Application of an endophytic Bacillus subtilis strain from Malus xifuensis and its metabolically active substances in the control of tomato black spot disease

Abstract

The present invention relates to an endophytic Bacillus subtilis (Bacillus sp.) A1 strain of Malus xifuensis and its metabolically active substances in the prevention and treatment of tomato black spot disease. The strain is deposited in the China Center for Type Culture Collection, with a preservation date of May 20, 2024, and a preservation number of CCTCC NO: M 20241010. This study found that the volatile substances produced by the A1 strain, including 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanone and 2-nonanol, can effectively inhibit the growth of tomato Alternaria mycelium, reduce the infection of tomato Alternaria to tomato fruits, and can be used to prevent and treat tomato black spot disease. Provides a new beneficial bacterial strain resource for the biological control of tomato black spot disease.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of an endophytic Bacillus sp. A1 strain of Malus fujianensis and metabolically active substances thereof in preventing and controlling tomato black spot disease. Background Art

[0002] Tomato black spot is one of the common fungal diseases in the planting and storage stages of tomatoes. The disease is caused by Alternaria alternaria of the Ascomycetes, and mainly harms tomato fruits, leaves and stems. The lesions on infected tomato fruits are brown or black, round to irregular in shape, slightly concave in the middle, and have obvious edges. The diseased fruit has one or several lesions of varying sizes, which can be connected into large lesions, on which velvety black hyphae and conidia grow. In the later stage, the diseased fruit will rot, seriously reducing the quality and commercial value of tomatoes. In addition, toxins produced by Alternaria infection of tomatoes, such as alternaria phenol (AOH), alternaria monomethyl ether (AME) and tenuisporic acid (TeA), have been detected in tomatoes and tomato products, endangering food safety. Integrated control technology based on chemical control is currently the main measure to control the damage of crops by Alternaria. However, the large-scale use of chemical pesticides has caused environmental pollution, affected water quality, and endangered the safety of humans and animals. Therefore, it is urgent to find new and effective green prevention and control strategies for tomato black spot.

[0003] The research on using Bacillus subtilis as a biocontrol bacterium has a long history. It is used to control powdery mildew, frost and dew disease, blight, gray mold and other diseases of various crops. Commercial preparations that have been successfully developed and put into production in my country include Baikang, Maifengning, Yabao, etc. The biocontrol mechanism of Bacillus subtilis is mainly competition and production of antibiotic antagonism. Bacillus subtilis has the same ecological site as some plant pathogens, and can play a biocontrol role by competing for ecological sites; at the same time, Bacillus subtilis can produce a variety of metabolites with antibacterial activity, such as subtilisin and iturin, which can inhibit or kill pathogens.

[0004] Bacillus subtilis has been disclosed to be used for preventing and controlling tomato leaf mold, gray mold (CN115011505A), and reports on other plant black spot diseases, such as cabbage black spot (CN108048357B) and pear black spot (CN112522127A). Although the black spot pathogens of other plants also belong to the genus Alternaria sp., the pathogens of black spot diseases of different plants are different, and the biocontrol mechanisms are also very different. There is no report that Bacillus subtilis and its metabolites can inhibit tomato Alternaria sp. and prevent and control tomato black spot. Summary of the invention

[0005] The technical problem to be solved by the present invention is the above-mentioned problem of insufficient prevention and control technology for infection of tomato fruit by Alternaria alternaria. An endophytic Bacillus subtilis (B. subtilis) A1 strain of Malus xifuensis is provided, and the volatile compounds produced by its metabolism can significantly inhibit the growth of Alternaria alternaria hyphae and effectively reduce the infection of tomato fruit by Alternaria alternaria.

[0006] The first object of the present invention is to provide a Bacillus subtilis A1 strain, characterized in that the strain was deposited in the China Center for Type Culture Collection on May 20, 2024, with a preservation number of CCTCCNO: M20241010.

[0007] The second object of the present invention is to provide a biocontrol agent containing the above-mentioned Bacillus subtilis A1 strain or the volatile substances produced by the A1 strain.

[0008] The third object of the present invention is to provide a biocontrol agent containing the volatile substances produced by the above-mentioned Bacillus subtilis A1 strain.

[0009] The fourth object of the present invention is to provide a use of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in inhibiting tomato Alternaria spp., or in preparing a product for inhibiting tomato Alternaria spp.

[0010] A fifth object of the present invention is to provide an application of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in preventing and controlling diseases caused by tomato Alternaria spp., or in preparing a product for preventing and controlling diseases caused by tomato Alternaria spp.

[0011] The sixth object of the present invention is to provide an application of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in preventing and controlling tomato black spot disease, or in the preparation of a product for preventing and controlling tomato black spot disease.

[0012] A seventh object of the present invention is to provide a drug for preventing and treating diseases caused by tomato Alternaria, wherein the drug contains Bacillus subtilis A1 strain and / or volatile substances produced by Bacillus subtilis A1 strain as active ingredients.

[0013] The eighth object of the present invention is to provide a tomato preservative containing volatile substances produced by Bacillus subtilis A1 strain.

[0014] The ninth object of the present invention is to provide a method for preventing and controlling diseases caused by tomato Alternaria.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The applicant team obtained an endophytic bacterium from the healthy leaves of Malus chinensis in the Quanshan Campus of Huainan Normal University through artificial separation and purification. The volatile compounds produced by the endophytic bacteria can significantly inhibit the growth of tomato Alternaria mycelium and significantly reduce the infection of tomato Alternaria to tomato fruits. Therefore, the present invention claims the following applications:

[0017] A Bacillus sp. A1 strain, which was deposited in the China Center for Type Culture Collection on May 20, 2024, with the accession number CCTCC NO: M20241010, classified and named Bacillus sp. A1, and the deposit address is: Wuhan University, Wuhan, China.

[0018] A biocontrol agent contains the above-mentioned Bacillus subtilis A1 strain or the volatile substance produced by the A1 strain.

[0019] A biocontrol preparation contains the volatile substances produced by the above-mentioned Bacillus subtilis A1 strain.

[0020] Disclosed is an application of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in inhibiting tomato Alternaria alternaria, or in preparing a product for inhibiting tomato Alternaria alternaria.

[0021] A use of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in preventing and controlling diseases caused by tomato alternaria, or in preparing a product for preventing and controlling diseases caused by tomato alternaria.

[0022] A use of a Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation in preventing and controlling tomato black spot disease, or in preparing a product for preventing and controlling tomato black spot disease.

[0023] A drug for preventing and treating diseases caused by tomato alternaria, wherein the drug takes Bacillus subtilis A1 strain and / or volatile substances produced by the Bacillus subtilis A1 strain as active ingredients.

[0024] A tomato preservative containing volatile substances produced by Bacillus subtilis A1 strain.

[0025] Specifically, the volatile substances produced by the Bacillus subtilis A1 strain contain one or more of 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanol, and 2-nonanone.

[0026] A method for preventing and controlling diseases caused by tomato alternaria.

[0027] Specifically, the Bacillus subtilis A1 strain or the above-mentioned biocontrol agent or the above-mentioned biocontrol preparation or the above-mentioned medicine is used to prevent and control the disease.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] The present invention isolates a Bacillus A1 strain from healthy leaves of Malus chinensis, and the volatile substances produced by the strain can inhibit the growth of tomato Alternaria mycelium and inhibit the infection of tomato Alternaria to tomato fruits. The antibacterial effects of 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanone and 2-nonanol in the volatile substances were further tested, among which 5 μL of 2-nonanone can reach 100%; 10 μL of 3-methylbutyric acid and 2-methylbutyric acid can also reach 100%. The application effects of 3-methylbutyric acid and 2-methylbutyric acid in the prevention and control of tomato black spot disease were tested separately, and it was found that both of them can significantly inhibit the infection of tomato Alternaria to the surface of tomato fruit, and have a significant prevention and control effect on the tomato black spot disease caused by them. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This graph shows the inhibitory effect of volatile compounds produced by strain A1 on the growth of tomato Alternaria mycelium.

[0031] Figure 2 This is the colony morphology of strain A1 on LB medium.

[0032] Figure 3 This is the clustering diagram of A1 strain based on 16S rDNA sequence.

[0033] Figure 4 This graph shows the effect of volatile antibacterial substances of strain A1 on the infection of tomato black spot pathogenic fungus on tomato fruit.

[0034] Figure 5 This is a graph showing the effects of four volatile organic compounds produced by the A1 strain on the mycelial growth of tomato black spot pathogen. 3-methyl-Butanoic acid is 3-methylbutanoic acid, 2-methyl-Butanoic acid is 2-methylbutanoic acid, 2-Nonanol is 2-nonanol, and 2-Nonanone is 2-nonanone.

[0035] Figure 6 This is a graph showing the control effect of 3-methylbutyric acid and 2-methylbutyric acid on tomato black spot disease. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Microorganisms used in the following examples:

[0039] Alternaria alternaria, laboratory collection

[0040] 3-Methylbutyric acid, Sigma, analytical grade

[0041] 2-Methylbutyric acid, Sigma, analytical grade

[0042] 2-Nonanol, Sigma, analytical grade

[0043] 2-Nonanone, Sigma, analytical grade

[0044] Example 1 Isolation and screening of biocontrol bacteria strains

[0045] 1. Experimental methods

[0046] (1) Collection of healthy Malus melongena leaves: Healthy Malus melongena leaves were collected on August 20, 2022, at the Quanshan Campus of Huainan Normal University, and were bagged and taken back to the laboratory for isolation of endogenous bacteria.

[0047] (2) Isolation of endophytic bacteria: Take the above healthy Malus chinensis leaves, rinse the leaf surface with running water, cut into appropriate small pieces with sterilized scissors, soak in 75% ethanol for 3 minutes, soak in 2% sodium hypochlorite for 5 minutes, soak in sterile water for 6 minutes, and then soak and rinse with sterile water for 6 times, and use the last sterile water to apply the plate as a control. Use sterile filter paper to absorb the surface moisture of the above treated leaf tissue block, put it into a sterile mortar and grind it, add 1mL sterile water to mix, take 100μL and apply it on the LB plate, culture at 28℃, pick a single colony and shake it in a 1.5mL centrifuge tube for culture, and obtain the biocontrol liquid to be tested.

[0048] (3) Biocontrol strain screening:

[0049] The double-plate interlocking method was used to screen for potential biocontrol strains that have antagonistic effects on tomato Alternaria. A tomato Alternaria block with a diameter of 5 mm was inoculated in the center of the PCA solid plate. 100uL of the biocontrol solution obtained in step (2) above was aspirated onto the LB solid plate, and the solution was evenly spread with a coating rod. The PCA plate and LB plate inoculated with the bacteria were uncapped and docked (PCA culture dish on top, LB culture dish on the bottom), the docking was sealed with a sealing film, and the plates were placed in a 28°C constant temperature incubator for light shielding (VOCs group). At the same time, a double-plate interlocking experiment was performed with an LB blank plate that was not inoculated with the biocontrol solution and a PCA plate inoculated with the pathogen as a control (CK group).

[0050] After 7 days of cultivation, the colony diameter of Alternaria alternata was measured by the cross method, and the inhibition rate was calculated.

[0051] 2. Experimental results

[0052] The endophytic bacterial strain numbered A1 was screened out (such as Figure 1 The volatile compounds produced by the extract have a significant inhibitory effect on the growth of tomato Alternaria mycelium, with an average inhibition rate of 68.8%.

[0053] 3. Conclusion

[0054] By isolating and culturing endophytic bacteria in healthy Malus fragrans leaves, and then using the double-plate method to screen out potential biocontrol strains with antagonistic effects on tomato Alternaria alternata, it was found that the volatile compounds produced by the endophytic bacterial strain A1 in the leaves of Malus fragrans had a significant antagonistic effect on the pathogen of tomato black spot disease (Alternaria alternata).

[0055] Example 2 Identification of biocontrol bacteria strain A1

[0056] 1. Morphological identification

[0057] A single colony of the A1 strain obtained in Example 1 was picked up with an inoculation loop, streaked onto LB medium, and cultured at 28°C for 24 hours to observe the growth of the colony. The bacteria were Gram-positive, and the colonies on the LB medium were off-white, convex in the middle, opaque, and with irregular edges (such as Figure 2 In liquid LB medium, it is diffusely turbid.

[0058] 2. Molecular identification and evolutionary analysis

[0059] 16S rDNA sequence analysis is a molecular biological identification method for bacterial identification. The present invention analyzes the evolutionary relationship of A1 strain by analyzing the 16S rDNA sequence. The results are as follows: Figure 3 As shown, strain A1 has the highest similarity with Bacillus subtilis. In the 16S rDNA gene sequence evolution tree, strain A1 and B. subtilis strain MANA18 (LN999780.1) are clustered into one branch with a support rate of 96.

[0060] 3. Conclusion

[0061] The biocontrol bacteria strain A1 was identified as Bacillus subtilis (B. subtilis) by morphological and 16S rDNA gene phylogenetic analysis. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on May 20, 2024, with the strain deposit number of CCTCC NO: M 20241010, and the classification name was Bacillus sp.A1. The deposit address is Wuhan University, Wuhan, China.

[0062] Example 3 Effect of volatile antibacterial substances produced by strain A1 on infection of tomato fruits by Alternaria alternata

[0063] 1. Experimental methods

[0064] Take 100uL of the bacterial solution of the A1 strain cultured overnight in LB liquid medium, and spread it evenly on the LB solid plate using a spreading rod.

[0065] After the healthy tomatoes of the same size were washed with clean water, they were washed twice with sterile water, and then the surface was disinfected with 75% ethanol solution. After the ethanol on the surface of the fruit evaporated, a tomato Alternaria cake with a diameter of 5 mm was placed on the surface of the tomato and placed in a 1.7L fresh-keeping box. At the same time, an LB plate (without the cover) coated with A1 bacterial solution was placed on the edge of the fresh-keeping box (VOCs group); a blank LB plate with the cover removed was used as the control (CK group). There were 3 fruits in each treatment group, and the experiment was repeated 3 times. The tomato lesion area was observed and measured after being placed at 28°C for 5 days.

[0066] 2. Experimental results

[0067] The results of the effects of volatile antibacterial substances of strain A1 on the infection of tomato black spot pathogens on tomato fruits are shown in Figure 4 As shown in the figure, the tomato surface of the CK group was infected with tomato Alternaria over a large area, while the VOCs group had only a small area of ​​wounds and no tomato Alternaria hyphae grew; the lesion area of ​​tomatoes in the VOCs group was significantly lower than that in the CK group.

[0068] 3. Conclusion

[0069] By measuring the effects of volatile compounds produced by the A1 strain on the infection of tomato fruit by Alternaria alternata, it was found that the A1 strain can produce volatile substances that can inhibit the growth of Alternaria alternata. These volatile compounds can significantly reduce the infection of tomato fruit by Alternaria alternata, thereby inhibiting the development of tomato black spot fungus.

[0070] Example 4 Production, collection and identification of volatile antibacterial substances of strain A1

[0071] 1. Production and collection of volatile antibacterial substances of strain A1:

[0072] The volatile organic compounds (VOCs) produced by Bacillus subtilis A1 strain were collected by headspace solid phase microextraction gas chromatography (SPME-GC-MS). A single colony of the A1 strain was picked into a headspace injection bottle containing 3 mL of LB solid medium and cultured at 28°C for 48 hours. The LB solid medium without inoculation under the same conditions was used as a control. The extraction head was 50 / 30μm DVB / CARon PDMS, and the A1 strain sample was subjected to headspace adsorption for 30 minutes at 50°C. After the extraction reached equilibrium under these conditions, it was immediately injected into the gas chromatography inlet, heated to 260°C, and then VOCs analysis and chromatographic analysis were performed.

[0073] 2. Determination of volatile antibacterial substances produced by strain A1:

[0074] VOCs analysis was performed using a CTC trinity autosampler. VOCs were separated in a DB-wax column (30m×0.25mm, 0.25μm). Start at 40°C and hold for 5min, then increase the oven temperature to 220°C at 5°C / min, and then to 250°C at 20°C / min and hold for 5min. The carrier gas was helium, and the inlet temperature was maintained at 260°C. The mass spectrometer was operated in electron ionization mode at 70eV, the ion source temperature was fixed at 230°C, the quadrupole temperature was 150°C, and continuous scanning from 20m / z to 400m / z was used. Volatile organic compounds (VOCs) were identified by comparing the mass spectrum and retention time with the mass spectrum of authentic reference compounds and the NIST2017 library. The relative content of each component was calculated by peak area normalization.

[0075] It was found that Bacillus subtilis A1 strain produced at least 40 volatile substances, and the specific information is shown in Table 1.

[0076] Table 1. Identification results of volatile organic compounds of strain A1

[0077]

[0078]

[0079] Example 5 Determination of the antibacterial activity of volatile substances produced by strain A1 against Alternaria alternata

[0080] 1. Experimental methods

[0081] Take out the tomato Alternaria strain from the 4°C refrigerator for activation and wait until it fills the plate for later use.

[0082] 5 μL and 10 μL of single pure 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanol and 2-nonanone were respectively dripped into the center of one side of the partitioned culture dish, and the treatment with the same amount of sterile water was used as the control; 7.5 mL of PCA medium was poured into the other side, and a 5 mm tomato Alternaria cake was inoculated in the center of the PCA medium, sealed with a sealing film, and cultured in a constant temperature incubator at 28°C for 5 days. Each treatment was repeated 3 times, and the colony diameters of tomato Alternaria in the treatment group and the control group plates were recorded, and the inhibition rate of volatile compounds on the growth of tomato Alternaria hyphae was calculated.

[0083] 2. Experimental results

[0084] The inhibitory effects of different volatile compounds produced by strain A1 on the mycelial growth of Alternaria alternata Figure 5 The study found that different concentrations of 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanol and 2-nonanone had an inhibitory effect on the growth of tomato Alternaria on PCA plates, and the colony diameters were all lower than those of the control group ( Figure 5 The petri dish in the figure shows the 5 μL treatment group of each compound). Among them, the antibacterial rates of the 5 μL group were: 3-methylbutyric acid 64.98%, 2-methylbutyric acid 55.48%, 2-nonanol 26.3%, 2-nonanone 100%; the antibacterial rates of the 10 μL group were: 3-methylbutyric acid 100%, 2-methylbutyric acid 100%, 2-nonanol 66.5%, 2-nonanone 100%.

[0085] 3. Conclusion

[0086] The antibacterial activity of the four volatile organic compounds produced by the above-mentioned Bacillus subtilis A1 strain against tomato Alternaria was determined through a separated culture dish experiment. It was found that 3-methylbutyric acid, 2-methylbutyric acid, 2-nonanol and 2-nonanone all had inhibitory effects on the growth of tomato Alternaria on the PCA plate, especially 2-nonanone, whose inhibition rate could reach 100% at a dosage of 5 μL; followed by 3-methylbutyric acid and 2-methylbutyric acid, whose inhibition rates could also reach 100% at a dosage of 10 μL.

[0087] Example 6 Application of volatile antibacterial substances of strain A1 in the prevention and treatment of tomato black spot disease

[0088] Taking two volatile substances with antibacterial activity, 3-methylbutyric acid and 2-methylbutyric acid, as examples, the protective effect of volatile antibacterial substances of strain A1 against tomato black spot disease was tested.

[0089] 1. Experimental methods

[0090] After the healthy tomatoes of the same size were washed with clean water, they were washed twice with sterile water, and then the surface was disinfected with 75% ethanol solution. After the ethanol on the surface of the fruit evaporated, a 5mm diameter tomato Alternaria cake was placed on the surface and placed in a 1.7L fresh-keeping box. At the same time, a 1.5mL centrifuge tube cap with 5μL and 10μL of single pure 3-methylbutyric acid and 2-methylbutyric acid was placed on the edge of the fresh-keeping box, and an equal amount of sterile water was added to the centrifuge tube cap as a control. There were 3 fruits in each treatment group, and the experiment was repeated 3 times. The results were observed after 5 days at 28℃.

[0091] 2. Experimental results

[0092] The two volatile compounds (3-methylbutyric acid and 2-methylbutyric acid) produced by strain A1 with strong antibacterial activity against tomato black spot disease are shown in Figure 2. Figure 6 As shown, the tomatoes in the control group showed that a large area of ​​Alternaria mycelium had grown, while the tomatoes in the treatment group showed no obvious signs of infection.

[0093] 3. Conclusion

[0094] The volatile compounds produced by Bacillus subtilis A1 strain can significantly inhibit the infection of tomato Alternaria alternata on the tomato surface, and have a significant preventive and control effect on tomato black spot disease caused by it.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Bacillus sp. A1 strain, characterized in that: The strain was deposited in the China Center for Type Culture Collection on May 20, 2024, with the deposit number CCTCC NO:M20241010.

2. A biocontrol agent, characterized in that: Contains the Bacillus subtilis A1 strain according to claim 1.

3. Use of the Bacillus subtilis A1 strain according to claim 1 or the bacterial agent according to claim 2 in inhibiting Alternaria alternata, or in preparing a product for inhibiting Alternaria alternata.

4. Use of the Bacillus subtilis A1 strain according to claim 1 and the bacterial agent according to claim 2 in preventing and controlling diseases caused by Alternaria alternata in tomatoes, or in preparing products for preventing and controlling diseases caused by Alternaria alternata in tomatoes.

5. Use of the Bacillus subtilis A1 strain according to claim 1 or the bacterial agent according to claim 2 in preventing and treating tomato black spot disease, or in preparing a product for preventing and treating tomato black spot disease.

6. A drug for preventing and treating diseases caused by tomato Alternaria alternata, characterized in that: The medicine uses the Bacillus subtilis A1 strain described in claim 1 as an active ingredient.

7. A tomato preservative, characterized in that: Contains the Bacillus subtilis A1 strain according to claim 1.

8. A method for preventing and controlling diseases caused by tomato Alternaria alternata, characterized in that: The Bacillus subtilis A1 strain according to claim 1, the bacterial agent according to claim 2, or the medicine according to claim 6 is used to prevent and control diseases.

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