Bacillus velezensis JNJK-1, microbial agent and application of bacillus velezensis JNJK-1 in edible fungus disease control

Abstract

The invention relates to the technical field of biological control, in particular to bacillus velezensis JNJK-1, a microbial agent and application of the bacillus velezensis JNJK-1 and the microbial agent in edible fungus disease control, the bacillus velezensis JNJK-1 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CMCC No.31075. The novel bacillus velezensis JNJK-1 is screened out for the first time, and the bacillus velezensis can be applied to biological control of edible mushroom diseases and has a broad-spectrum inhibition effect on pathogenic bacteria of edible mushrooms. Meanwhile, the inventor also finds that the bacillus velezensis JNJK-1, curcumin and sodium alginate are compounded into the microbial agent, so that the inhibition effect on the pathogenic bacteria of the edible fungi can be improved, and the microbial activity can be maintained.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to Bacillus velez JNJK-1, a microbial agent and applications thereof in the prevention and control of edible fungi diseases. Background Art

[0002] Bacillus Velezii was first reported and named in 2005. It is a new species of Bacillus that can produce a variety of secondary metabolites, has a broad spectrum of antibacterial activity and growth-promoting effects, and has broad application prospects as a new type of biocontrol resource. In terms of plants, it can be used as a symbiotic bacteria in the rhizosphere and leaves of plants, can secrete IAA, promote plant growth, inhibit pathogen infection, reduce the use of chemical drugs, has no residue, is non-pathogenic, is environmentally friendly, and can improve the growth performance and disease resistance of plants and animals.

[0003] At present, the use of Bacillus Velez subtilis to control plant diseases has become one of the research hotspots in the field of biocontrol: for example, the invention patent with application number CN202111166439.8 discloses a Bacillus Velez subtilis, microbial agent, microbial fertilizer and its application for controlling peanut white rot; for example, the invention patent with application number CN202211509366.2 discloses a Bacillus Velez subtilis and its screening method and application, and prepares Bacillus Velez subtilis BGB-121R Fertilizers and / or growth promoters; such as the invention patent with application number CN202310120885.8, which discloses a Bacillus Velez compound chitosan oligosaccharide fermentation agent and its application, including Bacillus Velez agent and chitosan oligosaccharide, and is used to treat tomato neck rot and root rot; such as the invention patent with application number CN202410845161.4, which discloses a Bacillus Velez and its culture method and application, and the Bacillus Velez TLF4-3 is used to prevent and control post-harvest Aspergillus rot of fruits and vegetables.

[0004] However, research on the use of Bacillus Velez in the prevention and treatment of edible fungi diseases is still relatively rare. The invention patent with application number CN202310760382.7 discloses a Bacillus Velez, a bacterial agent and its application in the prevention and treatment of pathogenic fungi diseases, which reports the application of the Bacillus Velez in the biological control of edible fungi diseases. In order to further deepen the application research of Bacillus Velez in the prevention and treatment of edible fungi diseases, the present invention uses a new type of Bacillus Velez to inhibit the growth and control of pathogenic bacteria that cause edible fungi diseases, providing excellent strain resources for the biological control of edible fungi diseases. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a Bacillus Velez JNJK-1, a microbial agent and the use thereof in the prevention and control of edible fungi diseases, so as to provide a new direction for the application of Bacillus Velez so as to improve its commercial value.

[0006] Based on the above-mentioned purpose, the present invention provides a Bacillus Velez subtilis JNJK-1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 25, 2024, with a deposit number of CMCC No.31075.

[0007] The present invention also provides a microbial agent, which comprises the Bacillus Velezii JNJK-1 described in claim 1.

[0008] The present invention also provides application of the Bacillus Velezii JNJK-1 in the prevention and treatment of edible fungi diseases.

[0009] Preferably, the edible fungi disease pathogens include one or more of Neurospora, Trichoderma, Mucor, Rhizopus nigricans, Cladosporium, Cercospora pseudoceras, and Pseudomonas.

[0010] Preferably, the edible fungi include one or more of shiitake mushrooms, oyster mushrooms, black fungus, button mushrooms, and morels.

[0011] Preferably, the microbial agent comprises the following raw materials in parts by weight: 100 parts of Bacillus Velez JNJK-1 fermentation broth, 0.1-0.3 parts of curcumin, and 0.5-1 parts of sodium alginate.

[0012] Preferably, the preparation method of the Bacillus Velez JNJK-1 fermentation broth is:

[0013] After activating the Bacillus Velezii JNJK-1 strain, inoculate it into LB liquid culture medium, and culture it on a shaker at 28-35°C and 180-220 r / min for 12-18 hours to obtain a bacterial suspension;

[0014] Then, the bacterial suspension was inoculated into PD medium at an inoculum rate of 2-3 wt%, and cultured on a shaker at 28-35 °C and 180-220 r / min for 45-50 h, and then diluted with sterile water until the number of viable spores was 5-10 × 10 6 CFU / mL, obtained from the fermentation broth of Bacillus velezensis JNJK-1.

[0015] The present invention also provides the use of the Bacillus Velezii JNJK-1 in the preparation of biological pesticides for preventing and controlling edible fungi diseases.

[0016] The present invention also provides application of the microbial agent in biological pesticides for preventing and controlling edible fungi diseases.

[0017] Beneficial effects of the present invention:

[0018] The present invention screens out a new type of Bacillus Velez JNJK-1 for the first time, and the Bacillus Velez can be applied to the biological control of edible fungi diseases, and has a broad-spectrum inhibitory effect on edible fungi pathogens. At the same time, the inventors also found that compounding Bacillus Velez JNJK-1 with curcumin and sodium alginate as a microbial agent can improve the inhibitory effect on edible fungi pathogens and maintain microbial activity. Combined with the experimental data, the reason for consideration is that Bacillus Velez JNJK-1 and curcumin have a synergistic antagonistic effect on Mucor. At the same time, sodium alginate is added to the microbial agent, and sodium alginate can be used as a cell protectant, which increases the number of live bacteria of Bacillus Velez JNJK-1 to a certain extent and maintains its microbial activity. In addition, sodium alginate has a biological immobilization effect on Bacillus Velez JNJK-1, which helps to improve the contact efficiency between Bacillus Velez JNJK-1 and pathogenic bacteria in actual edible fungi biological control applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The colony morphology of Bacillus velez JNJK-1 strain in Example 1 of the present invention;

[0021] Figure 2 This is a diagram showing the results of the evolutionary tree analysis of the Bacillus velez JNJK-1 strain in Example 1 of the present invention;

[0022] Figure 3 This is a diagram showing the antagonistic effect of Examples 2-4 of the present invention on Mucor edodes. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0024] Example 1

[0025] Isolation, screening and identification methods of Bacillus velez JNJK-1:

[0026] 1.1 Test materials:

[0027] Rhizosphere soil samples from pepper fields in Jining City, Shandong Province were selected for isolation and culture;

[0028] LB plate medium formula: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH = 7.0;

[0029] PDA culture medium formula: 200g potato, 20g glucose, 20g agar, 1L distilled water.

[0030] 1.2 Isolation and screening of bacterial strain JNJK-1

[0031] First, weigh 10 g of rhizosphere soil into 90 mL of sterile water and shake it on a constant temperature oscillator at 180 r / min for 30 min. Then put it into a clean bench and dilute it with 9 mL of sterile water. Then use a pipette to draw 100 μL from the dilution and aspirate it onto the prepared LB plate medium. The dilution multiples were 10 3 , 10 4 and 10 5 , and each concentration was repeated three times, and the plate was spread with a coating rod. After the coating was completed, the plate was inverted for marking, and finally placed in a 37°C constant temperature incubator for 12-24 hours. After the culture was completed, different colonies were selected for streaking purification based on the structure, size, color, gloss and texture of the single colony surface. The purified strains were numbered and stored in a 4°C refrigerator;

[0032] Then, taking pathogens as target bacteria, the plate confrontation culture method was used to screen antagonistic bacteria with antagonistic effects. Specifically, the mycelium of the pathogen was picked up with a sterilized inoculation loop and placed in the center of the PDA culture medium plate. A cross line was marked on the back of the PDA plate for partitioning. The antagonistic bacteria screened out from the soil were inoculated in four diagonal areas at an equal distance of 2.5 cm from the center of the plate according to the partitions. Each treatment was repeated 3 times. At the same time, the PDA plate not inoculated with antagonistic bacteria but only inoculated with pathogens was used as a control and placed in a 28°C incubator for culture for 7 days. When the pathogens in the control group filled the plate, the confrontation growth in the test group was observed, and the bacterium JNJK-1 with the best antagonistic effect was screened as the antagonistic bacteria in this study.

[0033] 1.3 Determination of the morphology and biological characteristics of the JNJK-1 strain

[0034] The JNJK-1 strain was cultured on PDA at room temperature for 7 days and its colony morphology was observed. Figure 1 As shown: The colony is milky white, round, with a smooth and slightly convex surface, 1.0-1.5 cm in size, and sticky in texture. The bacteria are Gram-positive aerobic bacteria.

[0035] The physiological and biochemical characteristics of the JNJK-1 strain are as follows: catalase (+), catalase (+), indole test (+), methyl red (-), VP test (-), nitrite reduction test (-), nitrate reduction test (+), glucose hydrolysis (+), starch hydrolysis (+), cellulose decomposition test (-), propionate test (-), citrate test (-), gelatin liquefaction (+), and ammonia production test (+).

[0036] The physiological and biochemical characteristics of the bacterial strain JNJK-1 were detected and searched in the retrieval tables in Bergey's Manual of Bacterial Identification (9th edition) and Manual of Systematic Identification of Common Bacteria, and it was determined that the physiological and biochemical characteristics of the bacterial strain JNJK-1 were basically consistent with the corresponding characteristics of Bacillus velez.

[0037] 1.4 Molecular Biological Identification

[0038] The screened bacterial strain JNJK-1 was amplified by PCR, and the amplified product was sequenced by 16SrDNA.

[0039]

[0040] The sequencing results were then compared and analyzed using BLAST software, that is, the bacterial JNJK-1 strain sequence was compared and analyzed with known sequences in GenBank, and a phylogenetic tree was constructed using MEGA5.0. The results of constructing the phylogenetic tree are shown in Figure 2. Figure 2 shown.

[0041] Figure 2 The results showed that the homology similarity between bacteria JNJK-1 and Bacillus velezensis reached 97%. Through morphological identification and molecular biological identification, the final identification result was that JNJK-1 strain was Bacillus velezensis.

[0042] The isolated and screened strain was named Bacillus velezensis JNJK-1 and was deposited in the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on June 25, 2024, with the deposit number CMCC No. 31075.

[0043] Example 2 Preparation of Bacillus Velez JNJK-1 fermentation broth

[0044] The Bacillus Velezii JNJK-1 strain isolated and screened in Example 1 was activated and inoculated into LB liquid culture medium, and cultured on a shaking table at 28° C. and 180 r / min for 12 h to obtain a bacterial suspension;

[0045] The bacterial suspension was then inoculated into PD medium at a 2wt% inoculum volume, cultured on a shaker at 28°C and 180 rpm for 45 h, and then diluted with sterile water until the number of viable spores was 5 × 10 6 CFU / mL, obtained from the fermentation broth of Bacillus velezensis JNJK-1.

[0046] Example 3 Preparation of microbial inoculants

[0047] The fermentation liquid of Bacillus velez JNJK-1 was prepared according to the preparation method of Example 2;

[0048] 100 parts of Bacillus Velez JNJK-1 fermentation liquid, 0.1 parts of curcumin and 0.5 parts of sodium alginate were weighed by weight, and mixed evenly to obtain a microbial agent.

[0049] Example 4 Preparation of microbial inoculants

[0050] The Bacillus Velezii JNJK-1 strain isolated and screened in Example 1 was activated, inoculated into LB liquid culture medium, and cultured on a shaking table at 35° C. and 220 r / min for 18 h to obtain a bacterial suspension;

[0051] The bacterial suspension was then inoculated into PD medium at a rate of 3 wt %, and cultured on a shaker at 35 °C and 220 r / min for 50 h. The medium was then diluted with sterile water until the number of viable spores was 1 × 10 7 CFU / mL, fermentation broth of Bacillus velez JNJK-1;

[0052] 100 parts of Bacillus Velez JNJK-1 fermentation liquid, 0.3 parts of curcumin and 1 part of sodium alginate were weighed by weight, and mixed evenly to obtain a microbial agent.

[0053] Determination of the antibacterial spectrum test of Examples 2-4

[0054] Test 1:

[0055] Mucor was isolated from the mushroom sticks infected with Mucor disease. After activation, a 5 mm diameter cake was punched out using a puncher and inoculated into the center of a PDA culture plate. A hole was then punched at a distance of 2.5 cm from the center of the pathogen using the cross method. The culture medium at the punched hole was taken out, and 10 μL of the fermentation liquid of Example 2 or the bacterial agent of Example 3 or 4 was added to each hole. An equal amount of sterile water was used as a control group. After culturing at 28°C for 5 days, the antibacterial effect was observed. The results are as follows: Figure 3 As shown:

[0056] Depend on Figure 3 It can be seen that compared with the control group, Examples 2-4 have obvious antagonistic effects on Mucor edodes, but the antagonistic effects of Examples 2-4 on Mucor increase successively. Compared with Example 2, it can be seen that the antibacterial effect of Example 3 with a small amount of curcumin added is better, indicating that Bacillus velez JNJK-1 and curcumin have a synergistic antagonistic effect on Mucor. At the same time, the mycelium growth diameter of Example 4 is smaller, proving that the antagonistic effect of the microbial agent on Mucor is positively correlated with the concentration of Bacillus velez JNJK-1 and the concentration of curcumin. Within a certain range, the higher the content of live spores, the better the antibacterial effect, and the higher the content of curcumin, the better the antibacterial effect.

[0057] At the same time, sodium alginate is added to the microbial agent. Sodium alginate can act as a cell protectant, which can increase the number of live bacteria of Bacillus Velez JNJK-1 to a certain extent and maintain its microbial vitality. In addition, sodium alginate has a biological immobilization effect on Bacillus Velez JNJK-1, which helps to improve the contact efficiency between Bacillus Velez JNJK-1 and pathogenic bacteria in actual edible fungus biological control applications.

[0058] Test 2:

[0059] Neurospora was isolated from Lentinus edodes fungus sticks infected with Neurospora disease. After activation, a 5 mm diameter bacterial cake was punched out using a puncher and inoculated into the center of a PDA culture plate. A hole was then punched at a distance of 2.5 cm from the center of the pathogen using a cross method. The culture medium at the punched hole was taken out, and 10 μL and 20 μL of the fermentation liquid of Example 2 or the bacterial agent of Example 3 or 4 were added to each hole. An equal amount of sterile water was used as a control group. After culturing at 28° C. for 7 days, the antibacterial effect was observed, the growth diameter of the pathogen was measured and recorded, and the antibacterial rate was calculated: antibacterial rate=100%×(control group pathogen colony diameter-test group colony diameter) / control group pathogen colony diameter. The antibacterial effect is shown in Table 1:

[0060] Table 1 Antagonistic effect of Examples 2-4 on Neurospora:

[0061] Antibacterial rate / % Example 2 Example 3 Example 4 10μL group 42.7 58.3 65.8 20μL group 65.5 80.9 87.6

[0062] As can be seen from Table 1, Examples 2-4 also have obvious antagonistic effects on Neurospora edodes, and also show the law that the antagonistic effects of Examples 2-4 on Neurospora increase in sequence, further verifying that Bacillus velez JNJK-1 and curcumin have synergistic antagonistic effects on edible fungi and pathogenic fungi, and further proving that the antagonistic effect of microbial agents on Mucor is positively correlated with the concentration of Bacillus velez JNJK-1 and the concentration of curcumin; at the same time, the antagonistic effects of the embodiments of the 20 μL dosage group are significantly better than those of the 10 μL dosage group, which also shows that increasing the dosage of the microbial agent within a certain range is also helpful to improve the antibacterial effect.

[0063] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0064] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus Velezii JNJK-1, characterized in that The Bacillus velezensis JNJK-1 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number CMCC No. 31075.

2. A microbial agent, characterized in that: It comprises the Bacillus velezensis JNJK-1 described in claim 1.

3. Use of the Bacillus Velez JNJK-1 according to claim 1 in the prevention and treatment of edible fungi diseases.

4. The use according to claim 3, characterized in that: The edible fungi disease pathogens include one or more of Neurospora, Trichoderma, Mucor, Rhizopus nigromaculus, Cladosporium, Cercospora pseudocerasus, and Pseudomonas.

5. The use according to claim 3 or 4, characterized in that: The edible fungi include one or more of shiitake mushrooms, oyster mushrooms, black fungus, button mushrooms, and morels.

6. A microbial agent according to claim 2, characterized in that: The invention comprises the following raw materials in parts by weight: 100 parts of fermentation liquid of Bacillus velez JNJK-1, 0.1-0.3 parts of curcumin and 0.5-1 parts of sodium alginate.

7. A microbial agent according to claim 6, characterized in that: The preparation method of the Bacillus Velez JNJK-1 fermentation broth is as follows: After activating the Bacillus Velezii JNJK-1 strain, inoculate it into LB liquid culture medium, and culture it on a shaker at 28-35°C and 180-220 r / min for 12-18 hours to obtain a bacterial suspension; Then, the bacterial suspension was inoculated into PD medium at an inoculum rate of 2-3 wt%, and cultured on a shaker at 28-35 °C and 180-220 r / min for 45-50 h, and then diluted with sterile water until the number of viable spores was 5-10 × 10 6 CFU / mL, obtained from the fermentation broth of Bacillus velezensis JNJK-1.

8. Use of the Bacillus Velez JNJK-1 according to claim 1 in the preparation of biological pesticides for preventing and controlling edible fungi diseases.

9. Use of the microbial agent according to claim 6 in biological pesticides for preventing and controlling edible fungi diseases.

Images