Bacillus tequilensis P61 and application of bacillus tequilensis P61 in prevention and treatment of kiwi fruit soft rot

Abstract

The invention discloses bacillus tequilensis P61 and application thereof in prevention and treatment of kiwi fruit soft rot, and belongs to the field of biological prevention and treatment. The bacillus tequilensis P61 provided by the invention is preserved in the China Center for Type Culture Collection on September 5, 2024, and the preservation number of the bacillus tequilensis P61 is CCTCC (China Center for Type Culture Collection) NO: M 20241916. According to the invention, the lipopeptide antibacterial substance is prepared according to the bacillus tequilensis P61. Experimental results show that the bacillus tequilensis P61 and lipopeptide antibacterial substances have an outstanding prevention and treatment effect on the soft rot of the kiwi fruits, have a remarkable bacteriostasis effect on pathogenic bacteria of the soft rot of the kiwi fruits such as botryosphaeria dothidea, alternaria alternata and phomopsis, remarkably reduce the damage rate of the kiwi fruits, are green and safe, are harmless to people and livestock, and are worthy of popularization and application. No pollution is caused to the environment. The invention provides a new biocontrol strain for biological prevention and control of kiwi fruit diseases, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biological control, and particularly to a strain of Bacillus tequilensis P61 and its application in controlling kiwifruit soft rot disease. Background Art

[0002] Kiwifruit (Actinidia chinensis Planch.) belongs to the genus Actinidia of the family Actinidiaceae, and is a vine fruit tree native to China with rich wild resources and wide geographical distribution. During the postharvest period, kiwifruit is vulnerable to infection by various pathogenic fungi, including fungal diseases such as fruit soft rot, gray mold, and blue mold, which seriously endanger the quality of kiwifruit. Among them, soft rot disease commonly occurs during the postharvest ripening period of kiwifruit. The symptoms of the disease mainly include depression of the fruit epidermis, soft rot of the internal pulp, and spreading in a conical shape towards the center of the fruit. The diseased part of the pulp is hollow and spongy, with a yellowish-brown center and a dark green water-soaked area around it. When severe, the fruit completely rots, emitting an alcohol smell, greatly affecting the quality of kiwifruit, losing its edible value, affecting storage and transportation, and causing huge economic losses. It has been reported at home and abroad that the main pathogens causing kiwifruit soft rot disease are Botryosphaeria dothidea, Phomopsis lithocarpus, Alternaria alternata, etc.

[0003] The kiwifruit soft rot disease develops rapidly and is difficult to control. At present, chemical control is still the main control method, but the overuse of pesticides will bring problems such as environmental pollution and insecurity to humans and livestock. Biological control has the advantages of no environmental pollution, no residue, and harmlessness to natural enemies. Applying it to control fruit tree diseases has increasingly become a research hotspot among scholars. Among them, Bacillus is one of the most widely used biological control microbial strain resources in the world. For example, Bacillus subtilis, Bacillus velezensis, Bacillus megaterlum, etc. have all been reported to be used to control postharvest diseases of kiwifruit. Screening out new, highly efficient and beneficial microbial strains suitable for kiwifruit will help to carry out green prevention and control of kiwifruit soft rot disease. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Bacillus tequilensis P61 and its application in controlling kiwifruit soft rot disease to solve the problems existing in the above-mentioned prior art. The present invention has found through research that Bacillus tequilensis P61 has good antagonistic effects against the pathogenic bacteria of kiwifruit soft rot disease, namely Botryosphaeria dothidea, Phomopsis lithocarpus, and Alternaria alternata, and is harmless to humans and livestock and has no pollution to the environment.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a strain of Bacillus tequilensis P61, and the preservation number of the Bacillus tequilensis P61 is CCTCC NO: M 20241916. It was deposited at the China Center for Type Culture Collection on September 5, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0007] The present invention provides a fermentation broth containing the above-mentioned Bacillus tequilensis P61.

[0008] The present invention also provides an application of the above-mentioned Bacillus tequilensis P61 or the above-mentioned fermentation broth in the production of lipopeptide antibacterial substances.

[0009] The present invention also provides a lipopeptide antibacterial substance, which is obtained by fermenting the above-mentioned Bacillus tequilensis P61 on Landy medium.

[0010] The present invention also provides an application of the above-mentioned Bacillus tequilensis P61, the above-mentioned fermentation broth or the above-mentioned lipopeptide antibacterial substance in the preparation of products for preventing and treating kiwifruit soft rot.

[0011] Preferably, the prevention and treatment of kiwifruit soft rot is achieved by inhibiting the growth of the pathogenic bacteria of kiwifruit soft rot;

[0012] The pathogenic bacteria include Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus.

[0013] The present invention also provides a biological agent for preventing and treating kiwifruit soft rot, which uses the above-mentioned Bacillus tequilensis P61, the above-mentioned fermentation broth or the above-mentioned lipopeptide antibacterial substance as the only active ingredient.

[0014] The present invention also provides an application of the above-mentioned Bacillus tequilensis P61, the above-mentioned fermentation broth, the above-mentioned lipopeptide antibacterial substance or the above-mentioned biological agent in the prevention and treatment of kiwifruit soft rot.

[0015] Preferably, the prevention and treatment of kiwifruit soft rot is achieved by inhibiting the growth of the pathogenic bacteria of kiwifruit soft rot;

[0016] The pathogenic bacteria include Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus.

[0017] The present invention also provides a method for preventing and controlling kiwifruit soft rot disease, which includes the step of applying the above-mentioned Bacillus tequilensis P61, the above-mentioned fermentation broth, the above-mentioned lipopeptide antibacterial substance or the above-mentioned biological agent to kiwifruit plants.

[0018] The present invention discloses the following technical effects:

[0019] The Bacillus tequilensis P61 provided by the present invention was deposited at the China Center for Type Culture Collection on September 5, 2024, with the deposit number CCTCC NO: M 20241916. The present invention prepared a lipopeptide antibacterial substance based on Bacillus tequilensis P61. The experimental results show that Bacillus tequilensis P61 and the lipopeptide antibacterial substance have outstanding prevention and control effects on kiwifruit soft rot disease, have significant antibacterial effects on kiwifruit soft rot pathogens such as Botryosphaeria dothidea, Alternaria alternata, and Phomopsis sp., significantly reduce the damage rate of kiwifruit fruits, and are green and safe, harmless to humans and livestock, and pollution-free to the environment. The present invention provides a new biocontrol strain for the biological control of kiwifruit diseases and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is an observation diagram of the antagonistic effect of Bacillus tequilensis P61 on kiwifruit soft rot pathogen;

[0022] Figure 2 It is an observation diagram of the colony morphology and cell morphology of Bacillus tequilensis P61;

[0023] Figure 3 It is a phylogenetic tree analysis result diagram of Bacillus tequilensis P61;

[0024] Figure 4 It is an observation diagram of the indoor control effect determination result of Bacillus tequilensis P61 on the soft rot disease of Guichang kiwifruit;

[0025] Figure 5 It is an observation diagram of the antibacterial effect of the crude extract of the lipopeptide antibacterial substance of Bacillus tequilensis P61 on kiwifruit soft rot pathogen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] The Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus used in the present invention are provided by the Plant Pathology Teaching and Research Section of the College of Agriculture, Guizhou University. Alternaria alternata has been disclosed in the literature "Wang Dahui, Zhao Rui, Liu Dandan, et al. Rapid detection technology of Botryosphaeria dothidea causing kiwifruit soft rot by loop-mediated isothermal amplification [J]. Acta Phytophylacica Sinica, 2022, 49(06): 1811 - 1812."; Botryosphaeria dothidea and Phomopsis lithocarpus have been disclosed in the literature "Shi Jinqiao, Long Youhua, Li Xiaoqian, et al. Isolation and identification of the pathogen causing soft rot of Guichang kiwifruit and indoor screening of its botanical fungicides [J]. Fujian Journal of Agricultural Sciences, 2019, 34(03): 331 - 337". The applicant undertakes to make the above biological materials available to the public within 20 years from the filing date of the present invention.

[0032] The Landy medium used in the present invention comprises the following components: 20 g of glucose, 5 g of sodium glutamate, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 1 g of KH2PO4, 0.15 g of FeSO4·7H2O, 0.005 g of MnSO4·H2O, 0.00016 g of CuSO4·5H2O, and pH 7.0.

[0033] Example 1

[0034] 1. Screening of Strain P61

[0035] 1.1 Sampling

[0036] In March 2022, rhizosphere soil of healthy kiwifruit plants was collected from the Beijing Garden Production Area of Zhongkang Company in Shidong Town, Xifeng County, Guiyang City, Guizhou Province (26.6°N, 106.9°E). The five-point sampling method was used to collect the soil, and the soil samples from the same plot were mixed as 1 soil sample. The soil collection tools were disinfected in advance to avoid the mixing of soil microorganisms. The soil samples were taken back to the laboratory to isolate Bacillus.

[0037] 1.2 Isolation and Purification of Bacillus

[0038] 1 g of each soil sample was placed into a test tube containing 9 mL of sterile water to obtain a soil suspension. The test tube was placed in a water bath at 85°C for 30 min to kill most of the non-spore bacteria. 1 mL of the soil suspension was transferred into a test tube containing 9 mL of sterile water to prepare a bacterial suspension with a concentration of 10 -2 . By analogy, bacterial suspensions with concentrations of 10 -3 , 10 -4 , and 10 -5 were obtained. 100 μL of the dilution solutions with different concentrations were respectively taken for LB plate coating, and each concentration was repeated 3 times and cultured at 37°C for 12 h. Observe the growth of colonies, and randomly pick different single bacterial colonies according to the size, morphology, color, etc. of the colonies grown on the plate, and purify and store them in a -80°C refrigerator.

[0039] 1.3 Screening of Antagonistic Bacillus

[0040] The plate confrontation method was used to screen the strains with obvious antagonistic effects against kiwifruit soft rot pathogens, observe whether an inhibition zone was produced, measure the colony diameter by the cross method, and calculate the inhibition rate. One strain of Bacillus with better antagonistic effect was selected and numbered P61 (Table 1). The calculation formula for the inhibition rate is:

[0041] Inhibition rate (%) = [(colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the treatment group] × 100%.

[0042] The tested kiwifruit soft rot pathogens are Botryosphaeria, Alternaria, and Phomopsis.

[0043] Table 1 Antagonistic effect of strain P61 against three kiwifruit soft rot pathogens

[0044]

[0045] As shown in Table 1 and Figure 1 as shown, the results showed that the inhibition rates of strain P61 against Botryosphaeria dothidea, Alternaria alternata, and Phomopsis sp. were 83.78%, 80.09%, and 86.78% respectively, showing good inhibitory effects against the three pathogens of kiwifruit soft rot.

[0046] 2. Identification of strain P61

[0047] 2.1 Morphological, physiological and biochemical characteristics of strain P61

[0048] Drop 10 μL of the bacterial liquid of strain P61 screened above on the LB solid medium, and observe the colony morphology after culturing at 37 °C for 3 d, as Figure 2 shown.

[0049] According to Bergey's Manual of Systematic Bacteriology, the physiological and biochemical characteristics of the strain were determined. The strain was a Gram-positive bacterium (G+), as Figure 2 shown. The physiological and biochemical characteristics were as follows: Strain P61 could not utilize citrate, failed to produce nitrate reductase, gelatin liquefaction was positive, amylase was positive, methyl red staining was negative, V-P reaction was negative, etc. (Table 2).

[0050] Table 2 Physiological and biochemical characteristics of strain P61

[0051]

[0052] Note: +: Positive reaction; -: Negative reaction.

[0053] 2.2 Molecular biological identification of strain P61

[0054] Culture the strain P61 screened above, and extract the genomic DNA using the Biomiga Bacterial Genomic DNA Extraction Kit. PCR amplification of the 16S rRNA and gyrA genes in the genome was carried out. After the PCR products were verified correctly, they were sent to Sangon Biotech in Shanghai for PCR product sequencing.

[0055] The sequencing results were analyzed by BLAST alignment in the NCBI database, and the maximum-likelihood algorithm was used to construct a multi-gene phylogenetic tree based on 16S rRNA and gyrA using the tree-building website CIPRES (https: / / www.phylo.org / portal2 / home.action), as Figure 3 shown.

[0056] Combined with the above morphological and some physiological and biochemical characteristics, the antagonistic strain P61 can be preliminarily determined to belong to Bacillus tequilensis, and is named Bacillus tequilensis P61.

[0057] 2.3 Preservation of Bacillus tequilensis P61

[0058] Bacillus tequilensis P61 was preserved in the China Center for Type Culture Collection on September 5, 2024, with the preservation number CCTCC NO: M 20241916, and the preservation address is Wuhan University, Wuhan, China.

[0059] 3. Control effect of Bacillus tequilensis P61 on postharvest disease of kiwifruit soft rot

[0060] The small holes pricked in the center of the kiwifruit surface were soaked in the bacterial suspension of Bacillus tequilensis P61, and then the spore suspensions of kiwifruit soft rot pathogens (Botryosphaeria dothidea, Alternaria alternata, Phomopsis sp.) were dropped into each kiwifruit wound. The positive control was kiwifruit treated with sterile water. All the treated fruits were placed in a constant temperature incubator at 25°C. There were 10 kiwifruits in each treatment, and the experiment was repeated 3 times. The lesion diameters were measured at 3 d, 6 d, and 9 d after inoculation, and the damage rate of the fruits was calculated.

[0061] Damage rate (%) = lesion diameter of the treatment group / (lesion diameter of the control group - wound diameter).

[0062] The treatment group used kiwifruits inoculated only with soft rot pathogens as the control, and kiwifruits inoculated only with soft rot pathogens used kiwifruits without inoculation with pathogens (CK) as the control to calculate the incidence rate and anti-corrosion effect.

[0063] As shown in Table 3 and Figure 4 The results showed that after treating kiwifruit fruits with Bacillus tequilensis P61, the fruits all showed a very low damage rate, with the damage rate between 15.50 - 25.05%, indicating that Bacillus tequilensis P61 has a good control effect on kiwifruit soft rot. In addition, after treatment with Bacillus tequilensis P61, the storage period of kiwifruit can be significantly increased, with good anti-corrosion effect.

[0064] Table 3 Control effect of Bacillus tequilensis P61 on kiwifruit

[0065]

[0066] 4. Bacteriostatic effect of the crude extract of lipopeptide antibacterial substances of Bacillus tequilensis P61 on kiwifruit soft rot pathogens

[0067] 4.1 Preparation of the crude extract of lipopeptide antibacterial substance from strain P61

[0068] Pick a fresh single colony of P61 and inoculate it into LB liquid medium. Incubate it overnight at 37 °C with shaking at 200 rpm to obtain the seed solution. Take 0.1 mL of the seed solution and inoculate it into 10 mL of LB liquid medium (in a 100 mL conical flask) at an inoculation amount of 1% and pre-culture for 6 h until OD 600 is approximately equal to 3.0. Then inoculate it into 100 mL of Landy medium (10 times dissolved oxygen) in a 1000 mL conical flask at a ratio of 1:100 (1 mL of the P61 culture solution pre-cultured for 6 h until OD 600 is approximately equal to 3.0: 100 mL of Landy medium) and culture it at 30 °C with shaking at 200 rpm for 36 h until it turns dark coffee color. Collect the bacterial suspension with a 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, and take the supernatant. Weigh 6 g of XAD16 adsorption resin and load it into a filter column. Wash the salt on the resin surface with 50 mL of deionized water. Add the supernatant obtained by centrifugation to this matrix and adsorb it in the adsorption resin for 30 min. Then wash the resin column with 50 mL of deionized water, add 14 ml of methanol to elute until the color becomes transparent, and collect the eluate. Dry the methanol sample with a rotary evaporator (dry until it turns dark yellow and adheres to the wall). First dissolve the dried sample in 100 μL of deionized water, and then add 1 mL of dimethyl sulfoxide (DMSO) to the sample (it can be stored at -20 °C for a long time).

[0069] 4.2 Antibacterial effect of the crude extract of lipopeptide antibacterial substance from strain P61 against the soft rot pathogen of kiwifruit

[0070] Filter the crude extract of lipopeptide antibacterial substance from P61 with a 0.22 μm sterile filter membrane to prepare a sterile crude extract. Using the Oxford cup method, operate under sterile conditions. Place the sterile Oxford cup into the prepared PDA medium, add 150 μL of the lipopeptide antibacterial substance crude extract to the Oxford cup. Use a 5 mm sterile punch to punch out the activated soft rot pathogens of kiwifruit Botryosphaeria dothidea, Alternaria alternata, and Phomopsis sp. and inoculate them on the PDA plate 1 cm away from the Oxford cup. After 7 d, measure the diameter of the pathogen by the cross method and calculate the inhibition rate. The calculation formula is shown in "1.3 Screening of antagonistic Bacillus".

[0071] As shown in Table 4 and Figure 5 as follows, the results show that the crude extract of lipopeptide antibacterial substance also has a strong growth inhibitory effect on the soft rot pathogen of kiwifruit.

[0072] Table 4 Antibacterial effect of the crude extract of lipopeptide antibacterial substance from Bacillus tequilensis P61

[0073]

[0074] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus tequilensis P61, characterized in that, The preservation number of the Bacillus tequilensis P61 is CCTCC NO: M 20241916. It was preserved in the China Center for Type Culture Collection on September 5, 2024, and the preservation address is Wuhan University, Wuhan, China.

2. A fermentation broth containing the Bacillus tequilensis P61 described in claim 1.

3. An application of the Bacillus tequilensis P61 described in claim 1 or the fermentation broth described in claim 2 in the production of lipopeptide antibacterial substances.

4. A lipopeptide antibacterial substance, characterized in that, The lipopeptide antibacterial substance is obtained by fermenting the Bacillus tequilensis P61 described in claim 1 on Landy medium.

5. An application of the Bacillus tequilensis P61 described in claim 1, the fermentation broth described in claim 2, or the lipopeptide antibacterial substance described in claim 4 in the preparation of a product for preventing and treating kiwifruit soft rot.

6. The application according to claim 5, characterized in that, The prevention and treatment of kiwifruit soft rot is achieved by inhibiting the growth of the pathogenic bacteria of the kiwifruit soft rot; The pathogenic bacteria include Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus.

7. A biological agent for preventing and controlling kiwifruit soft rot disease, characterized in that, Using the Bacillus tequilensis P61 described in claim 1, the fermentation broth described in claim 2, or the lipopeptide antibacterial substance described in claim 4 as the sole active ingredient.

8. An application of the Bacillus tequilensis P61 described in claim 1, the fermentation broth described in claim 2, the lipopeptide antibacterial substance described in claim 4, or the biological agent described in claim 7 in the prevention and treatment of kiwifruit soft rot.

9. The application according to claim 8, characterized in that, The prevention and treatment of kiwifruit soft rot is achieved by inhibiting the growth of the pathogenic bacteria of the kiwifruit soft rot; The pathogenic bacteria include Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus.

10. A method for preventing and controlling soft rot of kiwifruit, characterized in that, It includes the step of applying the Bacillus tequilensis P61 described in claim 1, the fermentation broth described in claim 2, the lipopeptide antibacterial substance described in claim 4, or the biological agent described in claim 7 to kiwifruit plants.

Images