A bactericidal composition comprising bacillus velezensis tcs001 and paenibacillus polymyxa hy96-2 and application thereof

Abstract

The present application provides a kind of bactericidal mixture, with active ingredient bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2, 10 billion CFU / milliliter bacillus velezensis TCS001 and 10 billion CFU / milliliter Paenibacillus polymyxa HY96-2 are 100% in the mass respectively, wherein the mass ratio of bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 is 4:1-1:4.The bactericidal composition of the present application provides a new alternative agent for the prevention and control of strawberry anthracnose, and bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 combination control strawberry anthracnose has synergistic effect.

Description

Technical Field

[0001] The present invention relates to the field of plant protection, in particular to a fungicide composition containing Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2 and an application thereof, particularly to the application of the fungicide composition in preventing and controlling strawberry anthracnose to increase strawberry seedlings and strawberry yield. Background Art

[0002] Bacillus velezensis is a novel biocontrol bacterium. Spanish researchers Ruiz-García et al. first isolated two strains, CR-14b and CR-502T, in 2005. These strains are capable of producing large quantities of lipopeptides and exhibit strong antibacterial activity. Bacillus velezensis is widely distributed and can survive adverse conditions such as drought and salinity. However, few related products exist, suggesting promising development prospects.

[0003] Bacillus velezensis TCS001 was isolated from Bohai Sea silt. The original strain was Bacillus velezensis CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited with the General Microbiology Center of the China General Culture Collection Administration (CGMCC) under the accession number CGMCC No. 8921 and was first published in Patent ZL201410168402.2. When grown on NA medium, TCS001 colonies are nearly round, light yellow, and opaque. Initially, the colonies are smooth with neat edges, but later develop wrinkles, slightly irregular edges, a central bulge, and a cloud-like appearance. Gram staining revealed that TCS001 is Gram-positive and rod-shaped. TCS001 has a certain inhibitory effect on cucumber gray mold (Botrytis cinerea), cucumber vine blight (Ascochyta citrullina), cucumber sclerotinia (Sclerotinia sclerotiorum), cucumber brown spot (Corynespora cassiicola), apple brown spot (Marssonina coronaria), wheat loose smut (Ustilago nuda), cotton wilt (Fusarium oxysporum), and banana leaf spot (Pseudocercospora musae). Among them, the inhibition rate against cucumber gray mold is the highest, reaching 87.66%.

[0004] In 1994, Ash C et al. used PCR probes to identify rRNA in several Bacillus species, assigning some of these species to a new genus, Paenibacillus. In the Manual Clinical Microbiology (1999) edited by Patrick R. Murry et al., spore-forming bacteria that produce swollen, oval-shaped bacteria were also subdivided from Bacillus spp. and given a new genus, Paenibacillus. Therefore, Bacillus polymyxa was assigned to the genus Paenibacillus and named Paenibacillus polymyxa.

[0005] Strain HY96-2, isolated from tomato rhizosphere soil in Nanchang, is Gram-positive and acid-fast negative. After culturing at 30°C for two days, the strain develops into straight or nearly straight rods containing a single elliptical spore with sparse peritrichous flagella, indicating motility, aerobic growth, and facultative anaerobic growth. It exhibits no soluble pigment on nutrient agar. Its cell wall contains meso-DAP (diaminopimelate)glycine and lacks characteristic sugars. 16S rDNA sequence analysis indicates that strain HY96-2 belongs to the genus Paenibacillus. The sequence homology between strain HY96-2 and Paenibacillus polymyxa is 99%. Paenibacillus polymyxa HY96-2 mainly prevents and controls diseases in three ways: (1) Paenibacillus polymyxa HY96-2 can colonize in the root surface soil, roots, lower stems, phloem and xylem of upper stems, and leaf surfaces of plants. It has good colonization ability in plants and controls diseases through site competition; (2) In addition to the good antibacterial effect of living bacteria on pathogens, Paenibacillus polymyxa HY96-2 can also produce metabolites such as fusin and phenolic acids, which have good antagonistic effects on pathogenic fungi and bacteria; (3) Paenibacillus polymyxa HY96-2 induces a significant increase in the activities of β-1,3-glucanase, PAL, and POD in the roots and stems of plants, which has the effect of inducing plant disease resistance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a sterilization mixture, which can not only have a synergistic effect on strawberry anthracnose, but also increase the number of strawberry runners and improve the yield of strawberry seedlings.

[0007] A technical solution provided by the present invention is a bactericidal composition, characterized in that it contains active ingredients Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2, with the mass of 1 billion CFU / ml of Bacillus Velez TCS001 and 1 billion CFU / ml of Paenibacillus polymyxa HY96-2 respectively being 100%, wherein the mass ratio of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 is 50:1-1:50; preferably, the mass ratio of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 is 4:1-1:4; particularly preferably, the mass ratio of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 is 1:1.

[0008] The total mass of the Bacillus Velez TCS001 and the Paenibacillus polymyxa HY96-2 accounts for 1-30% of the mass percentage of the sterilization composition; preferably, the total mass of the Bacillus Velez TCS001 and the Paenibacillus polymyxa HY96-2 accounts for 5-25% of the mass percentage of the sterilization composition; particularly preferably, the total mass of the Bacillus Velez TCS001 and the Paenibacillus polymyxa HY96-2 accounts for 10-20% of the mass percentage of the sterilization composition.

[0009] The bactericidal composition can be formulated into any dosage form permitted in agriculture; preferably, the dosage form of the bactericidal composition is a suspension, seed coating agent, wettable powder, water-dispersible granules, microcapsule suspension, coated granules, granules, water-soluble granules, water-soluble powder, aqueous emulsifiable concentrate, microemulsion, suspoemulsion, water emulsion, etc.

[0010] The fungicidal composition further comprises one or more other active ingredients. Preferably, the other active ingredients include insecticides, fungicides, herbicides, plant growth regulators, plant immunizers, and the like.

[0011] Another technical solution provided by the present invention is the use of the fungicide composition for preventing and controlling plant pathogens. Preferably, the plants include cereals, fruits, vegetables, and economic crops, and the pathogens include a wide range of plant pathogenic fungi such as Plasmodium, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes. Particularly preferably, the plant pathogen is strawberry anthracnose.

[0012] Another technical solution provided by the present invention is a method for controlling plant pathogens, characterized in that: a fungicidal composition is applied to the pathogens and / or their environment, or plants, plant parts, seeds, soil, areas, materials or spaces.

[0013] The Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 are administered simultaneously, separately, or sequentially.

[0014] Another technical solution provided by the present invention is the use of the bactericidal composition for promoting the growth of strawberry seedlings and / or increasing the number of strawberry seedlings.

[0015] The method of promoting the growth of strawberry seedlings includes increasing the number of runners of the strawberry seedlings.

[0016] Another technical solution provided by the present invention is a kit comprising a bactericidal composition containing active ingredients Bacillus velezensis TCS001 and Bacillus polymyxa and Paenibacillus polymyxa HY96-2.

[0017] Beneficial technical effects of the present invention

[0018] The present invention performs binary compounding of Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2, so that the obtained fungicidal mixture has a synergistic effect in preventing and treating strawberry anthracnose, providing a new alternative agent for the prevention and treatment of strawberry anthracnose.

[0019] The bactericidal composition provided by the present invention can increase the number of strawberry runners, ultimately increasing 30,000 strawberry seedlings per mu (the number of seedlings raised when TCS001 is sprayed is more than 90,000 per mu, and the number of seedlings raised when TCS001 is not sprayed is more than 60,000 per mu). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of affinity screening tests between Bacillus velezensis TCS001 and other biocontrol bacteria;

[0021] Figure 2 The effectiveness of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 against strawberry anthracnose;

[0022] Figure 3 Field control effects of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 as single agents and in combination against anthrax. DETAILED DESCRIPTION

[0023] Example 1

[0024] Affinity screening of Bacillus velezensis TCS001 with Paenibacillus polymyxa HY96-2, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus brevis.

[0025] Bacillus Velezii TCS001, Paenibacillus polymyxa HY96-2, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus brevis were streaked onto LB plate medium and cultured in the dark at 28°C for 48 hours to obtain single colonies. A single colony of the biocontrol strain that had been activated for 2 days was picked up with an inoculation loop and inoculated into MLB in a 150 / 250 mL conical flask. The culture was shaken at 25°C and 164 rpm for 24 hours, and the bacterial suspension was adjusted to OD 0. 630 The value is 0.1. 100 μL of the bacterial suspension of Bacillus Velez TCS001 (plate strain) was aspirated and placed on an LB culture medium plate, and evenly spread with a triangular applicator. Sterilized filter paper pieces (d = 5 mm) were placed in another biocontrol bacterial suspension (test strain), soaked for 10 seconds, and placed on the four vertices of a square 30 mm away from the center of the culture medium coated with the plate bacterial solution. A sterilized filter paper piece soaked in sterile water for 10 seconds was placed in the center of the plate as a control. Each treatment was repeated 3 times. After sealing the film, it was placed in a 28°C constant temperature incubator for dark incubation to observe whether an inhibition zone was produced. The presence of an inhibition zone means that the strains have no affinity, and the absence of an inhibition zone means that the two strains have affinity.

[0026] The results of the affinity screening test between Bacillus velezensis TCS001 and biocontrol bacteria are shown in Figure 1 No inhibition zones appeared around the filter paper discs of Paenibacillus polymyxa HY96-2 and Bacillus amyloliquefaciens, indicating that Bacillus Velez TCS001 was compatible with Paenibacillus polymyxa HY96-2 and Bacillus amyloliquefaciens. Inhibition zones appeared around the filter paper discs of Bacillus subtilis and Bacillus brevis, indicating that Bacillus Velez TCS001 was not compatible with Bacillus subtilis and Bacillus brevis.

[0027] Example 2

[0028] A confrontation test between Bacillus velezensis TCS001, Paenibacillus polymyxa HY96-2 and strawberry anthracnose.

[0029] A 5 mm diameter, actively growing strawberry anthracnose pathogen cake was seeded in the center of a PDA plate, mycelial side facing downward. Four single colonies of Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 were inoculated symmetrically 2 cm above and below the pathogen cake. A plate without single colonies of the pathogen served as a control. Each treatment was replicated three times. Incubate inverted at 26°C in the dark. When the diameter of the pathogen in the control group reached three-quarters of the plate diameter, the colony diameter was measured using the cross-hatch method.

[0030] The inhibition rate was calculated as follows: inhibition rate (%) = (colony diameter of the control group - colony diameter of the treated group) / (colony diameter of the control group - 5) × 100.

[0031] Test results ( Figure 2):The inhibition rates of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 against strawberry anthracnose pathogens were 72.18% and 63.36%, respectively.

[0032] Example 3

[0033] Screening of compound formulas of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 for controlling strawberry anthracnose

[0034] Select uniformly growing strawberry seedlings from plug trays for transplanting. Inoculate the seedlings with the fungus at the time of transplanting. Puncture the base of the strawberry stem with a sterile needle and spray the prepared spore suspension onto the puncture site. Apply the pesticide 24 hours after inoculation. Each treatment was replicated three times, with 10 seedlings per replicate and three unfolded leaves per seedling.

[0035] The disease classification standard is based on the main stem:

[0036] Level 0: no disease;

[0037] Level 1: The lesion area occupies less than 5% of the entire main stem;

[0038] Level 3: The lesion area occupies 6%-10% of the entire main stem;

[0039] Level 5: The lesion area occupies 11%-25% of the entire main stem;

[0040] Level 7: The lesion area occupies 26%-50% of the entire main stem;

[0041] Level 9: The area of ​​lesions occupies more than 51% of the entire main stem, or the plant dies.

[0042] Calculation method of drug efficacy: The drug efficacy is calculated in the following way.

[0043]

[0044]

[0045] ×100

[0046] Theoretical toxicity index (TTI) = toxicity index of A × content of A in the mixture (%)

[0047] + Toxicity index of B × content of B in the mixture (%)

[0048]

[0049]

[0050] If the co-toxicity coefficient is greater than 120, it indicates a synergistic effect; if it is less than 80, it indicates an antagonistic effect; if it is greater than 80 and less than 120, it indicates an additive effect.

[0051] Table 1 Screening of compound formulas for controlling strawberry anthracnose with Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2

[0052]

[0053]

[0054] The test results showed (Table 1) that the mixture of Bacillus velez TCS001 and Paenibacillus polymyxa HY96-2 had high activity against the test targets. When the ratio of Bacillus velez TCS001: Paenibacillus polymyxa HY96-2 was 50:1, 10:1 and 1:50, an additive effect was observed. When the ratio of Bacillus velez TCS001 and Paenibacillus polymyxa HY96-2 was 4:1-1:4, the co-toxicity coefficient was 122.03-157.91, showing a significant synergistic effect. When the ratio was 1:1, the co-toxicity coefficient was 157.91, and the synergistic effect was the most significant.

[0055] Example 4

[0056] Field efficacy test of a mixture of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 against anthracnose of strawberry during seedling stage

[0057] The test was carried out in Banqiao Village, Lin'an District, Hangzhou City. The strawberry variety was Hongyan. During the strawberry seedling stage, the first application of pesticide was made before the occurrence of anthracnose. Each application was repeated 7-10 days apart, for a total of 10 applications. The water consumption was 30 liters per mu. Each plot was 50 square meters, and each treatment was repeated 3 times. The control effect was investigated 7-10 days after the last application. Referring to "GB / T 17980.112-2004 Guidelines for Field Efficacy Tests of Pesticides (II) Fungicide Control of Anthracnose in Cucurbits", samples were taken at 5 points in each plot, 5 plants were investigated at each point, for a total of 25 plants, the disease conditions of the main stems and runners of strawberries in each plot were investigated, and the control effect was calculated. The disease grading standard is based on the main stem and runner:

[0058] Level 0: no disease;

[0059] Level 1: The lesion area occupies less than 5% of the entire main stem and runners;

[0060] Level 3: The lesion area occupies 6%-10% of the entire main stem and runners;

[0061] Level 5: The lesion area occupies 11%-25% of the entire main stem and runners;

[0062] Level 7: The lesion area occupies 26%-50% of the entire main stem and runners;

[0063] Level 9: The area of ​​lesions occupies more than 51% of the entire main stem and runners, or the plant dies.

[0064] Calculation method of drug efficacy: The drug efficacy is calculated in the following way.

[0065]

[0066]

[0067] Table 2 Field efficacy test results of Bacillus lesii TCS001, Paenibacillus polymyxa HY96-2 and their mixtures against strawberry anthracnose

[0068]

[0069]

[0070] Test results (Table 3 and Figure 3 ): 10 days after the last application, the control efficacy of the mixture of Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2 (TCS001:HY96-2 = 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4) at a dosage of 200 g / mu was 85.88%, 83.11%, 86.84%, 88.12%, 85.79%, 85.40% and 82.13%, respectively, all showing significant The control efficacy of two single agents—1 billion CFU / mL of Bacillus Velez TCS001 and 1 billion CFU / mL of Paenibacillus polymyxa HY96-2 at a dose of 250 g / mu—was significantly higher than that of the chemical agent 10% difenoconazole water-dispersible granules at 80 g / mu. The highest control efficacy, 88.17%, was achieved at a 1:1 ratio of Bacillus Velez TCS001:Paenibacillus polymyxa HY96-2. The control efficacy of single agents of 1 billion CFU / mL of Bacillus Velez TCS001 and 1 billion CFU / mL of Paenibacillus polymyxa HY96-2 at a dose of 250 g / mu was 76.82% and 73.24%, respectively, significantly higher than the 39.66% control efficacy of 10% difenoconazole water-dispersible granules at 80 g / mu.

[0071] During the trial, no adverse effects of the test agent on other pests and diseases and non-target organisms were observed, and it is safe for strawberries and bees.

[0072] In addition, the experiment found that Bacillus Velez TCS001, Paenibacillus polymyxa HY96-2, and their mixture increased the number of strawberry runners and seedlings. When the blank control had an average of 2.27 runners per plant, the number of runners treated with 10% difenoconazole water-dispersible granules was 2.20, while Bacillus Velez TCS001, Paenibacillus polymyxa HY96-2, and their mixture increased the number of strawberry runners by 3.18-3.73. The highest runner number was achieved when the mixture had a 1:1 ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2, with an increase of 64.32%.

[0073] At the final harvest in the demonstration area, it was measured that the number of strawberry seedlings increased by 30,000 per mu (more than 90,000 seedlings per mu when sprayed with TCS001, and more than 60,000 seedlings per mu when not sprayed with TCS001).

[0074] Table 3 Effects of Bacillus velezensis TCS001, Paenibacillus polymyxa HY96-2 and their mixture on strawberry runners

[0075]

[0076] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bactericidal composition, characterized in that The active ingredients include Bacillus velez TCS001 and Paenibacillus polymyxa HY96-2, with the mass of 1 billion CFU / ml of Bacillus velez TCS001 and 1 billion CFU / ml of Paenibacillus polymyxa HY96-2 respectively being 100%, and the mass ratio of Bacillus velez TCS001 to Paenibacillus polymyxa HY96-2 being 4:1-1:

4.

2. The bactericidal composition according to claim 1, characterized in that The total mass of Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 accounts for 5%-30% of the mass of the sterilization composition.

3. The bactericidal composition according to claim 1, characterized in that The dosage form of the bactericidal composition is a suspension, seed coating, wettable powder, water-dispersible granules, microcapsule suspension, coated granules, granules, water-soluble granules, water-soluble powder, aqueous emulsifiable concentrate, microemulsion, suspoemulsion, aqueous emulsion or effervescent tablet.

4. The bactericidal composition according to claim 1, characterized in that The fungicidal composition further comprises one or more other active ingredients, including insecticides, fungicides, herbicides, plant growth regulators, and plant immune activators.

5. Use of the fungicidal composition according to any one of claims 1 to 4 for preventing and controlling plant diseases, wherein the plant disease is strawberry anthracnose.

6. A method for preventing and treating strawberry anthracnose pathogens, characterized in that: The fungicidal composition according to any one of claims 1 to 4 is applied to strawberry anthracnose pathogens and / or their environment, or plants, plant parts, seeds, soil, areas, materials or spaces.

7. The method according to claim 6, characterized in that: Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 are administered simultaneously, separately, or sequentially.

8. Use of the bactericidal composition according to any one of claims 1 to 4 for promoting the growth of strawberry seedlings and / or increasing the number of strawberry seedlings.

9. The use according to claim 8, characterized in that: The method of promoting the growth of strawberry seedlings includes increasing the number of runners of the strawberry seedlings.

10. A kit, characterized in that The invention comprises the bactericidal composition according to any one of claims 1 to 4.

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