CN116024117B - Bacillus firmus for preventing and treating cotinus coggygria canker, biocontrol microbial inoculum and application thereof - Google Patents
Bacillus firmus for preventing and treating cotinus coggygria canker, biocontrol microbial inoculum and application thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
本发明涉及生物农药技术领域，具体涉及一种防治黄栌溃疡病的坚强芽孢杆菌及其生防菌剂和应用。坚强芽孢杆菌(Bacillus firmus)HG‑13c已于2022年07月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.25308；该菌株及其生防菌剂可用于抑制葡萄座腔菌生长和/或防治黄栌溃疡病。本发明提供的含有坚强芽孢杆菌HG‑13c的生防菌剂在大田实验中14天防效可达到61.48％，21天防效可达到65.02％；特别是与巨大芽孢杆菌BMJBN02复配使用时，14天防效可达到88.69％，21天防效可达到89.12％。The present invention relates to the technical field of biological pesticides, and in particular to a Bacillus firmus for preventing and treating cotinus canker and its biocontrol agent and application. Bacillus firmus HG‑13c has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on July 15, 2022, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 25308; the strain and its biocontrol agent can be used to inhibit the growth of Botrytis cinerea and/or prevent and treat cotinus canker. The biocontrol agent containing Bacillus firmus HG‑13c provided by the present invention can achieve a 14-day protection effect of 61.48% and a 21-day protection effect of 65.02% in field experiments; in particular, when used in combination with Bacillus megaterium BMJBN02, the 14-day protection effect can reach 88.69%, and the 21-day protection effect can reach 89.12%.
Description
Technical Field
The invention relates to the technical field of biological pesticides, in particular to bacillus firmus for controlling cotinus coggygria canker, and a biocontrol microbial inoculum and application thereof.
Background
In summer 2018, the cotinus coggygria is in a plurality of forest parks in the middle eastern region of Shandong province, symptoms mainly occur on trunks, the trees gradually wilt along with pathogen infection, dark brown ulcer symptoms appear under the infected part, the pathogenic plants are scattered or distributed in pieces, the red leaf landscapes of the cotinus coggygria are seriously influenced, and the disease is found to be similar to common cotinus coggygria in symptoms through pathogen identification, but the pathogen is the viticola (B.dothidea).
At present, the prevention and treatment of cotinus coggygria is mainly to strengthen the water and fertilizer management, treat the plants in time and spray chemical pesticides, but the prevention and treatment effect is not obvious, chemical pesticide residues cause serious environmental pollution problems, and an urgent need exists to find economic, green and safe prevention and treatment measures.
Disclosure of Invention
Aiming at the technical problem that the existing control method of the cotinus coggygria is not ideal in effect, the invention provides bacillus firmus for controlling cotinus coggygria canker, and a biocontrol microbial agent and application thereof. The bacillus firmus and biocontrol microbial inoculum with high-efficiency inhibition activity on the viticola are screened for the first time, and a better control effect on the cotinus coggygria canker is obtained in a field plot test.
In a first aspect, the present invention provides bacillus firmus (Bacillus firmus) HG-13c for preventing and treating cotinus coggygria, which has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 15 th month of 2022, with a deposit address of North Star Xiya 1, baiya 1, beijing, and a deposit number of CGMCC No.25308.
In a second aspect, the invention provides an application of bacillus firmus HG-13c in inhibiting the growth of Botrytis cinerea.
In a third aspect, the invention provides an application of bacillus firmus HG-13c in controlling cotinus coggygria.
In a fourth aspect, the present invention provides a biocontrol agent comprising Bacillus firmus HG-13 c.
Furthermore, the biocontrol microbial agent also contains bacillus megatherium (Bacillus megatherium) BMJBN, the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 30 days in 01 month 2015, the preservation address is the No. 3 of the West Song No.1 of North Star of the Korean area of Beijing, the preservation number is CGMCC No.10478, and the strain is disclosed in a composite microbial agent for preventing and treating downy mildew of fruits and vegetables in China patent No. 20151082386. X and a preparation method thereof.
Further, the preparation method of the biocontrol microbial agent comprises the steps of adding a carrier into microbial fermentation broth according to saturated adsorption quantity, drying after adsorption to obtain an adsorption carrier, mixing the adsorption carrier with a stabilizer, a dispersing agent and a wetting agent, crushing and sieving to obtain the biocontrol microbial agent.
Further, the carrier is at least one of diatomite and starch, the stabilizer is humic acid, the dispersing agent is polyvinyl alcohol, and the wetting agent is Tween 80.
Further, the mass ratio of the adsorption carrier, the stabilizer, the dispersing agent and the wetting agent is 70-85:10-15:5-10:1-5.
Furthermore, the dosage form of the biocontrol microbial agent can be wettable powder, water dispersible granules, suspending agents, suspoemulsion, aqueous emulsion or microemulsion.
Furthermore, when the biocontrol microbial agent is prepared, additives such as a binder, a defoaming agent, a disintegrating agent, an anti-freezing agent and the like can be added according to different dosage forms.
In a fifth aspect, the invention provides an application of the biocontrol microbial agent in inhibiting the growth of the Botrytis cinerea.
In a sixth aspect, the present invention provides an application of the biocontrol microbial agent in controlling cotinus coggygria.
The invention has the beneficial effects that:
The bacillus firmus HG-13c provided by the invention has a high-efficiency inhibition effect on the viticola, and can be used as a high-efficiency biocontrol bacterium for cotinus canker for developing a novel biological pesticide or biological microbial inoculum.
The biocontrol microbial inoculum containing bacillus firmus HG-13c provided by the invention has a 14-day control effect of 61.48%, a 21-day control effect of 65.02% in field experiments, and especially has a 14-day control effect of 88.69% and a 21-day control effect of 89.12% when being compounded with bacillus megaterium BMJBN 02.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
EXAMPLE 1 isolation and identification of Bacillus firmus HG-13c
1.1 Isolation of HG-13c Strain
(1) In an ultra-clean workbench, 10g of soil collected from a vegetable greenhouse in the region of Jinan City of Shandong province is placed in 100mL of sterile normal saline, and is subjected to shake flushing for 2h, and fully suspended to prepare bacterial suspension.
(2) After the bacterial suspension is diluted by sterile water in a gradient way, 100 mu L of bacterial suspension dilution liquid with each concentration is coated on an LB culture medium plate, the culture is carried out for 48 hours at the constant temperature of 28 ℃, and then single bacterial colonies with different shapes, sizes and colors on the plate are picked up, streaked and purified on the LB plate, and the serial numbers are obtained.
(3) Culturing purified strains with different numbers in LB liquid medium at 28 ℃ and 180rpm/min for 24 hours, respectively mixing 2mL of fermentation liquor into 15mL of PDA culture medium, pouring the fermentation liquor into a flat plate, inoculating the Botrytis cinerea at the central position of the flat plate for inhibition co-culture after solidification, and naming the obtained strain with strong antagonistic capability to Cotinus coggygria as HG-13C.
1.2 Identification of strains
(1) 16S rDNA sequencing
Extracting the genome DNA of HG-13c, using the genome DNA as a template, carrying out PCR amplification by using a bacterial 16S rDNA universal primer, recovering an amplification product, and sequencing to obtain a sequence shown as SEQ ID NO. 1.
(2) The sequences obtained by sequencing were subjected to homology comparison, and the homology of the strain HG-13c with Bacillus firmus was found to be 99.99%, and the strain HG-13c was identified as Bacillus firmus (Bacillus firmus) in combination with the detection of gram staining and physiological and biochemical characteristics of bacteria.
The bacillus firmus HG-13c is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 15 months of 2022, and has a preservation address of number 1, number 3, and a preservation number of CGMCC No.25308 in the Korean area North Star of Beijing city.
EXAMPLE 2 antagonism of Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c against Botrytis cinerea
(1) Respectively taking purified bacillus megatherium BMJBN and bacillus firmus HG-13C strains, culturing for 24 hours in LB liquid medium at 28 ℃ and 180rpm/min, taking 2mL of fermentation liquor, mixing into 15mL of PDA medium, pouring the fermentation liquor into a flat plate, and solidifying for later use.
Wherein, the bacillus megatherium BMJBN is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 30 months in 01 month 2015, the preservation address is North Star Xili No.1, 3 in the Korean region of Beijing city, and the preservation number is CGMCC No.10478.
(2) Punching holes on the edge of the cultured grape vine cavity colony by using a puncher with the diameter of 0.5cm to obtain a bacterial sheet for later use.
(3) And (3) picking the bacterial slices into the central positions of the PDA flat plates in the step (1) by using a sterile picking needle, setting the PDA flat plates which are only inoculated with the Botrytis cinerea bacterial slices as blank control, setting 3 repetitions of each treatment, culturing at 28 ℃, and observing the colony growth speed and the colony radius of the Botrytis cinerea.
After 3 days of culture, compared with a blank control, the growth inhibition rate of bacillus megatherium BMJBN02 on the Botrytis cinerea is 67.6%, and the growth inhibition rate of bacillus firmus HG-13C on the Botrytis cinerea is 79.3%.
EXAMPLE 3 control Effect of Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c on large Tian Huanglu ulcer disease
(1) Bacillus megaterium BMJBN and Bacillus firmus HG-13C strains are respectively inoculated into LB liquid culture medium and cultured for 24 hours at 28 ℃ and 180rpm/min to obtain seed liquid with the concentration of about 10 9 cfu/mL.
(2) A60 liter mechanically stirred stainless steel fermenter is used for fermenting bacillus megatherium BMJBN and bacillus firmus HG-13C respectively, the fermentation culture medium is molasses culture medium (molasses 100mL/L, sodium chloride 1g/L and urea 3 g/L), the liquid loading amount is 40L, the inoculation amount is 6%, the temperature is 28 ℃, the stirring speed is 300rpm/min, the ventilation amount is 3L/min, the fermentation time is 2 days, and the biomass reaches 2.10X10 10 cfu/mL.
(3) And (3) preparing finished powder, namely respectively adding carrier diatomite and starch into the fermentation broths of the two strains according to saturated adsorption amount, stirring once every 1h to enable the carriers to fully adsorb thalli, drying at low temperature after 24h to obtain an adsorption carrier, adding stabilizer humic acid, dispersing agent polyvinyl alcohol and wetting agent Tween 80, mixing according to the mass ratio of 78:12:7:3, crushing by a jet mill, and sieving by a 300-mesh sieve to obtain BMJBN and HG-13c wettable powder.
(4) Spraying 6L of wettable powder diluent (200X) per mu of smoke tree of 2-3 years old. Three treatments of independent spraying BMJBN of diluent, independent spraying HG-13c of diluent, mixed spraying BMJBN of diluent and HG-13c of diluent (the volume ratio of the two bacterial strain diluents is 1:1) with equal volume are respectively arranged, and meanwhile clear water spraying is used as a Control (CK). Each treatment was repeated with 3 cells and a second dose was given 7 days later.
The disease states were investigated 14 days and 21 days after the second application, five samples were taken every treatment, the number of plants and the disease level were recorded, and the disease feeling index and the relative control effect were calculated.
Wherein, the cotinus coggygria canker is classified into 5 grades:
grade 0, no disease occurrence of whole plant;
stage 1, leaf under 1/4 of the whole plant or leaf of 1 branch has wilting symptom;
2-grade, leaves less than 1/2 of the whole plant or leaves of 2-3 branches have wilting symptoms;
3, wilting 1/2-3/4 of the whole plant and falling off part of the leaves;
4-stage, leaf wilting and falling off of 3/4 or more of the whole plant or withering of the whole plant.
The calculation formula of the disease sensing index and the relative prevention and treatment effect is as follows:
As shown in Table 1 below, it can be seen that the wettable powders of Bacillus megaterium BMJBN and Bacillus firmus HG-13c can be used alone or in combination to prevent and treat Cotinus canker well under field conditions.
TABLE 1 control effects of treatments 14 days and 21 days after the second administration
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (10)
1. The bacillus firmus (Bacillus firmus) HG-13c for preventing and treating cotinus coggygria canker is characterized in that the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the month 07 of 2022, and the preservation address is North Star Xiyu No. 1,3 in the Korean region of Beijing city, and the preservation number is CGMCC No.25308.
2. Use of bacillus firmus HG-13c according to claim 1 for inhibiting the growth of plasmopara viticola.
3. Use of bacillus firmus HG-13c according to claim 1 for controlling cotinus coggygria.
4. A biocontrol agent comprising Bacillus firmus HG-13c according to claim 1.
5. The biocontrol microbial agent of claim 4, further comprising bacillus megatherium (Bacillus megatherium) BMJBN02, wherein the strain has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at a deposition address of Seagate North Star, west Song No.1, 3, and a deposition number of CGMCC No.10478, in the Korean region of Beijing.
6. The biocontrol microbial agent as claimed in claim 4 or 5, wherein the biocontrol microbial agent is prepared by adding a carrier into a microbial fermentation broth according to a saturated adsorption amount, drying after adsorption to obtain an adsorption carrier, mixing the adsorption carrier with a stabilizer, a dispersing agent and a wetting agent, crushing and sieving.
7. The biocontrol microbial agent of claim 6, wherein the carrier is at least one of diatomaceous earth and starch, the stabilizer is humic acid, the dispersant is polyvinyl alcohol, and the wetting agent is tween 80.
8. The biocontrol microbial agent of claim 6, wherein the mass ratio of adsorption carrier, stabilizer, dispersant to wetting agent is 70-85:10-15:5-10:1-5.
9. Use of the biocontrol agent of claim 4 or 5 for inhibiting the growth of plasmopara viticola.
10. The use of the biocontrol agent as defined in claim 4 or 5 for controlling cotinus coggygria ulcers.
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| CN104673720A (en) * | 2015-02-15 | 2015-06-03 | 山东省科学院生物研究所 | Bacillus megatherium and suspending agent for preventing and treating grape downy mildew |
| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
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| CN114015609B (en) * | 2021-11-25 | 2023-03-31 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Broad-spectrum antibacterial bacillus and application thereof |
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| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
| CN104673720A (en) * | 2015-02-15 | 2015-06-03 | 山东省科学院生物研究所 | Bacillus megatherium and suspending agent for preventing and treating grape downy mildew |
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