Bacillus firmus for preventing and treating cotinus canker and its biocontrol agent and application

Abstract

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

[0001] The invention relates to the technical field of biological pesticides, and in particular to Bacillus firmus for preventing and treating cotinus canker and a biological control agent and application thereof. Background Art

[0002] Since the summer of 2018, Cotinus coggygria canker has occurred in many forest parks in central and eastern Shandong Province. The symptoms mainly occur on the trunks. As the pathogen infects the trees, they gradually wither and the infected areas show dark brown ulcers under the skin. The diseased plants are scattered or distributed in patches, seriously affecting the red leaf landscape of Cotinus coggygria. Through pathogen identification, it was found that although the symptoms of this disease are similar to the common Cotinus coggygria wilt disease, the pathogen is Botrytis cinerea (B. dothidea).

[0003] At present, the prevention and control of Cotinus coggygria wilt mainly focuses on strengthening water and fertilizer management, timely handling of diseased plants and spraying of chemical pesticides. However, the prevention and control effect is not significant, and the residues of chemical pesticides have caused serious environmental pollution problems. There is an urgent need to find economical, green and safe prevention and control measures. Summary of the invention

[0004] In view of the technical problem that the existing prevention and control methods of cotinus coggygria wilt are not effective, the present invention provides a Bacillus firmus and a biocontrol agent for preventing and controlling cotinus coggygria canker and its application. The present invention screens for the first time Bacillus firmus and a biocontrol agent with high inhibitory activity against Botrytis cinerea, and obtains a good prevention and control effect on cotinus coggygria canker in field plot tests.

[0005] In the first aspect, the present invention provides a Bacillus firmus HG-13c for preventing and treating cotinus canker. The strain has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25308.

[0006] In a second aspect, the present invention provides a use of Bacillus firmus HG-13c in inhibiting the growth of Botrytis cinerea.

[0007] In a third aspect, the present invention provides a use of Bacillus firmus HG-13c in preventing and treating Cotinus coggygria canker.

[0008] In a fourth aspect, the present invention provides a biocontrol agent containing Bacillus firmus HG-13c.

[0009] Furthermore, the biocontrol agent also contains Bacillus megatherium BMJBN02, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on January 30, 2015. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 10478. The strain has been disclosed in the Chinese invention patent CN201510082386.X "Composite microbial agent for preventing and controlling downy mildew of fruits and vegetables and its preparation method".

[0010] Furthermore, the preparation method of the biocontrol agent is to add a carrier to the microbial fermentation liquid according to the saturated adsorption amount, dry it after the adsorption is completed to obtain an adsorption carrier, mix the adsorption carrier with a stabilizer, a dispersant and a wetting agent, crush it, and sieve it to obtain the biocontrol agent.

[0011] Furthermore, 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.

[0012] Furthermore, the mass ratio of the adsorption carrier, the stabilizer, the dispersant and the wetting agent is 70-85:10-15:5-10:1-5.

[0013] Furthermore, the formulation of the biocontrol agent can be a wettable powder, a water dispersible granule, a suspension, a suspoemulsion, an aqueous emulsion or a microemulsion.

[0014] Furthermore, when preparing the biocontrol agent, auxiliary agents such as binders, defoamers, disintegrants, antifreeze agents, etc. may be added depending on the dosage form.

[0015] In a fifth aspect, the present invention provides a use of the above-mentioned biocontrol agent in inhibiting the growth of Botrytis cinerea.

[0016] In a sixth aspect, the present invention provides a use of the above-mentioned biocontrol agent in preventing and treating Cotinus coggygria canker disease.

[0017] The beneficial effects of the present invention are:

[0018] The Bacillus firmus HG-13c provided by the invention has a highly effective inhibitory effect on Botrytis cinerea and can be used as a highly effective biocontrol bacterium for Cotinus coggygria canker to develop new biological pesticides or biological microbial agents.

[0019] The biocontrol agent containing Bacillus firmus HG-13c provided by the invention can achieve a 14-day control effect of 61.48% and a 21-day control effect of 65.02% in field experiments; in particular, when used in combination with Bacillus megaterium BMJBN02, the 14-day control effect can reach 88.69% and the 21-day control effect can reach 89.12%. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0021] Example 1 Isolation and Identification of Bacillus firmus HG-13c

[0022] 1.1 Isolation of HG-13c strain

[0023] (1) In a clean bench, 10 g of soil collected from a vegetable greenhouse in Shizhong District, Jinan City, Shandong Province was placed in 100 mL of sterile saline and shaken for 2 h to fully suspend and prepare a bacterial suspension.

[0024] (2) After the bacterial suspension was diluted with sterile water, 100 μL of each concentration of the bacterial suspension was spread on an LB medium plate and incubated at 28°C for 48 h. Then, single colonies with different shapes, sizes, and colors on the plate were picked and streaked on the LB plate for purification and numbering.

[0025] (3) Purified strains with different numbers were cultured in LB liquid medium at 28°C and 180 rpm / min for 24 h, and 2 mL of fermentation broth was taken and mixed into 15 mL of PDA medium. The plates were poured and solidified, and then Botrytis cinerea was inoculated in the center of the plate for inhibitory co-culture. The resulting strain with strong antagonism to Fusarium wilt was named HG-13c.

[0026] 1.2 Strain identification

[0027] (1) 16S rDNA sequencing

[0028] The genomic DNA of HG-13c was extracted and used as a template to perform PCR amplification using bacterial 16S rDNA universal primers. The amplified product was recovered and sequenced. The obtained sequence is shown in SEQ ID NO:1.

[0029] (2) The sequences obtained by sequencing were compared for homology, and it was found that the homology between strain HG-13c and Bacillus firmus was 99.99%. Combined with bacterial Gram staining and physiological and biochemical characteristics detection, strain HG-13c was identified as Bacillus firmus.

[0030] The Bacillus firmus HG-13c was deposited in the General Microbiology Center of the China Culture Collection Administration on July 15, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25308.

[0031] Example 2 Antagonistic Effects of Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c on Botrytis cinerea

[0032] (1) Purified Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c strains were cultured in LB liquid medium at 28° C. and 180 rpm / min for 24 h, and 2 mL of the fermentation broth was mixed into 15 mL of PDA medium, poured into plates, and solidified for later use.

[0033] Among them, the Bacillus megaterium BMJBN02 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 30, 2015. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.10478.

[0034] (2) Use a 0.5 cm diameter puncher to punch holes at the edge of the cultured grape colony to obtain bacterial slices for later use.

[0035] (3) Use a sterile pick to pick the above bacterial slices into the center of each PDA plate in (1). At the same time, set a PDA plate inoculated with only the Botrytis cinerea bacterial slice as a blank control. Set up 3 replicates for each treatment and culture at 28°C to observe the colony growth rate and colony radius of Botrytis cinerea.

[0036] After 3 days of culture, compared with the blank control, the growth inhibition rate of Bacillus megaterium BMJBN02 on Botrytis cinerea was 67.6%, and the growth inhibition rate of Bacillus firmus HG-13C on Botrytis cinerea was 79.3%.

[0037] Example 3 Control effect of Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c on field cotinus canker

[0038] (1) Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c were inoculated into LB liquid culture medium respectively, and cultured at 28°C and 180 rpm / min for 24 h to obtain seed solution with a concentration of about 10 9 cfu / mL.

[0039] (2) Six 60-liter stainless steel fermenters with mechanical stirring were used to ferment Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c, respectively. The fermentation medium was molasses medium (molasses 100 mL / L, sodium chloride 1 g / L, urea 3 g / L), the liquid volume was 40 L, the inoculum size was 6%, the temperature was 28°C, the stirring speed was 300 rpm / min, the ventilation volume was 3 L / min, the fermentation time was 2 days, and the biomass reached 2.10 × 10 10 cfu / mL.

[0040] (3) Preparation of finished powders: Carrier diatomaceous earth and starch were added to the fermentation broth of the two strains according to the saturated adsorption capacity, respectively. The mixture was stirred every 1 h to allow the carrier to fully adsorb the bacteria. After 24 h, the adsorption carrier was obtained by low-temperature drying. The stabilizer humic acid, the dispersant polyvinyl alcohol, and the wetting agent Tween 80 were added and mixed in a mass ratio of 78:12:7:3. The mixture was crushed with a jet mill and passed through a 300-mesh sieve to obtain wettable powders of BMJBN02 and HG-13c.

[0041] (4) Spray 2-3 year old Cotinus coggygria with 6 L of wettable powder dilution (200×) per mu. Three treatments were set up: spraying BMJBN02 dilution alone, spraying HG-13c dilution alone, and spraying BMJBN02 and HG-13c dilutions (the volume ratio of the two strain dilutions was 1:1). At the same time, spraying with clear water was used as a control (CK). Three plots were set up for each treatment as replicates, and the second application of the drug was carried out 7 days later.

[0042] The disease condition was investigated 14 days and 21 days after the second application of the pesticide. Samples were taken at five points in each treatment, the number of diseased plants and the disease level were recorded, and the disease susceptibility index and relative control effect were calculated.

[0043] Among them, Cotinus coggygria canker disease is divided into 5 levels:

[0044] Level 0, no disease in the whole plant;

[0045] Level 1: less than 1 / 4 of the leaves of the whole plant or one branch have wilting symptoms;

[0046] Level 2: less than 1 / 2 of the leaves of the whole plant or 2-3 leaves of branches have wilting symptoms;

[0047] Level 3: 1 / 2-3 / 4 of the leaves of the whole plant wilt and some leaves fall off;

[0048] Level 4: More than 3 / 4 of the leaves wilt and fall off or the entire plant dies.

[0049] The calculation formulas for disease susceptibility index and relative control effect are as follows:

[0050]

[0051]

[0052] The results are shown in Table 1 below. It can be seen that under field conditions, the wettable powders of Bacillus megaterium BMJBN02 and Bacillus firmus HG-13c applied alone or in combination can effectively prevent and control Cotinus coggygria canker.

[0053] Table 1 The efficacy of each treatment 14 days and 21 days after the second application

[0054]

[0055] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. A Bacillus firmus HG-13c for preventing and treating cotinus canker, characterized in that: The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection on July 15, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25308.

2. Use of the Bacillus firmus HG-13c as claimed in claim 1 in inhibiting the growth of Botrytis cinerea.

3. Use of the Bacillus firmus HG-13c as claimed in claim 1 in preventing and treating Cotinus coggygria canker.

4. A biocontrol agent containing the Bacillus firmus HG-13c as claimed in claim 1.

5. The biocontrol agent according to claim 4, characterized in that It also contains Bacillus megatherium BMJBN02, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 30, 2015. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 10478.

6. The biocontrol agent according to claim 4 or 5, characterized in that The preparation method of the biocontrol agent comprises the following steps: adding a carrier to the microbial fermentation liquid according to the saturated adsorption amount, drying after the adsorption is completed to obtain an adsorption carrier, mixing the adsorption carrier with a stabilizer, a dispersant and a wetting agent, crushing and sieving to obtain the biocontrol agent.

7. The biocontrol agent according to claim 6, characterized in that 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 agent according to claim 6, characterized in that The mass ratio of the adsorption carrier, the stabilizer, the dispersant and the wetting agent is 70-85:10-15:5-10:1-5.

9. Use of the biocontrol agent according to claim 4 or 5 in inhibiting the growth of Botrytis cinerea.

10. Use of the biocontrol agent according to claim 4 or 5 in preventing and treating Cotinus coggygria canker.

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