Bactericidal composition comprising bacillus velezensis TCS001 and use thereof

Abstract

Provided in the present invention is a bactericidal composition comprising active components, i.e. Bacillus velezensis TCS001, boscalid and Paenibacillus polymyxa HY96-2, wherein the mass ratio of Bacillus velezensis TCS001 to boscalid is 100:1-1:100, and the mass ratio of Bacillus velezensis TCS001 to Paenibacillus polymyxa HY96-2 is 50:1-1:50. The bactericidal composition of the present invention provides a new alternative agent for the prevention and control of gray mold on strawberries, namely, the problem of low prevention efficacy of Bacillus velezensis TCS001 alone is ameliorated, and the anti-disease immune function of Bacillus velezensis TCS001 is also fully exerted. Moreover, by means of mixing with boscalid, the development of boscalid resistance can be delayed or reduced, and an unexpected synergistic effect is produced. The bactericidal composition of the present invention provides a new alternative agent for the prevention and control of anthracnose on strawberries, and the combination of Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 has a synergistic effect in the prevention and control of anthracnose on strawberries.

Description

A bactericidal composition containing Bacillus velezensis TCS001 and its application Technical Field

[0001] The present invention relates to the field of plant protection, and in particular to a fungicide composition containing Bacillus velezensis TCS001, boscalid, and Paenibacillus polymyxa HY96-2. The fungicide composition is particularly applicable to the effective prevention and control of gray mold of strawberries to improve strawberry traits, and to the prevention and control of anthracnose of strawberries to increase strawberry seedlings and strawberry yields. Background Art

[0002] Strawberry gray mold is currently a major disease in strawberry production. It occurs in all strawberry-growing regions of my country, particularly in southern China where the fruit harvest coincides with the spring rainy season, making the disease more severe. Strawberry gray mold often causes flower and fruit rot, significantly impacting strawberry yield and quality. In recent years, pesticide resistance has developed, resulting in yield reductions of approximately 10% in typical years and up to 40% in endemic years. Currently, chemical agents are commonly used to control strawberry gray mold, which carries the risk of pesticide residues. There is an urgent need to develop a highly effective, low-toxic pesticide.

[0003] 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.

[0004] Bacillus velezensis TCS001 was isolated from Bohai Sea mud. The original strain was Bacillus marineus 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 (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 surface is smooth with neat edges, but later, the surface becomes wrinkled, with slightly irregular edges and a central bulge, which diffuses in a cloud-like pattern. Gram staining showed that the Bacillus Velezii TCS001 strain is Gram-positive and rod-shaped; it has a certain inhibitory effect on cucumber gray mold, cucumber vine blight, cucumber sclerotinia, cucumber brown spot, cotton wilt and banana leaf spot, among which the inhibition rate against cucumber gray mold is the highest, reaching 87.66%.

[0005] Boscalid is a mitochondrial respiratory inhibitor and a succinate dehydrogenase inhibitor (SDHI). It works by inhibiting succinate-CoA reductase in the mitochondrial electron transport chain, similar to other amide and benzamide fungicides. It affects the entire growth cycle of pathogens, with a particularly strong inhibitory effect on spore germination. Boscalid has a broad spectrum of activity and preventive properties, showing activity against nearly all fungal diseases. It is highly effective against powdery mildew, gray mold, root rot, sclerotinia, and various rots. It is not prone to cross-resistance and is also effective against bacteria resistant to other agents. It is primarily used to control diseases of rapeseed, grapes, fruit trees, vegetables, and field crops.

[0006] Boscalid is expensive and carries the risk of resistance. Bacillus velez TCS001, a biopesticide, exhibits antibacterial and disease-inducing properties, posing no resistance risk, but is generally ineffective against strawberry gray mold. Therefore, blending boscalid with Bacillus velez TCS001 can lower the price of strawberry gray mold control agents, reduce boscalid usage, and delay the development of boscalid resistance. While leveraging the Bacillus velez TCS001's disease-inducing properties, it can improve overall control effectiveness.

[0007] In 1994, Ash C et al. used PCR probes to identify rRNA in several Bacillus species and assigned some of them 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 assigned a new genus, Paenibacillus. Therefore, Bacillus polymyxa was assigned to the genus Paenibacillus and named Paenibacillus polymyxa.

[0008] 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.

[0009] Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a fungicide mixture, which can not only have a synergistic effect on strawberry gray mold, but also increase the fruit weight, vitamin C, total sugar and organic acid content of strawberries, thereby improving the quality of strawberries.

[0011] A technical solution provided by the present invention is a fungicide composition, characterized in that it contains active ingredients boscalid and Bacillus velez TCS001, with 1 billion CFU / ml of Bacillus velez TCS001 as 100%, wherein the mass ratio of Bacillus velez TCS001 to boscalid is 100:1-1:100; preferably, the mass ratio of Bacillus velez TCS001 to boscalid is 100:1-10:1; particularly preferably, the mass ratio of Bacillus velez TCS001 to boscalid is 30:1.

[0012] The total mass of the Bacillus velez TCS001 and boscalid accounts for 1-50% of the mass percentage of the fungicide composition; preferably, the total mass of the Bacillus velez TCS001 and boscalid accounts for 5-30% of the mass percentage of the fungicide composition; particularly preferably, the total mass of the Bacillus velez TCS001 and boscalid accounts for 10-20% of the mass percentage of the fungicide composition.

[0013] 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.

[0014] 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.

[0015] 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 gray mold.

[0016] 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.

[0017] The Bacillus velezensis TCS001 and boscalid are applied simultaneously, separately, or sequentially.

[0018] Another technical solution provided by the present invention is the use of the bactericidal composition for improving strawberry traits.

[0019] The strawberry traits include one or more of fruit weight, vitamin C, total sugar and organic acid.

[0020] Another technical solution provided by the present invention is a kit comprising the fungicide composition containing Bacillus velezensis TCS001 and boscalid.

[0021] Another technical problem to be solved by the present invention is to provide a sterilization mixture that 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 are administered simultaneously, separately, or sequentially.

[0029] 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.

[0030] The method of promoting the growth of strawberry seedlings includes increasing the number of runners of the strawberry seedlings.

[0031] 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.

[0032] Beneficial technical effects of the present invention

[0033] The present invention utilizes a binary compound of Bacillus velezensis TCS001 and boscalid to create a fungicide mixture that exhibits synergistic efficacy in preventing and controlling gray mold in strawberries, increasing fruit weight, vitamin C, total sugar, and organic acid content, and improving strawberry quality. This provides a novel alternative agent for preventing and controlling gray mold in strawberries, leveraging the anti-disease immune function of biological agents to enhance prevention efficacy while also reducing the development of boscalid resistance.

[0034] 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, thereby providing a new alternative agent for the prevention and treatment of strawberry anthracnose.

[0035] 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

[0036] Figure 1 Results of affinity screening tests between Bacillus velezensis TCS001 and other biocontrol bacteria;

[0037] Figure 2: Effects of Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 against strawberry anthracnose;

[0038] Figure 3 Field effects of Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2 after single-dose and combined use. DETAILED DESCRIPTION

[0039] Example 1

[0040] Affinity screening of Bacillus velezensis TCS001 and boscalid

[0041] Preparation of Bacillus velez TCS001 seed liquid: Prepare 1000 mL of LB solid medium (27 mL*30 bottles) and 150 mL of LB liquid medium, and sterilize them; inoculate a single colony of Bacillus velez TCS001 into the LB liquid medium and culture in a shaking incubator at 25°C, 164 rpm, for 16 h.

[0042] Preparation of toxic culture medium: Dissolve boscalid stock in DMF to a 10% stock solution for later use. Dilute to 10 times the test dose with sterile water containing 1‰ Tween-80. Quantitatively pipette 3 mL of the solution into 27 mL of LB medium, starting from the lowest concentration and increasing to the highest. Shake thoroughly and pour equal amounts into two 9 cm diameter Petri dishes to prepare drug-containing plates of the appropriate concentration. Perform two replicates for each treatment.

[0043] Affinity screening: dilute the shaken Bacillus Velez TCS001 seed solution to 1*10 6 CFU / mL, pipette 40 μL to coat the drug-containing culture medium and control group plates, culture in a 28°C incubator in the dark, and observe the growth of TCS001.

[0044] When the concentration of boscalid was 400 mg / L, the colony count of Bacillus velezensis TCS001 was equivalent to that of the blank control, indicating that boscalid had affinity with Bacillus velezensis TCS001.

[0045] Example 2

[0046] Toxicity determination of the complex of Bacillus velezensis TCS001 and boscalid against gray mold of strawberry in laboratory

[0047] Bacillus Velez TCS001, boscalid, and the mixture of Bacillus Velez TCS001 and boscalid were diluted to the test dose. Healthy strawberry fruits were used as test materials. The strawberry fruits were immersed in the liquid for 20 seconds. Each treatment was repeated 3 times, with 9 fruits in each repeat. The holes were uniformly pierced with 5mm×5mm. 5 spores / mL of a spore suspension of Botrytis cinerea. 24 hours later, the treated strawberry fruits were sprayed with the spore suspension. The fruits were then placed in a high-humidity environment (90-95% relative humidity) at 22°C. Four days later, the lesion diameter was measured and the inhibition rate (%) was calculated.

[0048] Inhibition rate (%) = (blank control colony diameter - drug-treated colony diameter) / blank control colony diameter × 100.

[0049] E0=(X+Y)-X×Y / 100

[0050] X: inhibition rate of A when the dosage is P;

[0051] Y: inhibition rate of B when the dosage is Q;

[0052] E0: Theoretical inhibition rate of A+B when the dosage is (P+Q);

[0053] E: represents the actual inhibition rate of each treatment.

[0054] The Colby method was used to evaluate the synergistic effect of drug mixing, that is, when E-E0>5%, it indicated a synergistic effect; when E-E0<-5%, it indicated an antagonistic effect; and when the E-E0 value was between ±5%, it indicated an additive effect.

[0055] As can be seen from Table 1, the inhibitory effect of the composition containing boscalid and Bacillus velez TCS001 on strawberry gray mold pathogens is significantly better than using boscalid or Bacillus velez TCS001 alone, indicating that there is a synergistic effect between boscalid and Bacillus velez TCS001. When Bacillus velez TCS001 and boscalid are treated separately, the inhibition rates on strawberry gray mold pathogens are only 32.97% and 34.44%-79.26%. When the two are mixed, the inhibition rate on strawberry gray mold pathogens is significantly increased. When the ratio of boscalid to Bacillus velez TCS001 is 1:6-1:100, the inhibition rate on strawberry gray mold pathogens is 61.48%-87.41%, which is a synergistic effect, but the synergistic effects are different in different ratios, and the synergistic effect of 1:30 is the most significant.

[0056] Table 1 Indoor toxicity test of the complex of Bacillus velezensis TCS001 and boscalid against gray mold of strawberry

[0057] Example 3

[0058] Field efficacy test of the combination of Bacillus velezensis TCS001 and boscalid in controlling gray mold of strawberry

[0059] Bacillus Velez TCS001 was mixed with boscalid in a weight ratio to conduct a field trial on the control of gray mold in strawberries. The strawberry variety was Hongyan. The plots were arranged in random blocks with an area of ​​10m 2 Each treatment was replicated three times. The first application occurred during the strawberry flowering period, before the onset of gray mold, and the second application occurred 7-10 days later, for a total of two applications. During the survey, samples were collected at five diagonal points in each plot, with 50 strawberry fruits collected at each point. The number of diseased fruits was counted, and the total number of fruit and the number of diseased fruit were recorded separately. The control efficacy was calculated according to the following formula. Twenty mature fruits were harvested from each treatment, and the weight of each fruit was measured. The total sugar, organic acid, and vitamin C content of the strawberries were also determined.

[0060] As shown in Table 2, the combination of Bacillus velezil TCS001 and boscalid exhibited excellent control efficacy against strawberry gray mold, reaching 85.29%-89.70% efficacy. This significantly outperformed both boscalid and Bacillus velezil TCS001 alone, demonstrating significant synergy. The highest efficacy of 89.70% was achieved at a boscalid:Bacillus velezil TCS001 ratio of 1:30, consistent with laboratory results.

[0061] Table 2 Field test results of the combination of Bacillus velezensis TCS001 and boscalid for the control of strawberry gray mold

[0062] As shown in Table 3, the combination of boscalid and Bacillus velez TCS001 has excellent control effects on strawberry gray mold. It can also increase the weight of individual strawberry fruits, increase the content of total sugar, vitamin C, and organic acids, improve yield and quality, and also have a better taste. The strawberry single fruit weight, total sugar, vitamin C, and organic acid contents at a boscalid:Bacillus velez TCS001 ratio of 1:6-1:40 were all higher than those in the blank control and boscalid treatment.

[0063] Table 3 Effects of the mixture of boscalid and Bacillus velezensis TCS001 on strawberry quality

[0064] Example 4

[0065] Affinity screening of Bacillus velezensis TCS001 with Paenibacillus polymyxa HY96-2, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus brevis.

[0066] 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 with an inoculating 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. 630The value is 0.1. 100 μL of the Bacillus Velez TCS001 bacterial suspension (plate strain) was aspirated and placed on an LB 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 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 formed. The presence of an inhibition zone indicates that the strains have no affinity, and the absence of an inhibition zone indicates that the two strains have affinity.

[0067] The results of the affinity screening test for Bacillus Velez TCS001 with biocontrol bacteria are shown in Figure 1. No inhibition zones appeared around the filter paper discs for Paenibacillus polymyxa HY96-2 and Bacillus amyloliquefaciens, indicating that Bacillus Velez TCS001 has affinity with these two bacteria. However, inhibition zones appeared around the filter paper discs for Bacillus subtilis and Bacillus brevis, indicating that Bacillus Velez TCS001 has no affinity with these two bacteria.

[0068] Example 5

[0069] A confrontation test between Bacillus velezensis TCS001, Paenibacillus polymyxa HY96-2 and strawberry anthracnose.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Example 6

[0074] Screening of compound formulas of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 for controlling strawberry anthracnose

[0075] 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.

[0076] The disease classification standard is based on the main stem:

[0077] Level 0: no disease;

[0078] Level 1: The lesion area occupies less than 5% of the entire main stem;

[0079] Level 3: The lesion area occupies 6%-10% of the entire main stem;

[0080] Level 5: The lesion area occupies 11%-25% of the entire main stem;

[0081] Level 7: The lesion area occupies 26%-50% of the entire main stem;

[0082] Level 9: The area of ​​lesions occupies more than 51% of the entire main stem, or the plant dies.

[0083] Calculation method of drug efficacy: The drug efficacy is calculated in the following way.

[0084] Theoretical toxicity index (TTI) = toxicity index of A × content of A in the mixture (%)

[0085] + Toxicity index of B × content of B in the mixture (%)

[0086] 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.

[0087] Table 1 Screening of compound formulas for controlling strawberry anthracnose with Bacillus velezensis TCS001 and Paenibacillus polymyxa HY96-2

[0088] 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.

[0089] Example 7

[0090] Field efficacy test of a mixture of Bacillus Velez TCS001 and Paenibacillus polymyxa HY96-2 against anthracnose of strawberry during seedling stage

[0091] 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:

[0092] Level 0: no disease;

[0093] Level 1: The lesion area occupies less than 5% of the entire main stem and runners;

[0094] Level 3: The lesion area occupies 6%-10% of the entire main stem and runners;

[0095] Level 5: The lesion area occupies 11%-25% of the entire main stem and runners;

[0096] Level 7: The lesion area occupies 26%-50% of the entire main stem and runners;

[0097] Level 9: The area of ​​lesions occupies more than 51% of the entire main stem and runners, or the plant dies.

[0098] Calculation method of drug efficacy: The drug efficacy is calculated in the following way.

[0099] Table 2 Field efficacy test results of Bacillus lesii TCS001, Paenibacillus polymyxa HY96-2 and their mixtures against strawberry anthracnose

[0100] 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 82.1 3%, significantly higher than the control efficacy of two single doses of Bacillus Velez TCS001 (1 billion CFU / mL) and Paenibacillus polymyxa HY96-2 (1 billion CFU / mL) at a dose of 250 g / mu. It was particularly superior to the control efficacy of the chemical agent 10% difenoconazole water-dispersible granules (80 g / mu). The highest control efficacy was 88.17% at a 1:1 ratio of Bacillus Velez TCS001:Paenibacillus polymyxa HY96-2. The control efficacy of single doses of Bacillus Velez TCS001 (1 billion CFU / mL) and Paenibacillus polymyxa HY96-2 (1 billion CFU / mL) at a dose of 250 g / mu was 76.82% and 73.24%, respectively, significantly higher than the control efficacy of 10% difenoconazole water-dispersible granules (54.44%) at 80 g / mu.

[0101] 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.

[0102] 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%.

[0103] 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).

[0104] Table 3 Effects of Bacillus velezensis TCS001, Paenibacillus polymyxa HY96-2 and their mixture on strawberry runners

[0105] 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 boscalid, or active ingredients Bacillus velez TCS001 and Paenibacillus polymyxa HY96-2, based on 1 billion CFU / ml of Bacillus velez TCS001 as 100%, the mass ratio of Bacillus velez TCS001 to boscalid is 100:1-1:100; preferably, the mass ratio of Bacillus velez TCS001 to boscalid is 100:1-10:1; particularly preferably, the mass ratio of Bacillus velez TCS001 to boscalid is 30:1, or, based on the mass of 1 billion CFU / ml of Bacillus Velez TCS001 and 1 billion CFU / ml of Paenibacillus polymyxa HY96-2 being 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 boscalid accounts for 1-50% of the mass percentage of the sterilization composition; preferably, the total mass of Bacillus velez TCS001 and boscalid accounts for 5-30% of the mass percentage of the sterilization composition; particularly preferably, the total mass of Bacillus velez TCS001 and boscalid accounts for 10-20% of the mass percentage of the sterilization composition; 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, 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 immunizers, and the like.

5. Use of the fungicide composition according to any one of claims 1 to 4 for 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 oomycetes, chytrids, ascomycetes, smut fungi, rust fungi, and deuteromycetes; particularly preferably, the plant pathogen is strawberry gray mold.

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: The Bacillus Velezii TCS001, boscalid and Paenibacillus polymyxa HY96-2 are applied simultaneously, separately or sequentially.

8. Use of the bactericidal composition according to any one of claims 1 to 4 for improving strawberry traits, or 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 strawberry traits include one or more of fruit weight, vitamin C, total sugar and organic acid; and the method for 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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