Cronobacter composition for preventing and treating plant fungal diseases

Abstract

The invention relates to a Cronobacter composition for preventing and treating plant fungal diseases and application thereof. The Cronobacter sp. C1 and the Cronobacter sp. C7 provided by the invention have the preservation numbers of CGMCC (China General Microbiological Culture Collection Center) No. 24172 and CGMCC No. 24173 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, respectively. The two strains are separated from roots of disease-resistant varieties of cucumbers, and viable bacteria and metabolites of the two strains have a strong inhibition effect on various pathogenic fungi. The bacterial composition is separated from plant roots, has strong affinity with plants, short strain fermentation culture time and low production cost, has more advantages than other bacterial agents, is beneficial to application in the field of plant disease control, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of agricultural, forestry and horticultural crop plant protection, and particularly relates to a Cronobacter combination having a strong inhibitory effect on plant fungal diseases and its metabolites. Technical Background

[0002] Soil microorganisms can be divided into several major groups, bacteria, fungi, and actinomycetes. Bacteria are an important microbial group for the prevention and control of plant fungal diseases. Many antibiotics and other metabolites produced by bacteria have a certain degree of control effect on plant fungal diseases, but there are currently few studies on the use of Cronobacter to prevent and control plant fungal diseases. Martínez-Rodríguez and Mora-Amutio (2014) isolated endophytic Cronobacter from Agave tequilana and found that it can produce IAA, has nitrogen fixation, phosphorus solubilization and growth promotion functions, but did not report its antibacterial effect. Sahana et al. (2018) reported that Cronobacter can produce extracellular polysaccharides, which can be used to prepare human dermal fibroblasts. There are no reports on the prevention and control of plant fungal diseases by Cronobacter. Obtaining new biocontrol bacteria with independent intellectual property rights in my country that can prevent and control a variety of plant fungal diseases has important practical significance for supplementing biocontrol resources and preventing and controlling diseases.

[0003] In the long-term coexistence of microorganisms with disease-resistant plant varieties, mutual benefit is achieved, and highly active antagonistic microorganisms can be obtained from the disease-resistant varieties. Our research group analyzed the rhizosphere and root bacterial communities of the disease-resistant cucumber varieties Chuanlu 11 and Zhongnong 106 and the susceptible variety Zhongnong 6, and isolated two differential bacteria from the roots of the disease-resistant cucumber varieties, which were identified as Cronobater sp. These two strains of bacteria have strong inhibitory effects on a variety of fungal pathogens, including the cucumber wilt pathogen Fusarium oxysporum f.sp.cucumerinum, the bean root rot pathogen Fusarium solani, the pepper wilt pathogen Phytophythora capsici, the cabbage wilt pathogen Fusarium oxysporum, the gloeosporioides Colletotrichum gloeosporioides, and the tomato gray mold pathogen Botrytis cinerea, and their metabolites also have strong antibacterial effects; these two strains can also migrate with the fungal hyphae and inhibit the production of fungal hyphae spores; the bacterial combination of these two strains (V∶V=1∶1) has a strong inhibitory effect on cucumber wilt, with a prevention efficiency of up to 57.0%. The two strains of bacteria exist in the roots of cucumbers and have the advantage of preferentially occupying the root space. The concentration of the bacterial combination is very low, at 1×10 6CFU / g can play its disease prevention role; the fermentation cycle is very short, fermentation can be completed in 20 hours, and the production cost is low, so it is a bacterial combination with great application potential. Summary of the invention

[0004] The purpose of the present invention is to provide a Cronobacter combination having a preventive and therapeutic effect on plant fungal diseases and its application.

[0005] The Cronobacter combination provided by the present invention is specifically composed of Cronobacter sp. C1 and Cronobacter sp. C7. The strains have been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 21, 2021, with the deposit numbers CGMCC No. 24172 and CGMCC No. 24173.

[0006] The present invention also protects a bacterial agent whose active ingredients are the Cronobacter sp. C1 and Cronobacter sp. C7.

[0007] The microbial agent may further include an adsorption carrier; the adsorption carrier may be at least one of a seedling medium, diatomaceous earth, peat, vermiculite, rice husk powder, straw powder, perlite and calcium carbonate.

[0008] More specifically, in one embodiment of the present invention, the adsorption carrier is specifically formed by mixing the seedling matrix according to a certain volume-to-weight ratio. Further, the bacterial agent is formed by mixing the fermentation liquid of Cronobacter sp. C1 and Cronobacter sp. C7 with the adsorption carrier according to a ratio of 1 mL: 4 kg to 1 mL: 6 kg (e.g., 1 mL: 4 kg). In one embodiment of the present invention (Example 5), a large amount of fermentation broth is used to prepare the fermentation broth of Cronobacter sp. C1 and Cronobacter sp. C7, wherein the number of viable bacteria of Cronobacter sp. C1 and Cronobacter sp. C7 is 4 to 6 billion / mL (such as 4 billion / mL) of the fermentation broth, wherein C1 and C7 are mixed at a ratio of 1:1 (V / V). The solvent of the large amount of fermentation broth is water, and the solutes and concentrations are as follows: beef extract 0.8%, yeast powder 0.5%, glucose 0.5%, peptone 1.0%, sodium chloride 0.05%; wherein % represents the mass percentage. Correspondingly, in the prepared bacterial agent, the number of live bacteria of Cronobacter sp. C1 and Cronobacter sp. C7 is 4 to 6 billion / g (such as 4 billion / g) of the bacterial agent; and the water content of the bacterial agent is within 5 to 8%.

[0009] The preparation method of the bacterial agent also belongs to the protection scope of the present invention.

[0010] The preparation method of the bacterial agent may specifically include the following steps:

[0011] (1) culturing the Cronobacter sp. C1 and Cronobacter sp. C7 to obtain fermentation broth of the Cronobacter sp. C1 and Cronobacter sp. C7; in the fermentation broth, the number of live bacteria of the Cronobacter sp. C1 and Cronobacter sp. C7 is 4 to 6 billion / mL (e.g., 4 billion / mL);

[0012] (2) The fermentation liquid obtained in step (1) and the adsorption carrier are mixed in a ratio of 1 mL: 4 Kg to 1 mL: 6 Kg (e.g., 1 mL: 4 Kg) or the bacterial sludge obtained after centrifugation of the fermentation liquid and the adsorption carrier are mixed in a ratio of 1 L: 4 Kg to 1 L: 6 Kg (e.g., 1 L: 4 Kg) to obtain the bacterial agent.

[0013] In step (1), the solvent of the culture medium for culturing the Cronobacter sp. C1 and Cronobacter sp. C7 is water, and the solutes and concentrations are as follows: beef extract 0.8-1.0%, yeast powder 0.3-0.5%, glucose 0.5-0.7%, peptone 1.0-1.2%, sodium chloride 0.03-0.05%; wherein % represents the mass percentage.

[0014] More specifically, the solvent of the culture medium is water, and the solutes and concentrations are as follows: 0.8% beef extract, 0.5% yeast powder, 0.5% glucose, 1.0% peptone, and 0.05% sodium chloride; wherein % represents the mass percentage.

[0015] The fermentation broth of Cronobacter sp. C1 and Cronobacter sp. C7 also belongs to the protection scope of the present invention.

[0016] The invention also provides a product for preventing and controlling plant fungal diseases.

[0017] The use of the Cronobacter sp. C1 and Cronobacter sp. C7 or the bacterial agent or the fermentation broth in the following (1) or (2) also falls within the protection scope of the present invention:

[0018] (1) Prevent and control plant diseases caused by infection by pathogenic fungi, or combined infection by bacteria and fungi, or fungi and nematodes.

[0019] (2) Prepare products for preventing and controlling diseases described in (1).

[0020] The CGMCC No. 24172 of Cronobacter sp. C1 and the CGMCC No. 24173 of Cronobacter sp. C7 provided by the present invention have the following advantages:

[0021] 1. This strain is isolated from the roots of plants, is more adaptable to the rhizosphere environment of plants, and grows rapidly.

[0022] 2. The strain is isolated from disease-resistant varieties, which is more conducive to its application in the field.

[0023] 3. The strain has a short fermentation cycle, low cost, low dosage of bacterial agents, and is easy to mass produce and commercialize.

[0024] The Cronobacter sp. C1CGMCC No. 24172 and Cronobacter sp. C7 CGMCC No. 24173 provided by the present invention are a Cronobacter combination isolated by the inventor from the roots of the disease-resistant cucumber variety Zhongnong 106 in April 2021, which has antagonistic effects on multiple pathogenic fungi.

[0025] The characteristics of these two strains are: strains C1 and C7 are yellow and have wrinkles on LB medium (tryptone 10g, NaCl 10g, yeast extract 5g, agar 18g, distilled water 1L, pH 7.0); on 1 / 2 PDA medium (yeast extract 4g, malt extract 10g, anhydrous glucose 4g, agar 18g, distilled water 1L, pH 7.3), the colonies are milky white and rich. After identification, the two strains were Cronobacter sp., named C1 and C7 respectively. This bacterial combination can migrate with the hyphae of the wilt pathogen and colonize in the roots of cucumbers, thereby reducing the incidence and severity of wilt disease. Its concentration is 1×10 6 CFU / g, the disease prevention effect can reach 57.0%. When C1 and C7 strains are fermented, they grow fast and can be fermented within 20 hours. After the fermentation, the number of live bacteria can reach 4-6 billion / mL. The concentration used is 1×10 6 CFU / g is much lower than other bacterial agents, which greatly reduces the cost of use. Therefore, C1 and C7 are a very potential bacterial combination, and thus the present invention is proposed.

[0026] Collection Instructions

[0027] Bacterial species name: Cronobacter

[0028] Latin name: Cronobacter sp.

[0029] Strain ID: C1

[0030] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0031] Abbreviation of depository institution: CGMCC

[0032] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0033] Date of deposit: December 21, 2021

[0034] CGMCC Registration Number: CGMCC No.24172

[0035] Bacterial species name: Cronobacter

[0036] Latin name: Cronobacter sp.

[0037] Strain ID: C7

[0038] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0039] Abbreviation of depository institution: CGMCC

[0040] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0041] Date of deposit: December 21, 2021

[0042] CGMCC Registration Number: CGMCC No.24173 DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] Example 1. Isolation and screening of Cronobacter sp.

[0046] (1) Isolation of Cronobacter C1 and Cronobacter C7 strains

[0047] The Cronobacter sp. C1 and Cronobacter sp. C7 of the present invention are obtained by separation from cucumber roots, as follows:

[0048] First, prepare a WA-N culture medium plate, and set the inoculation position of the wilt pathogen cake 1 cm away from the edge of the culture dish. Inoculate an 8mm wide bacterial inoculation strip 2 cm away from the inoculation position of the wilt pathogen cake. Bacterial inoculation method: Use a cell scraper to scrape 10μL of bacterial suspension into a 2cm wide bacterial strip. Each bacterial suspension stock solution and 10-fold dilution are repeated 3 times. Use a culture dish that is not inoculated with bacterial suspension and only inoculated with wilt pathogens as a control. Culture at 26℃ for 3 days. After the bacteria grow well, inoculate the wilt pathogen cake with a diameter of 5mm on the culture medium. The culture dish is placed at 20℃ for 15 days, and the growth of pathogenic fungi hyphae is observed. Single colonies that have an inhibitory effect on wilt pathogen hyphae are isolated and purified on 1 / 10 TSA culture medium.

[0049] Among them, the components and proportions of the WA-N medium are: NaCl 5g, KH2PO4 1g, (NH4)2SO4 0.1g, agar powder 18g, deionized water 1L, pH 6.7. The components and proportions of the 1 / 10TSA medium are: trypsin 1.5g, yeast extract 0.05g, KH2PO4 0.05g, NaCl 2.5g, agar 18.0g, deionized water 1L, pH 7.0.

[0050] Results: The above method was used to isolate and pick bacteria, and 37 pure bacterial strains were obtained by streaking.

[0051] (2) Inhibitory effects of Cronobacter C1 and C7 on cucumber wilt pathogen

[0052] The antagonistic effect of the isolated bacterial strains on Fusarium wilt was determined by the confrontation culture method. Four 1 cm bacterial lines were streaked and inoculated at 1 cm from the edge of the 1 / 2 PDA culture medium plate. After culturing at 26°C for 2 days, a fresh Fusarium wilt cake was punched out with a 5 mm diameter puncher and transferred to the center of the 1 / 2 PDA plate. The culture was continued for 5-7 days. The inhibitory effect of bacteria on the growth of pathogen hyphae was observed and the radius of the pathogen colony was measured. The results were repeated 3 times, and the plate without bacteria was used as the control. The inhibition rate was expressed as (R1-R2) / R1, where R1 is the colony radius of the normal growth of the pathogen (mm), and R2 is the radius from the edge of the inhibition zone to the center of the pathogen colony (mm).

[0053] The components and proportions of the 1 / 2 PDA culture medium are: 100 g potato, 10 g glucose, 18 g agar powder, and 1 L deionized water.

[0054] Results: The confrontation culture method was used to determine the antagonistic activity of the isolated strains against Fusarium wilt, and Cronobacter C1 and C7 were obtained. These two strains not only had obvious antibacterial effects, with inhibition rates of 54.58±1.82% and 55.71±2.84%, respectively, but also changed the colony morphology and color of Fusarium wilt. The edge of the colony became petal-shaped and the colony turned red, indicating that its mechanism of action was different from that of other bacteria.

[0055] (3) Inhibitory effects of Cronobacter strains C1 and C7 on various pathogenic fungi

[0056] The inhibitory effects of strains C1 and C7 on various pathogenic fungi were determined according to the method in Example 1 (2). The results (Table 1) show that strains C1 and C7 have a certain degree of inhibitory effect on Fusarium solani, Phytophthora capsici, Fusarium wilt of cabbage, Colletotrichum gloeosporioides, and Botrytis cinerea. The inhibitory rate of C1 on Fusarium solani reached 61.34%, and that of C7 reached 46.02%, followed by Colletotrichum gloeosporioides and Fusarium wilt of cabbage, with an inhibitory rate of more than 50.0%, and the inhibitory rate on Phytophthora capsici and Botrytis cinerea was slightly weaker. Strain C7 had a significant inhibitory effect on Colletotrichum gloeosporioides, Fusarium wilt of cabbage, and Botrytis cinerea, reaching more than 50.0%.

[0057] Table 1 Inhibitory effects of Cronobacter C1 and C7 on plant pathogenic fungi

[0058]

[0059] Example 2, Identification of Cronobacter C1 and C7 strains

[0060] The genomic DNA of strains C1 and C7 were extracted, and the 16S rRNA gene sequences were amplified with universal primers 27F and 1492R and sequenced.

[0061] 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′; 1492R: 5′-TAC GGC TAC CTT GTT ACGACT T-3′. The 16S rRNA gene sequences of strains C1 and C7 have been submitted to GenBank, with accession numbers ON248529 and ON248530, respectively. In view of the above molecular biological characteristics identification results, strains C1 and C7 isolated and purified in Example 1 (1) were identified as Cronobacter sp. These two strains of Cronobacter sp. were deposited in the General Microbiology Center of China National Microbiological Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 21, 2021. The deposit numbers of strains C1 and C7 are CGMCC No. 24172 and CGMCC No. 24173, respectively.

[0062] Example 3: Inhibitory effects of Cronobacter C1 and C7 metabolites on Fusarium wilt

[0063] Sterile cellophane was spread on 1 / 2 PDA medium (100g potato, 10g glucose, 18g agar powder, 1L deionized water), strains C1 and C7 were streaked on the cellophane, and cultured at 28℃ for 2d and 5d, respectively. One group removed the cellophane and inoculated a 5mm diameter Fusarium wilt fungus cake in the center of the culture medium, and the other group inoculated a 5mm Fusarium wilt fungus cake directly into the center of the culture medium covered with cellophane. Only Fusarium wilt was inoculated as a control, and each treatment was repeated 3 times. All plates were placed in a 28℃ incubator, and fungal growth was observed after continuing to culture for 5d.

[0064] The results (Table 2) showed that when strains C1 and C7 were cultured for 2 days, there was no significant difference in the inhibitory effect of the group with cellophane removed and the group with cellophane on Fusarium wilt, and the inhibitory effect of the metabolites of the two strains on 2 days was higher than that on 5 days; when strains C1 and C7 were cultured for 5 days, the inhibitory effect of the group with cellophane removed on Fusarium wilt was slightly lower than that of the group with cellophane. This result shows that the metabolites produced by strains C1 and C7 have important antibacterial effects.

[0065] Table 2 Inhibitory effects of live strains C1 and C7 and their metabolites on Fusarium wilt

[0066]

[0067] Note: Different lowercase letters in the same column indicate significant differences among different treatments (p < 0.05)

[0068] Example 4: Cronobacter C1 and C7 migrate along with the growth of Fusarium wilt hyphae

[0069] Place a sterilized ordinary glass slide in a 9 cm culture dish, and slowly pour WA-N culture medium into the culture dish until the glass slide is covered by 1-2 mm. Then apply 8 mm C1 and C7 bacterial strips (concentration of 5×10 6 CFU / mL), and cultured at 28℃ for 2 days. After the bacteria grew well, the wilt bacteria cake with a diameter of 5mm was inoculated on the culture medium and cultured together at 20℃. When the hyphae grew to the other side of the slide, the hyphae that had passed through the bacteria band was cut with a sterile scalpel, and the migration of C1 and C7 with the hyphae of the wilt bacteria was observed under a microscope. The treatment without bacteria was used as a control.

[0070] Under the microscope, it was observed that C1 and C7 were able to migrate with the hyphae of the wilt pathogen, and most of the bacterial bodies gathered near the hyphae; within the 10 observed fields of view, the number of spores produced by the wilt pathogen was significantly reduced compared with the control, which was reduced by about 10 times; the migration distance of bacteria C1 and C7 with the fungal hyphae (i.e., the distance from the right side of the bacterial belt) was 19.7±1.5mm and 18.3±1.2mm.

[0071] Example 5: Disease prevention effect of the combination of Cronobacter C1 and C7 on cucumber wilt

[0072] Preparation of bacterial suspension: Inoculate bacteria C1 and C7 into LB liquid medium (tryptone 10 g, NaCl 10 g, yeast extract 5 g, distilled water 1 L, pH 7.0), culture at 180 r / min, 28 °C for 14 h, centrifuge at 12000 r / min for 2 min, remove the supernatant, resuspend in phosphate buffer, spread on LB solid medium for counting, and the concentration of bacterial stock solution reaches 4 × 10 9 ~6×10 9 CFU / mL, for future use.

[0073] According to the ratio of 1mL:4Kg to 1mL:6Kg, C1 and C7 bacterial solutions (V:V=1:1) were adsorbed onto the seedling matrix to make the bacterial load in the seedling matrix 1×10 6 CFU / g. First, fill the flower pot (diameter 8.8cm×height 9.6cm) with sterilized (121℃, 1h) substrate, 90g / pot; then fill with seedling substrate that has been adsorbed with C1 and C7 bacterial solution, 90g / pot. The humidity of the seedling substrate is controlled at about 50%. Dig a trench 1.5cm away from the left and right sides of the flower pot to plant the seedlings, and sprinkle 2g of 1×10 4 CFU / g of substrate, sow the germinated seeds with a hypocotyl of 1 cm into the furrow, and use a substrate with a bacterial load of 1×10 4 CFU / g, and a substrate with a water content of 50%. The experiment was divided into 2 treatments (Table 2), with 6 seedlings per pot and 5 replicates per treatment. The substrate treated with the same amount of sterile water was sprinkled at the control furrow. One week after the emergence of the seedlings, when the cotyledons were fully expanded, a 5mm wilt pathogen cake was inoculated 1cm away from each seedling. After the inoculation, a transparent breathable cover was covered on the top of the flowerpot to keep the flowerpot moisturized and sterile. The transparent cover was uncovered after 5 days. Water was replenished in time during the seedling stage to keep the substrate moisture content at 52%-55%. The disease situation was investigated after 4 weeks.

[0074] The results of the potted plant experiment (Table 2) show that the combination of bacteria C1 and C7 has a good disease prevention effect on cucumber wilt. The incidence rate of the susceptible variety ZN6 inoculated with cucumber wilt bacteria was 56.67%, and the disease index was 25.0; after adding the combination of C1 and C7 bacterial solution to the substrate, the incidence rate and disease index decreased significantly, with a significant difference from the control, and the prevention effect could reach 57.0%.

[0075] Table 2 Disease prevention effect of Cronobacter bacteria combination on cucumber wilt

[0076]

[0077] Note: The letters after the data indicate significant differences, p≤0.05.

Claims

1. Cronobacter sp. C1 and Cronobacter sp. C7 are deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit numbers CGMCC No. 24172 and CGMCC No. 24173 respectively.

2. A bacterial agent, characterized in that The active ingredients of the bacterial agent are Cronobacter sp. C1 and Cronobacter sp. C7 as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent, and the number of live bacteria of Cronobacter sp. C1 and Cronobacter sp. C7 in the liquid bacterial agent is 4 to 6 billion / mL.

4. The bacterial agent according to claim 2, characterized in that: The bacterial agent is a solid bacterial agent, which is a mixture of the fermentation liquid of Cronobacter sp. C1 and Cronobacter sp. C7 and an adsorption carrier in a ratio of 1 mL: 4 kg to 1 mL: 6 kg, or a mixture of the bacterial sludge after centrifugation of the fermentation liquid and the adsorption carrier in a ratio of 1 L: 4 kg to 1 L: 6 kg. The adsorption carrier can be one or more of a seedling substrate, diatomaceous earth, peat, vermiculite, rice husk powder, straw powder, perlite and calcium carbonate. The method for preparing the bacterial agent according to claim 4 comprises the following steps: (1) culturing Cronobacter sp. C1 and Cronobacter sp. C7 according to claim 1 to obtain fermentation broth of Cronobacter sp. C1 and C7; (2) The fermentation liquid obtained in step (1) and the adsorption carrier are mixed in a ratio of 1 mL: 4 kg to 1 mL: 6 kg, or the bacterial sludge obtained after centrifugation of the fermentation liquid obtained in step (1) and the adsorption carrier are mixed in a ratio of 1 L: 4 kg to 1 L: 6 kg to obtain the bacterial agent.

5. The method for preparing the bacterial agent according to claim 3, characterized in that: The culture medium solvent for culturing Cronobacter sp. C1 and Cronobacter sp. C7 according to claim 1 in the step (1) is water, and the culture medium solutes and concentrations are as follows: beef extract 0.8-1.0%, yeast powder 0.3-0.5%, glucose 0.5-0.7%, peptone 1.0-1.2%, sodium chloride 0.03-0.05%; wherein % represents the mass percentage.

6. Use of Cronobacter sp. C1 and Cronobacter sp. C7 according to claim 1, the bacterial agent according to any one of claims 2 to 4, and the bacterial agent prepared by the method of claim 5 in plant disease control.

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