Biocidal composition containing acid-resistant bacterial strain and use thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a biological sterilization composition containing acid-resistant strains and application thereof. The biological sterilization composition containing acid-resistant strains is characterized in that an effective component is compounded from a microbial active component and a chemical active component; the microbial active component is prepared from acid-resistant strains TXB1-10, and the chemical active component is benzene ether carbendazol; the taxonomic name of the acid-resistant strain TXB1-10 is Sinomonas atrocyanea, and the strain is preserved in the China Center for Type Culture Collection on December 28, 2020, with a preservation number of CCTCC No: M2020984. When the strain TXB1-10 is compounded with benzene ether carbendazol after being prepared into a microbial active component, the strain exhibits a synergistic effect on inhibiting the growth of tomato botrytis cinerea mycelium, and compared with a single effective component, the strain can improve the prevention and treatment effect on tomato botrytis cinerea, and can provide support for developing biological pesticides for preventing and treating tomato botrytis cinerea.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a biological bactericidal composition containing an acid-resistant strain and its application. Background Technology

[0002] Tomato gray mold, caused by Botrytis cinerea, is a serious global disease that primarily occurs during the flowering and fruiting stages, damaging flowers, fruits, leaves, and stems. This disease not only causes significant damage during plant growth but also continues to cause serious harm during harvesting, storage, and transportation. For a long time, the main method for controlling tomato gray mold has been the spraying of chemical fungicides. However, due to the rapid reproduction and high variability of the tomato gray mold fungus, it easily develops resistance to chemical agents. The long-term use of single-ingredient chemical agents has led to varying degrees of resistance to many existing chemical agents, resulting in a gradual decrease in control effectiveness. Therefore, the development of new fungicides is essential.

[0003] With increasing awareness of pesticide resistance and food safety issues, the use of biocontrol technology to prevent and control plant diseases has become a research hotspot. Biocontrol offers advantages such as wide availability of production sources, safety against non-target organisms, low toxicity, good environmental compatibility, and sustained action against harmful targets. Existing technologies have revealed the control effects of Bacillus amyloliquefaciens, Pseudomonas fluorescens, and Bacillus subtilis against tomato gray mold. Studies have also reported that the combination of Pseudomonas fluorescens YG-1 and the chemical agent boscalid has a synergistic effect in controlling tomato gray mold, reducing the amount of fungicide required.

[0004] Strain TXB1-10 is a bacterium screened from the selenium-rich soil of Tengxian County, Guangxi Province. It significantly increases the content of exchangeable selenium in the soil and has an activating effect on soil selenium. The screening and identification of this strain are disclosed in patent application CN201811201689.9, "A Method for Cultivating a Strain for Highly Efficient Soil Selenium Conversion." Additionally, patent application CN202110734139.9, "A Special Microbial Fertilizer for Selenium-Enriched Vegetables and Its Preparation Method," also discloses the selenium-enriching effect of this strain. However, there are currently no reports on its application in crop diseases.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-fungicide composition containing an acid-resistant strain and its application, which has the potential to be developed into a bio-fungicide and can provide support for the biological control of crop diseases.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide an acid-resistant strain TXB1-10, the taxonomic name of which is *Sinomonas atrocyanea*, which was deposited at the China Center for Type Culture Collection on December 28, 2020, with accession number CCTCC No: M2020984.

[0009] A second object of the present invention is to provide the use of the acid-resistant strain TXB1-10 in the prevention and control of fungal diseases in crops.

[0010] Preferably, the fungal diseases of the crop include gray mold.

[0011] A third objective of this invention is to provide a biological bactericidal composition, the effective component of which is a compound of microbial active ingredients and chemical active ingredients; wherein the microbial active ingredients are prepared from the acid-resistant strain TXB1-10 as described in claim 1; and the chemical active ingredients are difenoconazole.

[0012] Preferably, the method for preparing the microbial active ingredient is as follows:

[0013] S1. Single colonies were obtained by purifying and isolating the acid-resistant strain TXB1-10 by streak plating;

[0014] S2. The single colony obtained in S1 was inoculated into LB liquid medium and cultured at 37℃ and 180 r / min in a shaker for 12 h. The bacterial count in the culture medium was then adjusted to 5.0 × 10⁻⁶ using LB liquid medium. 7 The cfu / g yields the active microbial component.

[0015] Preferably, the LB liquid culture medium is formulated as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L distilled water, and pH adjusted to 7.0.

[0016] Preferably, the mass ratio of the microbial active ingredient to the chemical active ingredient is 1-35:10-1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The bacterial count of strain TXB1-10 of the present invention in LB liquid medium is 5.0 × 10⁻⁶. 7 At cfu / g, its EC50 response to tomato gray mold is... 50 The concentration was 131.4641 mg / L, and strain TXB1-10 has the potential to be developed into a fungicide.

[0019] (2) When the microbial active ingredient of strain TXB1-10 of the present invention is combined with difenoconazole, it shows a synergistic effect on inhibiting the growth of mycelium of tomato gray mold. Compared with a single active ingredient, it can improve the control effect of tomato gray mold and provide support for the development of biological agents for the control of tomato gray mold.

[0020] Preservation Information

[0021] Sinomonas atrocyanea strain TXB1-10, with accession number CCTCC No: M 2020984, accession date December 28, 2020, is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. Detailed Implementation

[0022] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0023] Example 1 Virulence test of strain TXB1-10 against the pathogen of tomato gray mold

[0024] The strain TXB1-10 used in this embodiment has been disclosed in patent application number CN202110734139.9, entitled "A Selenium-Enriched Vegetable-Specific Microbial Fertilizer and Its Preparation Method." This embodiment only studies the inhibitory effect of strain TXB1-10 on the pathogen of tomato gray mold.

[0025] 1. Test strain

[0026] The pathogen of tomato gray mold was collected from tomato plants infected with tomato gray mold in the tomato experimental field of Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences. It was isolated and purified in the laboratory and preserved in PDA medium.

[0027] The formula for PDA culture medium is as follows: 200g potato, 10g glucose, 20g agar powder, distilled water to a final volume of 1L, and pH adjusted to 7.0.

[0028] 2. Test reagents

[0029] Microbial active ingredients: Single colonies were obtained by purifying and isolating strain TXB1-10 by streak plating; the obtained single colonies were inoculated into LB liquid medium and cultured in a shaker at 37℃ and 180 r / min for 12 h, and then the bacterial count of the culture medium was adjusted to 5.0 × 10⁻⁶ with LB liquid medium. 7 The cfu / g yields the active microbial component.

[0030] The LB liquid culture medium formula is as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L distilled water, and pH adjusted to 7.0.

[0031] 3. Test Methods

[0032] The mycelial growth rate method was used. The microbial active ingredient was diluted with 0.1% Tween-80 aqueous solution, and five mass concentration gradients were set up in a proportional manner. 9 mL of pre-melted PDA medium was added to a sterile Erlenmeyer flask. 1 mL of the drug solution was then quantitatively pipetted from low to high concentration and added to the respective Erlenmeyer flasks. After thorough mixing, the mixture was poured into 9 cm diameter petri dishes to prepare drug-containing plates of the corresponding concentrations. A blank control without the drug was also included, with 10 replicates for each treatment.

[0033] Using a punch, cut 5mm diameter mycelial discs from the edge of the test strain colony and inoculate them into the center of both the drug-containing plate and the blank control plate. Cover the plates and incubate at 25℃. When the diameter of the blank control colony approaches 2 / 3 of the plate diameter, measure the colony diameter using the cross-sectional method and calculate the inhibition rate of the drug treatment on the mycelial growth of the target pathogen.

[0034]

[0035] 4. Data Analysis

[0036] Data statistical analysis was performed using DPS software. Linear regression was conducted with the logarithm of the agent concentration as x and the corresponding mycelial growth inhibition rate probability value as y to derive the virulence regression equation and the EC50 of the agent against the target pathogen. 50 The values ​​are shown in Table 1.

[0037] Table 1. Virulence determination of microbial active ingredients against the pathogen of tomato gray mold.

[0038]

[0039] Table 1 shows that the microbial active ingredients have an EC50 effect on the pathogen of tomato gray mold. 50 The concentration was 131.4641 mg / L, and strain TXB1-10 has the potential to be developed into a fungicide.

[0040] Example 2 Compatibility test of chemical agents with strain TXB1-10

[0041] 1. Test chemical reagents

[0042] 97% Tebuconazole technical grade (Jiangsu Fengdeng Crop Protection Co., Ltd.)

[0043] 95% difenoconazole technical grade (Jiangsu Youjia Plant Protection Co., Ltd.)

[0044] 98% Chlorothalonil Technical Grade (Inner Mongolia Bailing Technology Co., Ltd.)

[0045] 95% Thiophanate-methyl technical grade (Shandong Huayang Pesticide & Chemical Group Co., Ltd.)

[0046] 98.5% iprodione technical grade (Zhejiang Heben Technology Co., Ltd.)

[0047] 98% imazalil technical grade (Shaoxing Donghu High-Tech Co., Ltd.)

[0048] 2. Test Methods

[0049] The test chemical reagent was first dissolved in dimethyl sulfoxide, then diluted with 0.1% Tween-80 aqueous solution to prepare solutions with concentrations of 0.05, 0.1, 0.5, 1.0, and 2.0 mg / L. 9 mL of pre-melted PDA culture medium was added to a sterile Erlenmeyer flask. 1 mL of the solution was then quantitatively pipetted from low to high concentration and added to the respective Erlenmeyer flasks. After thorough mixing, the solution was poured into 9 cm diameter petri dishes to prepare plates containing the corresponding drug concentrations. A treatment with an equal volume of water was included as a blank control. Each treatment was replicated five times.

[0050] 100 μL of the microbial active ingredient prepared in Example 1 was evenly spread on the surface of the drug-containing plate and the blank control plate using a pipette. The plates were then incubated in a constant temperature incubator at 37°C. The bacterial growth was detected by plate dilution method after 5 days, and the number of colonies was counted and recorded.

[0051] Results: At concentrations of 0.05–0.5 mg / L, all six chemical fungicides showed good compatibility with strain TXB1-10, and strain TXB1-10 could grow normally in all concentrations. At 1.0 mg / L, strain TXB1-10 ceased growth on agar plates containing tebuconazole, chlorothalonil, and iprodione. At 2.0 mg / L, strain TXB1-10 ceased growth on agar plates containing thiophanate-methyl and prochloraz. At this concentration, strain TXB1-10 could still grow on agar plates containing difenoconazole, but the number of colonies was significantly reduced compared to lower concentrations. Therefore, difenoconazole was selected as the chemical agent in the compound formulation for strain TXB1-10.

[0052] Example 3 Virulence determination of strain TXB1-10 against the pathogen of tomato gray mold

[0053] 1. Test strain

[0054] The pathogen of tomato gray mold was collected from tomato plants infected with tomato gray mold in the tomato experimental field of Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences. It was isolated and purified in the laboratory and preserved in PDA medium.

[0055] The formula for PDA culture medium is as follows: 200g potato, 10g glucose, 20g agar powder, distilled water to a final volume of 1L, and pH adjusted to 7.0.

[0056] 2. Test reagents

[0057] Microbial active ingredients: Single colonies were obtained by purifying and isolating strain TXB1-10 by streak plating; the obtained single colonies were inoculated into LB liquid medium and cultured in a shaker at 37℃ and 180 r / min for 12 h, and then the bacterial count of the culture medium was adjusted to 5.0 × 10⁻⁶ with LB liquid medium. 7 The cfu / g of the microbial active ingredient is obtained. The LB liquid culture medium is formulated as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L distilled water, adjusted to pH 7.0.

[0058] Chemically active ingredient: 95% difenoconazole technical grade (Jiangsu Youjia Plant Protection Co., Ltd.)

[0059] 3. Experimental Method (Mycelial Growth Rate Method)

[0060] Microbial active ingredients were directly diluted with 0.1% Tween-80 aqueous solution to prepare single-agent stock solutions; difenoconazole technical material was first dissolved in dimethyl sulfoxide and then diluted with 0.1% Tween-80 aqueous solution to prepare single-agent stock solutions; multiple formulations were set up, and each single agent and formulation mixture was set with 5 mass concentration gradients according to the proportional method. All agents were prepared and used immediately.

[0061] Add 9 mL of pre-melted PDA medium to a sterile Erlenmeyer flask. Quantitatively add 1 mL of drug solution from low to high concentration to the Erlenmeyer flasks, shake well, and pour into 9 cm diameter petri dishes to prepare drug-containing plates of the corresponding concentrations. A blank control without drug is also set up, with 10 replicates for each treatment.

[0062] Using a punch, cut 5mm diameter mycelial discs from the edge of the test strain colonies and inoculate them into the center of drug-containing plates and blank control plates. After covering the plates, incubate them at 25℃. When the diameter of the blank control colonies is close to 2 / 3 of the plate diameter, measure the colony diameter using the cross-sectional method and calculate the inhibition rate of different drug treatments on the mycelial growth of the target pathogen.

[0063]

[0064] 4. Data Analysis

[0065] Data statistical analysis was performed using DPS software. Linear regression was conducted with the logarithm of the agent concentration as x and the corresponding mycelial growth inhibition rate probability value as y to derive the virulence regression equation and the EC50 of the agent against the target pathogen. 50The synergistic effect was evaluated using the Wadlley method.

[0066] SR ≥ 1.5 indicates a synergistic effect; SR ≤ 0.5 indicates an antagonistic effect; 0.5 < SR < 1.5 indicates an additive effect. The results are shown in Table 2.

[0067] Table 2. Virulence of the combination of microbial active ingredient (A) and difenoconazole (B) against tomato gray mold.

[0068]

[0069] As shown in Table 2, the combination of microbial active ingredients and difenoconazole showed a synergistic effect in inhibiting the growth of tomato gray mold mycelium. Compared with the single active ingredient, it can improve the control effect on tomato gray mold and provide support for the development of biological agents for the control of tomato gray mold.

[0070] Example 4 Acid resistance test of strain TXB1-10

[0071] 4.1 Acid resistance test of culture medium

[0072] The nutrient agar medium formula is as follows: 3g beef extract, 10g peptone, 5g sodium chloride, 15g agar, and distilled water to a final volume of 1L. The pH of the nutrient agar medium is adjusted with HCl and NaOH, respectively, to create six pH gradient treatments: 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. After sterilization at 120℃ for 20 min, the medium is poured into 9cm diameter petri dishes, 20mL per dish. 10μL of TXB1-10 bacterial suspension in the logarithmic growth phase is pipetted onto a pH gradient plate and incubated at 37℃ for 48 h. The growth of strain TXB1-10 is then observed.

[0073] The results showed that strain TXB1-10 could grow on nutrient agar plates with pH 4.5-7.0.

[0074] 4.2 Acid resistance test in soil

[0075] Soil 1: pH 5.88

[0076] Soil 2: pH 4.86

[0077] Each petri dish contains 50g of sterile soil, inoculated with 5mL of TXB1-10 bacterial suspension in the logarithmic growth phase, 10mL of sterile water, and stirred evenly. Water is added periodically to control the soil moisture content to about 30%. Samples are taken at 0, 7, 14, 21, 35, and 49 days for bacterial counting.

[0078] The results showed that strain TXB1-10 maintained growth at 10 in soil 1. 7 -108 Quantity level; growth maintained at 10 in soil 2. 8 -10 9 Quantitative level. The colonization ability of strain TXB1-10 in soil 2 was stronger than that in soil 1, and strain TXB1-10 had a stronger acid resistance.

[0079] The exemplary embodiments have been chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various exemplary embodiments of the invention, as well as various alternatives and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A bio-bacterial composition containing acid-resistant bacterial strains, characterized in that, Its active ingredients are a combination of microbial and chemically active components; wherein the microbial active components are prepared from the acid-resistant strain TXB1-10, and the bacterial count of the microbial active components is 5.0 × 10⁻⁶. 7 cfu / g, wherein the chemically active ingredient is difenoconazole; the mass ratio of the microbial active ingredient to the chemically active ingredient is 1-35:10-1; The acid-resistant strain TXB1-10 is taxonomically named *Sinonovacula davidii* (Dark Sinonovacula). Sinomonas atrocyanea It was deposited at the China Center for Type Culture Collection on December 28, 2020, with accession number CCTCC No: M 2020984.

2. The bio-bactericidal composition containing acid-resistant strains according to claim 1, characterized in that, The preparation method of the microbial active ingredient is as follows: S1. Single colonies were obtained by purifying and isolating the acid-resistant strain TXB1-10 by streak plating; S2. The single colonies obtained in S1 were inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 12 h with shaking. The bacterial count in the culture medium was then adjusted to 5.0 × 10⁻⁶ using LB liquid medium. 7 The cfu / g yields the active microbial component.

3. The bio-bactericidal composition containing acid-resistant strains according to claim 2, characterized in that, The LB liquid culture medium formula is as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L distilled water, and pH adjusted to 7.

0.

4. The use of the bio-bacterial composition containing acid-resistant strains as described in any one of claims 1-3 in the control of tomato gray mold.

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