria, pepper colletotrichum gloeosporioides and sorghum spot bacteria (see figure 5). The two strains have potential biological control effect on the diseases.
3. Identification of biocontrol strain antibacterial substance
Strain GUAL210 produces both siderophore and phosphatase (see FIG. 6), which are associated with the growth of plants by Bacillus. The bacillus colonizes plant root systems and generates a complex biomembrane structure, can effectively occupy favorable sites on root surfaces, limit the invasion of pathogenic bacteria hyphae, can induce plants to generate resistance to pathogenic bacteria, and achieves the effects of preventing diseases and increasing yield.
Application of Bacillus belgii GUAL210 in preventing and treating plant diseases
1. Tobacco black shank control field test
The test varieties are: and (4) cloud smoke 87. Test site: guizhou Anlong. 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 belgii GUAL210, using a 30% pesticide-metalaxyl-hymexazol water agent (Zhongnong Rihua (Tianjin) agricultural chemical Co., Ltd.) as a positive control, using a greenish kang (greenish kang is a microecological preparation developed by Zhongnong greenkang (Beijing) Co., Ltd.) for improving the soil microecological disease control and growth promotion and yield increase effects), using a blank control without any pesticide treatment, totally 3 treatments, repeating for 3 times, 30 cigarettes in each cell, and randomly arraying all the treatments into a block. 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 108And (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 27 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 Anlong area tobacco Black shank in the field plot control
As can be seen from Table 2, the bacillus has certain control effect on the tobacco black shank, and the control effect is equivalent to that of onychomycosis and hymexazol. The hymexazol is a main medicament for preventing and treating the tobacco black shank in the prior production. 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: guizhou Anlong. 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 beiLeisi GUAL210 is used for processing, the zhongshengmycin (the zhongshengmycin is a novel agricultural antibiotic developed and succeeded by the biological control of Chinese academy of agricultural sciences, is an antibiotic produced by Hainan variety of streptomyces lavendulae, belongs to N-glycoside alkaline water-soluble substance, the processing dosage form of the bacterium is a protective bactericide with wider bactericidal spectrum, has contact killing and osmosis functions, the zhongshengmycin has high activity to bacterial diseases and partial fungal diseases of crops, has a certain yield-increasing effect, is a common biological bactericide in the prior production, has obvious effect of preventing and treating solanaceous bacterial wilt) as a positive control, pseudomonas fluorescens (a commercialized reagent) is adopted (Pseudomonas fluorescens is a widely distributed gram-negative rod-shaped bacterium, has strong environmental adaptability, and has beneficial effects of inhibiting various pathogenic bacteria, helping plants to absorb nutrition, promoting plant growth and the like.It is used as a specific medicine for preventing and treating bacterial diseases such as bacterial wilt, soft rot, bacterial angular leaf spot, rot and ginger blast. The disadvantages are that the shelf life is short, the number of live bacteria is reduced and the activity of the bacteria is reduced after 6 months of production, the efficacy is greatly reduced, the bacteria can not be used as bactericide, but can be mixed into compost to play a little role in fertilizer efficiency. The bacillus can generate stress-resistant spores, if the water content of the product is less than 5%, the product can reach the shelf life of 2-3 years at present, and can also be stored for a long time, which is the main reason for the bacillus to be used as a main biological agent), the blank control is not treated by any medicament, 3 treatments are carried out, 30 cigarettes are repeated for 3 times, 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 2 times when the tobacco seedlings are transplanted; the Bacillus concentration is 108And (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 27 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 to start disease initiation, and then the blank control is carried out for 3 times before harvesting, the disease incidence of tobacco bacterial wilt of all treated cells is investigated, and the disease severity is investigated 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 field plot control of tobacco bacterial wilt in Anlong region
Experimental results show that the bacillus has good control effect on tobacco bacterial wilt.
3. Field test for controlling pepper anthracnose
And (3) test varieties: codonopsis pilosula and Wu. Test site: flower stream district in Guiyang city.
And (3) experimental design: treatment with Bacillus belgii GUAL210 for commercial controlAgent Lukangwei (Zhongnong Lukang (Beijing) Biotechnology Co., Ltd.) as a positive control, a blank control without any agent treatment, 4 treatments, 3 repetitions, 30 peppers per cell, all treatments 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 108And (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.
In the field investigation time, blank control is selected to start disease initiation and then the disease initiation is carried out for 2 times, the disease incidence of pepper anthracnose in all treatment districts is investigated, and the disease severity 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.
TABLE 4 field plot control of Bacillus vs. Capsicum anthracnose
Experimental results show that the bacillus has obvious control effect on pepper anthracnose and has better effect than commercial biocontrol agents.
Bacillus belgii can produce various antibacterial substances to inhibit phytopathogens. It has been found that the antifungal activity of Bacillus is derived from cyclic lipopeptide antibiotics, whereas the antibacterial activity may be due to polyketides and bacteriocins produced by the ribosome synthesis pathway. These substances can inhibit the growth of bacteria and fungal hyphae and spore germination. However, the genome difference of different bacilli is large, so the bacteriostatic substances produced by the bacilli separated by the invention need to be further researched.
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)
1. Bacillus beleisi capable of preventing and treating tobacco black shank, tobacco bacterial wilt and pepper anthracnoseBacillus velezensis ) GUAL210, preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018871.
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