Bactericidal composition containing bacillus velezensis TCS001 and paenibacillus polymyxa HY96-2 and application thereof

Abstract

The invention provides a bactericidal mixture, which contains active ingredients of bacillus velezensis TCS001 and paenibacillus polymyxa HY96-2, the mass of the bacillus velezensis TCS001 and the mass of the paenibacillus polymyxa HY96-2 are respectively 100% according to 100 billion CFU / ml of bacillus velezensis TCS001 and 100 billion CFU / ml of paenibacillus polymyxa HY96-2, and the mass ratio of the bacillus velezensis TCS001 to the paenibacillus polymyxa HY96-2 is (50: 1)-(1: 50). The bactericidal composition disclosed by the invention provides a novel alternative medicament for preventing and treating strawberry anthracnose, and the bacillus velezensis TCS001 and the paenibacillus polymyxa HY96-2 are combined for preventing and treating strawberry anthracnose and have a synergistic effect.

Description

Technical Field

[0001] The 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, and in particular to the application of the fungicide composition in preventing and controlling strawberry anthracnose to increase the yield of strawberry seedlings and strawberries. Background Art

[0002] Bacillus velezensis is a new type of biocontrol bacteria. In 2005, Spanish scholars Ruiz-García et al. isolated two strains, CR-14b and CR-502T, which can synthesize a large amount of lipopeptides and have strong antibacterial activity. Bacillus velezensis is widely distributed and can survive in adverse conditions such as drought and salinity. There are few related products, and the development prospects are broad.

[0003] Bacillus velezensis TCS001 was isolated from Bohai silt. The original strain was marine Bacillus CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No.8921, and was first published in Patent ZL201410168402.2. After growing on NA medium, the single colony morphology of TCS001 is nearly round, light yellow, and opaque; the surface of the colony is smooth and the edges are neat in the early stage of culture, and the surface is wrinkled in the later stage, the edges are slightly irregular, there is a bulge in the middle, and it spreads in a cloud-like manner around. Gram staining shows that the TCS001 strain is Gram-positive and rod-shaped. TCS001 has certain inhibitory effects on cucumber gray mold (Botrytis cinerea), cucumber vine blight (Ascochyta citrullina), cucumber sclerotinia (Sclerotinia sclerotiorum), cucumber brown spot (Corynesporacassiicola), apple brown spot (Marssonina coronaria), wheat loose smut (Ustilago nuda), cotton wilt (Fusarium oxysporum), banana leaf spot (Pseudocercospora musae), among which 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 of some species of Bacillus, and classified some of them into a new genus Paenibacillus. In Manual Clinical Microbiology (1999) compiled by Patrick R. Murry et al., spore-forming bacteria that produce swelling and oval shapes were also subdivided from Bacillus spp. and given a new genus named Paenibacillus. Therefore, Bacillus polymyxa was also classified into Paenibacillus spp. and called Paenibacillus polymyxa.

[0005] The strain HY96-2 was isolated from the rhizosphere soil of tomato in Nanchang. It is Gram-positive and negative for acid-fast staining. After culturing at 30℃ for 2 days, the bacteria are straight or nearly straight rods, containing only one oval spore, and have sparse peritrichous flagella. They can move, grow aerobically and facultatively anaerobic, and have no soluble pigment on nutrient agar. The cell wall contains meso-DAP (diaminopimelic acid) glycine and no characteristic sugars. The results of 16s rDNA sequence analysis show that the strain HY96-2 belongs to the genus Paenibacillus. The sequence homology of the 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 fusarium oxytocin 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 plants to produce disease resistance. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a bactericidal 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, and the mass ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2 is 50:1-1:50, based on the mass of 1 billion CFU / ml Bacillus Velez TCS001 and 1 billion CFU / ml Paenibacillus polymyxa HY96-2 being 100% respectively; preferably, the mass ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2 is 4:1-1:4; particularly preferably, the mass ratio of Bacillus Velez TCS001 to 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, a seed coating agent, a wettable powder, a water-dispersible granule, a microcapsule suspension, a coated granule, a granule, a water-soluble granule, a water-soluble powder, an aqueous emulsifiable concentrate, a microemulsion, a suspoemulsion, an aqueous 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 controlling plant pathogenic bacteria. Preferably, the plants include cereals, fruits, vegetables, and economic crops. The pathogens include a wide range of plant pathogenic fungi such as Plasmodium, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes. Particularly preferably, the plant pathogenic bacteria are strawberry anthracnose bacteria.

[0012] Another technical solution provided by the present invention is a method for controlling plant pathogens, characterized in that the 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 Velez TCS001 and Bacillus 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 bactericidal mixture has a synergistic effect in preventing and controlling strawberry anthracnose, thereby providing a new alternative agent for preventing and controlling strawberry anthracnose.

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

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

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

[0022] Figure 3 Field control effects of Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2 as single agents and in combination against anthracnose. 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 on LB plate culture medium, and single colonies were obtained after culturing at 28°C in the dark for 48 hours. 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 with a liquid volume of 150 / 250mL conical flask, and cultured in a shaking incubator at 25°C and 164r / min for 24 hours. The bacterial suspension was adjusted to OD 0. 630 The value is 0.1. Pipette 100 μL of the bacterial suspension of Bacillus Velez TCS001 (plate strain) on the LB medium plate, and spread it evenly with a triangular applicator. Put sterilized filter paper (d=5 mm) in another biocontrol bacterial suspension (test strain), soak for 10 seconds, and place it on the four vertices of the square 30 mm away from the center of the culture medium coated with the plate bacterial solution. Place the sterilized filter paper soaked in sterile water for 10 seconds in the center of the plate as a control. Each treatment was repeated 3 times. After sealing the film, place it in a 28°C constant temperature box for dark culture to observe whether there is an inhibition zone. The presence of an inhibition zone means that the strain has 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 velez TCS001 and biocontrol bacteria are shown in Figure 1 , no inhibition zone appeared around the filter paper 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 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 Velez TCS001, Paenibacillus polymyxa HY96-2 and strawberry anthracnose.

[0029] A 5mm diameter, vigorously growing strawberry anthracnose pathogen cake was inoculated in the center of the PDA plate, with the mycelium facing down. Four single colonies of Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2 were inoculated symmetrically up and down and left and right at a distance of 2cm from the pathogen. The plate without single colonies of biocontrol bacteria was used as the control. Each treatment was repeated 3 times. Incubate inverted in the dark at 26℃. When the diameter of the pathogen in the control group grew to 3 / 4 of the diameter of the culture dish, the diameter of the pathogen colony was measured by the cross method.

[0030] The inhibition rate was calculated by the following formula: 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 formula of Bacillus Velezii TCS001 and Paenibacillus polymyxa HY96-2 for controlling strawberry anthracnose

[0034] Select the strawberry seedlings with the same growth in the plug tray for transplanting, and inoculate them during transplanting. When inoculating, puncture the base of the strawberry stem with a sterilized needle, then spray the prepared spore suspension to the punctured position, and apply the medicine 24 hours after inoculation. Each treatment was repeated 3 times, with 10 strawberry seedlings in each repeat, and 3 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 lesion area 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 manner.

[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 mixed pair 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 exhibited. 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 most significant.

[0055] Example 4

[0056] Field efficacy test of mixed strains of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 in controlling strawberry anthracnose during seedling stage

[0057] The test was conducted in Banqiao Village, Lin'an District, Hangzhou City. The strawberry variety was Hongyan. The first application of pesticides was made during the strawberry seedling stage before the occurrence of anthracnose, and each application was made once every 7-10 days, for a total of 10 applications. The water consumption was 30 liters / 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 of pesticides. According to "GB / T 17980.112-2004 Pesticide Field Efficacy Test Guidelines (II) Fungicide Control of Melon Anthracnose", 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 as the unit:

[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 covers 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 covers 26%-50% of the entire main stem and runners;

[0063] Level 9: The lesion area covers 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 manner.

[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%, all significantly The control effect of two single agents, 1 billion CFU / ml Velez TCS001 and 1 billion CFU / ml Paenibacillus polymyxa HY96-2 at a dosage of 250 g / mu, was significantly better than the control effect of the chemical agent 10% difenoconazole water dispersible granules 80 g / mu, among which the control effect was the highest at 88.17% when the ratio of Velez TCS001: Paenibacillus polymyxa HY96-2 was 1:1. The control effects of single agents of 1 billion CFU / ml Velez TCS001 and 1 billion CFU / ml Paenibacillus polymyxa HY96-2 at a dosage of 250 g / mu were 76.82% and 73.24%, respectively, which were significantly higher than the control effect of 10% difenoconazole water dispersible granules 80 g / mu (39.66%).

[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 mixtures can increase the number of strawberry runners and the number of strawberry seedlings. When the average number of runners per plant in the blank control was 2.27, the number of runners treated with 10% difenoconazole water-dispersible granules was 2.20, while Bacillus Velez TCS001, Paenibacillus polymyxa HY96-2 and their mixtures can increase the number of strawberry runners to 3.18-3.73. In the mixture, the number of runners was the largest when Bacillus Velez TCS001: Paenibacillus polymyxa HY96-2 was 1:1, with an increase rate 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 in the area sprayed with TCS001, and more than 60,000 seedlings per mu in the area not sprayed with TCS001).

[0074] Table 3 Effects of Bacillus Velezii 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 invention contains active ingredients Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2, and based on the mass of 1 billion CFU / ml of Bacillus Velez TCS001 and 1 billion CFU / ml of Paenibacillus polymyxa HY96-2 as 100%, the mass ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2 is 50:1-1:50; preferably, the mass ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2 is 4:1-1:4; particularly preferably, the mass ratio of Bacillus Velez TCS001 to Paenibacillus polymyxa HY96-2 is 1:

1.

2. The bactericidal composition according to claim 1, characterized in that The total mass of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 accounts for 1-50% of the mass percentage of the sterilization composition; preferably, the total mass of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 accounts for 5-30% of the mass percentage of the sterilization composition; particularly preferably, the total mass of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 accounts for 10-20% of the mass percentage of the sterilization composition.

3. The bactericidal composition according to claim 1, characterized in that The bactericidal composition can be formulated into any dosage form permitted in agriculture; preferably, the dosage form of the bactericidal composition is a suspension, a seed coating agent, a wettable powder, a water-dispersible granule, a microcapsule suspension, a coated granule, a granule, a water-soluble granule, a water-soluble powder, an aqueous emulsifiable concentrate, a microemulsion, a suspoemulsion, an aqueous emulsion, an effervescent tablet, etc.

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

5. Use of the fungicide composition according to any one of claims 1 to 4 for preventing and controlling plant diseases, preferably, the plants include cereals, fruits, vegetables, and economic crops, and the pathogens include a wide range of plant pathogenic fungi such as oomycetes, chytrids, ascomycetes, smut fungi, rust fungi, and ascomycetes, and particularly preferably, the plant disease is strawberry anthracnose.

6. A method for controlling plant pathogenic bacteria, characterized in that: The fungicidal composition of claims 1 to 4 acts on pathogenic bacteria 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 Velez 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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