Bacillus velezensis NJ157 and application thereof

Abstract

The invention provides bacillus velezensis NJ157 and application thereof, and relates to the technical field of biological pesticides. The bacillus velezensis NJ157 can resist saline and alkaline, has a good inhibition effect on ralstonia solanacearum, rhizoctonia solanacearum, fusarium moniliforme and phytophthora capsici, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot. The bacillus velezensis NJ157 disclosed by the invention also has relatively strong capability of producing protease, cellulase and IAA (Indoleacetic Acid), and has a relatively good growth promoting effect on tomatoes, and a fungicide prepared from the biocontrol bacterium does not cause pollution to the ecological environment in a use process, and is pollution-free. By using the biocontrol bacterium disclosed by the invention, the dosage of other chemical pesticides can be reduced, the prevention and control cost of tomato diseases is reduced, the tomato growth is promoted, and the biocontrol bacterium has a good popularization value.

Description

Technical Field

[0001] The invention relates to the technical field of biological pesticides, and in particular to a strain of Bacillus velez NJ157 and an application thereof. Background Art

[0002] The production mode of greenhouse vegetables has the characteristics of low investment, low energy consumption, intensive, and year-round cultivation, which leads to the increasingly serious continuous cropping obstacles and high incidence of diseases, which has become a bottleneck problem restricting the sustainable utilization of greenhouse vegetables in my country. For example, tomato gray mold caused by tomato gray Botrytis infection caused by Fusarium oxysporum infection, tomato late blight caused by Phytophthora, etc. At present, the prevention and control of pathogenic microorganisms mainly includes physical control, chemical control and biological control, and most of them still use traditional chemical control, such as the use of organophosphate pesticides, organoarsenic, and benzene pesticides. Although these chemicals are effective quickly, long-term use will cause these pathogens to increase their resistance to drugs, and will endanger human health and pollute the environment, which is not in line with the concept of sustainable development. Biological control is a new prevention and control method that has emerged in recent years. Because there is no environmental pollution, no drug residues, and it can meet the requirements of pollution-free vegetable production, there are more and more studies in this area, and biocontrol bacteria are also favored by more and more people.

[0003] At present, most of the newly identified unknown microorganisms are isolated from common habitats, and the number of new bacterial species is decreasing. Due to the unique geological and climatic characteristics of the Antarctic geographical environment, a natural environment with low temperature, high salt, and strong radiation has been formed, which has created unique molecular biological mechanisms and physiological and biochemical characteristics of polar microorganisms in terms of genetic composition, enzymatic characteristics, and metabolic regulation. It is the birthplace of chemical substances with novel structures and unique functions in the world. Polar microorganisms have great potential in the application of biological pesticides. Therefore, the discovery of different types of strains from polar microorganisms for biological control of plant diseases is of great significance to the development and utilization of polar microbial resources. However, there have been no reports of salt-alkali-tolerant strains isolated from Antarctica that have biological control functions. Summary of the invention

[0004] The present invention aims to provide a strain of Bacillus Velez NJ157 and its application. The Bacillus Velez NJ157 is isolated from the soil of a penguin colony near the seismic station of the Great Wall Station in Antarctica, is resistant to low temperature and salt and alkali, and can inhibit a variety of pathogens and tomato diseases, thus solving the problem of single function of strains isolated from common habitats in the prior art.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Bacillus Velezii NJ157, which is characterized in that it is deposited in the General Microbiology Center of China Culture Collection Administration, with a deposit number of CGMCC No. 31984, a deposit date of September 14, 2024, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a method for preparing the bacterial liquid of the Bacillus Velez NJ157, wherein the Bacillus Velez NJ157 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

[0008] Preferably, the fermentation temperature is 28-32° C., the fermentation speed is 180-220 rpm, and the fermentation time is 22-26 h.

[0009] The present invention also provides the use of the Bacillus Velezii NJ157 in all or part of the following (1) to (6):

[0010] (1) Application in inhibiting pathogenic oomycetes, pathogenic bacteria and pathogenic fungi;

[0011] (2) Application in inhibiting tomato diseases;

[0012] (3) Application in promoting tomato growth in severe saline-alkali land;

[0013] (4) Application in decomposing protein or cellulose;

[0014] (5) Application in the preparation of tomato growth promoter in severe saline-alkali soil;

[0015] (6) Application in the preparation of bacterial agents for decomposing protein or cellulose.

[0016] Preferably, the pathogenic oomycetes include Phytophthora capsici; the pathogenic bacteria include Pseudomonas syringae and Ralstonia solanacearum; and the pathogenic fungi include Botrytis cinerea, Fusarium moniliforme, Fusarium solani and Rhizoctonia solani.

[0017] Preferably, the tomato diseases include bacterial wilt, root rot and fruit rot.

[0018] The present invention also provides a microbial agent, which comprises the bacterial suspension of Bacillus Velez NJ157 according to claim 1.

[0019] Preferably, the spore concentration in the bacterial suspension is 1 to 9×10 7 CFU / mL.

[0020] The invention also provides a method for promoting tomato growth in heavy saline-alkali land, wherein the tomato plants are irrigated with the microbial agent.

[0021] By adopting the above technical scheme, the present invention has the following beneficial effects: the Bacillus Velez NJ157 of the present invention is salt-alkali resistant, and has a good inhibitory effect on Ralstonia solanacearum, Rhizoctonia solani, Fusarium moniliforme and Phytophthora capsici, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot. The Bacillus Velez NJ157 of the present invention also has a strong ability to produce protease, cellulase and IAA, and has a good growth-promoting effect on tomatoes. The bacterial agent prepared by the biocontrol bacteria will not pollute the ecological environment during use and is pollution-free. The use of the biocontrol bacteria of the present invention can reduce the amount of other chemical pesticides, reduce the cost of preventing and controlling tomato diseases, promote tomato growth, and has good promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of Bacillus velez NJ157.

[0023] Figure 2 This is a diagram showing the inhibitory effect of Bacillus Velez NJ157 on pathogens, wherein A is Ralstonia solanacearum, B is Pseudomonas syringae, C is Botrytis cinerea, D is Phytophthora capsici, E is Fusarium moniliforme Sheld, and F is Rhizoctonia solani.

[0024] Figure 3 Figure 2 is the result of the salt stress tolerance test of Bacillus Velez NJ157, and Figure AB are the plate phenotype result diagrams of salt tolerance (11%, 13%) of Bacillus Velez NJ157, FZB42, 168, and DMW1, respectively.

[0025] Figure 4 Figure 2 is the result of the alkali stress resistance test of Bacillus Velez NJ157, and AC is the alkali resistance (pH=8, 9, 10) plate phenotype result of Bacillus Velez NJ157, FZB42, 168, and DMW1, respectively.

[0026] Figure 5 This is the HPLC-MS identification result of secondary metabolites of Bacillus velez NJ157.

[0027] Figure 6This is a graph showing the antibacterial effect test results of the crude extract of secondary metabolites of Bacillus velez NJ157; A is Ralstonia solanacearum, B is Pseudomonas syringae, C is Phytophthora capsici, D is Rhizoctonia solani, and E is Botrytis cinerea.

[0028] Figure 7 This is a graph showing the detection results of protease, cellulase, amylase and indoleacetic acid (IAA) produced by Bacillus Velez NJ157, wherein A is protease, B is cellulase, C is amylase, and D is indoleacetic acid.

[0029] Figure 8 This is a diagram showing the disease prevention effect of Bacillus Velez NJ157 on bacterial wilt infection on tomato plants.

[0030] Fig. 9 This is a diagram showing the disease prevention effect of Bacillus Velez NJ157 against pepper phytophthora (P. capsici) infection on tomato plants.

[0031] Fig.10 This is a diagram showing the disease prevention effect of Bacillus Velez NJ157 against pepper phytophthora (P. capsici) infection on tomato fruits.

[0032] Fig.11 This is a diagram showing the disease prevention effect of Bacillus Velez NJ157 against Fusarium solani infection in tomato fruit.

[0033] Fig.12 This is a diagram showing the effect of Bacillus Velez NJ157 on the promotion of tomato seed germination. A shows the effect of Bacillus Velez NJ157 on tomato seed germination; BC shows the effect of NJ157 on the root length and shoot length of tomato seed germination.

[0034] Fig.13 Figure 1 is the effect diagram of Bacillus Velez NJ157 on the growth promotion of tomato plants, A is the effect diagram of Bacillus Velez on tomato plant height and root length; B and B are statistical diagrams showing that Bacillus Velez can increase tomato plant height, root length, chlorophyll content, stem diameter, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight.

[0035] Fig.14Figure 5 is the effect diagram of Bacillus Velez NJ157 on the growth of tomato plants under salt stress environment. A is the effect diagram of Bacillus Velez NJ157 on tomato plant height and root length under salt stress environment; BI: Statistical diagram of Bacillus Velez NJ157 being able to increase tomato plant height, root length, chlorophyll content, stem diameter, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight under salt stress environment.

[0036] Biological Deposit Description

[0037] The Bacillus velezensis NJ157 provided by the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31984, a deposit date of September 14, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1. Analysis of the taxonomic status of Bacillus velez NJ157 based on the core genome

[0040] The Velez Bacillus NJ157 strain was inoculated into liquid LB medium, shaken at 200 rpm and 37°C for 12 hours, and the cells were collected by centrifugation. The genomic DNA was extracted using a bacterial extraction kit (Beijing Zhuangmeng International Biogene Technology Co., Ltd.), and then the 16SrDNA gene was PCR amplified using forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No.3) and reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No.4). The PCR conditions were: 95°C, 5min; 95°C, 15s; 56°C, 1min; 72°C, 1min; 32 cycles; 72°C, 10min, and stored at 4°C. After observing the amplification of the 16SrRNA fragment by gel electrophoresis, the PCR stock solution was sent for sequencing. The 16SrRNA gene sequence is shown in SEQ ID No.1.

[0041] 16SrRNA gene sequence:

[0042]

[0043] PCR amplification and sequence determination of gyrB gene

[0044] Forward primer gyrB-F:

[0045] 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'

[0046] (SEQ ID No.5);

[0047] Reverse primer gyrB-R:

[0048] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'

[0049] (SEQ ID No.6), the gyrB gene PCR reaction conditions are the same as above. The length of the gyrB partial gene sequence is about 1500 bp, and the result is shown in the sequence table SEQ ID No.2.

[0050] gyrB gene sequence:

[0051]

[0052] The results of 16S rDNA and gyrB identification showed that strain NJ157 belonged to Bacillus velezensis. Secondly, according to the genome sequence of NJ157, based on the 31 housekeeping genes of all samples, the 19 strains closest to the species level were selected, and the phylogenetic tree was constructed using the MEGA6.0 software selection method. The results showed that the evolutionary distance between strain NJ157 and Bacillus velezensis was the closest. The above results show that strain NJ157 belongs to Bacillus velezensis.

[0053] Example 2. Determination of antibacterial ability of Bacillus velez NJ157

[0054] The antibacterial ability of Bacillus Velez NJ157 was preliminarily determined, and several representative pathogenic bacteria, pathogenic fungi and pathogenic oomycetes were selected. Among them, the pathogenic bacteria included Ralstonia solanacearum and Pseudomonas syringae; the pathogenic fungi included Botrytis cinerea, Fusarium moniliforme Sheld and Rhizoctonia solani; the pathogenic oomycetes included Phytophthora capsici.

[0055] Determination of the antibacterial ability of Bacillus Velez NJ157 against pathogenic bacteria: First, activate the pathogenic bacteria. Pick a single colony of pathogenic bacteria into 20 mL of the corresponding culture solution and culture it overnight at 30°C and 200 rpm. Heat the LB or NA solid culture medium, and when it cools to the same temperature as human body temperature, add the bacterial solution to the culture medium at a content of 1%, mix it thoroughly and pour it into the bacteria-containing plate. Add 5 μL of Bacillus Velez bacterial solution (OD 600 =2.0), LB culture medium was used as control treatment, and after being fully dried, they were inverted in a constant temperature incubator at 30°C. The antibacterial effect was observed after 24 h, and the diameter of the transparent antibacterial zone was measured.

[0056] Determination of the antibacterial ability of Bacillus Velez NJ157 against pathogenic fungi: From the original pathogenic fungus culture plate, use a sterile puncher with a diameter of about 0.5 cm to punch a certain number of bacterial cakes at the outermost edge of the colony, then transfer the bacterial cakes (the side containing hyphae) to the center of a new PDA culture plate, and culture them in an incubator at a suitable temperature for 10-18 hours. After new hyphae grow around the bacterial cakes, they can be used for later use. Then place a sterilized filter paper at the same distance from the bacterial cakes, and drop 5 μL of Bacillus Velez bacterial solution (OD600 =2.0), LB culture medium was used as blank control, and the mixture was placed back into the 25°C incubator after drying. The antibacterial effect was observed after 48 hours. The results are as follows Figure 2 The results showed that Bacillus Velez NJ157 had good antagonistic effects on a variety of plant pathogenic fungi and bacteria, among which Ralstonia solanacearum, Rhizoctonia solani and Fusarium moniliforme Sheld had the best inhibitory effects. The above results showed that Bacillus Velez NJ157 had a good biocontrol effect and could be used as a biocontrol strain for subsequent research.

[0057] Example 3. Determination of stress resistance of Bacillus velez NJ157

[0058] Antarctica is a polar permafrost environment. Bacillus that survive and grow and reproduce normally in this environment may have a strong ability to resist adverse stress. Therefore, the stress tolerance ability of Bacillus Velez NJ157 was tested.

[0059] 1. Determination of salt stress tolerance of Bacillus velez NJ157

[0060] Different salt stresses (salt gradient: 11%, 13% NaCl concentration) were simulated to determine the ability of Antarctic strains NJ157, model strains 168, FZB42 and DMW1 to resist salt stress. Single colonies of the test strains were picked into LB liquid medium and cultured overnight at 37°C and 200r / min shaking incubator for 24h. When the bacterial solution concentration reached OD 600 =2.0, transfer 5 μL of bacterial solution to LB solid medium with different salt gradients (11%, 13%), and then put the medium into a 37°C incubator. After 5 days, observe the colony growth of each strain under different salt gradients. Biological replication was performed 3 times. The results are as follows Figure 3 The results showed that the Antarctic strain NJ157 could grow under salt stress conditions with a salt content of 13%, while the model strain FZB42 could only tolerate salt stress of 11% NaCl. Its ability to tolerate salt stress was higher than that of the model strain FZB42.

[0061] 2. Determination of the ability of Bacillus velez NJ157 to tolerate alkaline stress

[0062] Different alkaline stresses (pH gradient: 8, 9, 10) were simulated to determine the ability of Bacillus Velezii NJ157, model strain 168, FZB42 and DMW1 to resist alkaline stress. Single colonies of the test strains were picked into LB liquid medium and cultured overnight at 37°C and 200 r / min in a shaking incubator for 24 hours. 600=2.0, transfer 5 μL of bacterial solution to LB solid medium with different alkaline gradients (pH 8, 9, 10), and then put the medium into a 37°C incubator. After 5 days, observe the colony growth of each strain under different salt gradients. Biological replication was performed 3 times. The results are as follows Figure 4 The results showed that Bacillus Velez NJ157 could grow under strong alkaline stress conditions of pH 10 and had a strong ability to resist alkaline stress.

[0063] Example 4. Extraction, identification and antibacterial activity determination of secondary metabolites of Bacillus velez NJ157

[0064] 1. Extraction and identification of secondary metabolites of Bacillus velez NJ157

[0065] Activate Bacillus Velezii NJ157 on LB solid medium and place in a 37°C incubator until a single colony grows; pick a single colony of NJ157 and transfer it to 20 mL LB liquid culture medium, shake culture at 37 ° C, 200 rpm overnight; the next day, transfer the bacterial solution of Bacillus Velez NJ157 to 200 mL of LB liquid culture medium at a ratio of 1%, and shake culture at 37 ° C, 200 rpm for 24 hours; add 5 mL of resin (XAD-16N) to the NJ157 bacterial solution, and continue to shake culture for 24 hours; the next day, centrifuge the bacterial solution at 8000 rpm for 10 minutes to collect the bacteria; dissolve the bacteria with 30 mL of methanol, and then continue to shake culture at 37 ° C, 200 rpm for 4 hours, centrifuge the bacterial suspension at 8000 rpm for 10 minutes, collect the supernatant and filter it with filter paper, use a rotary evaporator to concentrate the supernatant to 2 mL, and then use a 0.22 μm organic filter to filter the crude extract, and use a high performance liquid chromatography-mass spectrometry (HPLC-MS) to identify and detect the production of lipopeptides. The results are as follows Figure 5 The results of HPLC-MS showed that Bacillus velezensis NJ157 could produce iturin, fengycin and surfactin as secondary metabolites.

[0066] 2. Determination of the antibacterial effect of secondary metabolites of Bacillus velez NJ157

[0067] From the original pathogenic fungus culture plate, use a sterile cork puncher with a diameter of about 0.5 cm to punch a certain amount of bacterial cakes at the outermost edge of the colony, then transfer the bacterial cakes (the side containing hyphae) to the center of the new PDA culture plate, and culture them in an incubator at a suitable temperature for 10-18 hours. When new hyphae grow around the bacterial cakes, they can be used for later use. After that, use a sterile cork puncher with a diameter of 0.5 cm to punch holes at the same distance from the bacterial cakes, and drop 50 μL of secondary metabolites of Bacillus Velezii strains in the center of the holes. Methanol solution is used as a control, and the plates are returned to the incubator after drying. The antibacterial effect is observed after 48 hours. The results are as follows: Figure 6 The results of the plate confrontation experiment showed that the secondary metabolites of Bacillus velezensis NJ157 had good antagonistic effects on a variety of plant pathogenic fungi and bacteria, among which the best inhibitory effects were on Ralstonia solanacearum, R. solani and B. cinerea.

[0068] 3. Determination of the ability of Bacillus velez NJ157 to produce extracellular hydrolases

[0069] Qualitative detection of protease production of Bacillus Velez NJ157 on skim milk agar plate: Determine whether Bacillus Velez has the ability to produce protease in agar medium containing skim milk powder (20g skim milk powder, 20g agar, pH7.0, sterilized at 115℃ for 10min). Pick a single colony of Bacillus Velez into LB liquid medium and culture it overnight at 37℃ and 200r / min for 12h; drop 5μL (OD 600 = 1.0) was placed in a 37°C constant temperature incubator for incubation. After 24-96 hours, the colonies were observed for the formation of a transparent ring. Figure 7 As shown in A.

[0070] Determination of cellulase production capacity of Bacillus Velez NJ157 using CMC-Na medium: Pick a single colony of Bacillus Velez NJ157 into LB liquid medium and culture it overnight at 37°C and 200 r / min for 12 hours. Place sterile filter paper discs with a diameter of 4 mm in a "cross" shape on the CMC-Na medium plate; draw 5 μL of bacterial suspension (OD 600 =1.0), inoculate in the center of the filter paper, seal the plate, and place it in a 37°C constant temperature incubator for culture; after 2 days, take out the culture dish, inject Gram iodine staining solution until the surface of the plate is submerged, let it stand for 4 minutes, then pour out the staining solution, and observe whether a transparent circle is formed around the colony. If a transparent hydrolysis circle appears around the bacteria, it means that the strain can produce the corresponding enzyme. If no transparent hydrolysis circle appears, it means that the strain cannot produce the enzyme. The results are as follows Figure 7 As shown in B.

[0071] Starch agar medium was used to detect the amylase activity of Bacillus Velez NJ157: the colonies of Bacillus Velez were picked and placed in LB liquid medium, cultured overnight at 37°C, 200 r / min for 12 h, and 5 μL of bacterial suspension (OD 600 =1.0) to a starch-containing culture medium and cultured in a 37°C constant temperature incubator; after 2 days, take out the culture dish, inject Gram iodine staining solution to submerge the surface of the plate, let it stand for 4 minutes, then pour out the staining solution, observe and record the experimental results. Figure 7 As shown in C.

[0072] The results showed that strain NJ157 was able to produce a large transparent hydrolysis zone in skimmed milk powder medium and CMC-Na medium, which indicated that it was able to produce protease and cellulase.

[0073] 4. Determination of IAA production capacity of Bacillus velez NJ157

[0074] Preparation of Salkowski colorimetric solution: Weigh 0.81g of FeCl3 and dissolve it in 10mL of ddH2O, fully dissolve it and mix it evenly to prepare a 0.5mol / L mother solution for later use. Prepare a 35% perchloric acid solution, then add the 0.5mol / L FeCl3 mother solution at a ratio of 1:50, mix evenly and set aside.

[0075] First, shake the bacteria in LB medium at 37°C and 180rpm to obtain the strain mother solution. Then transfer the mother solution to LB liquid medium containing 100mg / L tryptophan at a content of 1%. After culturing at 37°C and 180rpm for 24h, take 1mL of the bacterial solution and centrifuge at 12000rpm to obtain the supernatant. Add an equal volume of Salkowski colorimetric solution to the supernatant, mix well, and react in a dark environment for 30 minutes. If the solution turns red, it means that the bacteria can produce IAA. Use the same volume of LB liquid containing 100mg / L tryptophan as a blank control. The results are as follows: Figure 7 As shown in D. The results showed that Bacillus velez NJ157 can produce IAA, and the IAA production capacity of strain NJ157 is similar to that of the model strain FZB42, indicating that it has great potential in promoting plant growth and development and has the potential to be developed into a biocontrol agent.

[0076] Example 5. Determination of the ability of Bacillus Velezii NJ157 to control tomato diseases

[0077] 1. Determination of the control ability of Bacillus velez NJ157 against tomato bacterial wilt

[0078] Place the sterilized seedling medium into a 50-hole seedling tray, place one tomato seed with similar growth status in each hole, plant it into the soil with the roots facing down using tweezers, and cover with a thin layer of soil. After two weeks of cultivation at 28°C, keep one tomato seedling with consistent growth in each hole of the seedling tray, and use a 350mL plastic cup for transplanting. After one month of cultivation in a greenhouse at 28°C, select tomato seedlings with similar growth status for disease prevention experiments. Activate Bacillus Velez NJ157 and model bacteria FZB42 on LB solid culture medium. Use a sterile toothpick to pick up a single colony and place it in 20mL of LB liquid culture medium, and shake it overnight at 37°C and 200rpm. The next day, transfer at 1%, and after culturing for 4-5 hours, measure the OD value of the bacterial solution. 600 Adjust to 0.5(10 7 CFU / mL) for later use. The tomato seedlings were irrigated with roots. Twelve tomato seedlings were irrigated with 10 mL of Bacillus Velezii solution per treatment. Sterile water was used as a control. The tomato seedlings were placed in a 30°C greenhouse. After 7 days, the same root irrigation treatment was performed again. At the same time, the root injury treatment was performed. Each tomato seedling was irrigated with 10 mL of OD 600 = 0.1 Ralstonia solanacearum bacterial solution. After 5 days, the disease conditions of the tomato plants were observed and recorded and the incidence rate was calculated. The results are as follows Figure 8 And as shown in Table 1.

[0079] Incidence rate (%) = (number of diseased plants / total number of plants) × 100%

[0080] Disease classification survey standards:

[0081] 0: no wilting;

[0082] 1: 1%-25% leaf area wilting;

[0083] 2: 26%-50% leaf area wilting;

[0084] 3: 51%-75% leaf area wilting;

[0085] 4: 76%-100% leaf area wilts or dies.

[0086] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0087] Control effect = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0088] Table 1 Control effect of Bacillus velezensis NJ157 on tomato bacterial wilt

[0089]

[0090] Note: The same letters in each column indicate that the difference is not significant (P>0.05), while the different letters indicate that the difference is significant (P<0.05).

[0091] The experimental results showed that treatment of tomato plants with Bacillus Velez NJ157 reduced the incidence of tomato bacterial wilt, with the tomato incidence rate being 61.11%, 22.22% lower than that of the blank control group. In addition, the disease index of tomatoes treated with Bacillus Velez NJ157 was significantly reduced, and the control effect on tomato bacterial wilt reached 56.18%. NJ157 has a good control effect on bacterial wilt.

[0092] 2. Determination of the ability of Bacillus Velezii NJ157 to control tomato root rot

[0093] Place the sterilized seedling medium into a 50-hole seedling tray, place a tomato seed with similar growth status in each hole, plant it into the soil with the roots facing down using tweezers, and cover with a thin layer of soil. After two weeks of cultivation at 25°C, keep one tomato seedling with consistent growth in each hole of the seedling tray, and use a 350mL plastic cup for transplanting. After one month of cultivation in a greenhouse at 25°C, select tomato seedlings with similar growth status for disease prevention experiments. Activate Bacillus Velez NJ157 and model bacteria FZB42 on LB solid culture medium. Use a sterile toothpick to pick up a single colony and place it in 20mL of LB liquid culture medium, and shake it overnight at 37°C and 200rpm. The next day, transfer at 1%, and after culturing for 4-5 hours, measure the OD value of the bacterial solution. 600 Adjust to 0.5(10 7 CFU / mL) for later use. The tomato seedlings were irrigated with roots. Each treatment had 12 tomato seedlings. Each tomato seedling was irrigated with 10mL of Bacillus Velezii solution. Sterile water was used as a control. The same root irrigation treatment was performed again after 7 days. At the same time, the root injury treatment was performed. Four Phytophthora capsici fungus discs were placed at the root zone of each tomato seedling and infected with the pepper Phytophthora capsici pathogen. After 5 days, the disease condition of the tomato plants was observed and recorded, and the incidence rate was calculated. The results are as follows: Fig. 9 And as shown in Table 2.

[0094] Disease classification survey standards:

[0095] 0: no wilting;

[0096] 1: 1%-25% leaf area wilting;

[0097] 2: 26%-50% leaf area wilting;

[0098] 3: 51%-75% leaf area wilting;

[0099] 4: 76%-100% leaf area wilts or dies.

[0100] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0101] Control effect = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0102] Table 2 The control effect of Bacillus Velez NJ157 on tomato root rot

[0103]

[0104] Note: The same letters in each column indicate that the difference is not significant (P>0.05), while the different letters indicate that the difference is significant (P<0.05).

[0105] The experimental results showed that the incidence of tomato root rot was reduced after tomato plants were treated with Bacillus Velez NJ157. The incidence of tomato treated with Bacillus Velez NJ157 was the lowest, at 55.55%, which was 25% lower than the blank control group. The disease index of tomato root rot treated with Bacillus Velez NJ157 was significantly reduced, and the control effect on tomato root rot was the best, reaching 54.33%.

[0106] 3. Determination of the disease protection ability of Bacillus velez NJ157 against Phytophthora capsici on tomato fruits

[0107] Activate Bacillus velezensis NJ157 and model bacteria FZB42 on LB solid medium. Pick a single colony with a sterile toothpick and place it in 20 mL LB liquid medium. Shake and incubate at 37°C, 200 rpm overnight. The next day, transfer to 1% and incubate for 4-5 hours. 600 Adjust to 0.5(5×10 7 CFU / mL) for later use. Activate the preserved Phytophthora capsici on the PDA plate, and use a gun tip to obtain a bacterial disc for later use. Use 75% anhydrous ethanol to disinfect the surface of tomato fruits of basically the same size. Place the disinfected tomato fruits upside down in different treatment solutions and soak for 30 minutes, then take them out and place them upright in a perforated tray (a tray with a layer of sterile water at the bottom). Dry the different treatment solutions on the surface of the tomatoes naturally, use a sterile syringe to gently poke a hole on the top of the tomato fruit, fit the P. capsici bacterial disc tightly into the pore of the tomato fruit, and wrap the bacterial disc with absorbent cotton for water retention. Wrap it with plastic wrap and place it in a greenhouse at 25°C. After 4 days, observe the disease condition of the tomato fruit and conduct a disease grade survey. The disease grade survey standards are shown in Table 3, and the survey results are shown in Table 3. Fig.10 And as shown in Table 4.

[0108] Table 3 Disease classification survey standards

[0109]

[0110]

[0111] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0112] Control effect = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0113] Table 4 Control effect of Bacillus Velez NJ15 on Phytophthora capsici on tomato fruits

[0114]

[0115] Note: The same letters in each column indicate that the difference is not significant (P>0.05), while the different letters indicate that the difference is significant (P<0.05).

[0116] The experimental results showed that treatment of tomato plants with Bacillus Velez NJ157 inhibited the occurrence of P. capsici in tomato fruits. The disease index of tomato fruits treated with strain NJ157 was significantly reduced to 29.17%, and it had the best control effect on tomato bacterial wilt, reaching 58.82%.

[0117] 4. Determination of the disease protection ability of Bacillus velezensis NJ157 against Fusarium solani (fruit rot) on tomato fruits

[0118] Activate Bacillus velezensis NJ157 and model bacteria FZB42 on LB solid medium. Pick a single colony with a sterile toothpick and place it in 20 mL LB liquid medium. Shake and incubate at 37°C, 200 rpm overnight. The next day, transfer to 1% and incubate for 4-5 hours. 600 Adjust to 0.5(5×10 7CFU / mL) for later use. Activate the preserved Fusarium solani on the PDA plate, and use a gun tip to obtain a bacterial disc for later use. Use 75% anhydrous ethanol to disinfect the surface of tomato fruits of basically the same size. Place the disinfected tomato fruits upside down in different treatment solutions and soak for 30 minutes, then take them out and place them upright in a perforated tray (a tray with a layer of sterile water at the bottom). Allow the different treatment solutions on the surface of the tomatoes to dry naturally. Use a sterile syringe to gently poke a hole on the top of the tomato fruit, fit the obtained F. solani bacterial disc tightly into the hole of the tomato fruit, and wrap the bacterial disc with absorbent cotton to retain water. Wrap the fruit with plastic wrap, place it in a greenhouse at 25°C, and observe the subsequent disease development. Calculate the diseased area of ​​F. solani on the tomato fruit, and the results are as follows Fig.11 And as shown in Table 5.

[0119] Table 5 The control effect of Bacillus Velezii NJ157 on fruit rot on tomato fruit

[0120]

[0121] Note: The same letters in each column indicate that the difference is not significant (P>0.05), while the different letters indicate that the difference is significant (P<0.05).

[0122] The experimental results showed that treatment of tomato plants with Bacillus Velez NJ157 inhibited the occurrence of F. solani on tomato fruits. The disease index of tomato fruits treated with Bacillus Velez NJ157 was significantly lower than that of tomato fruits treated with CK. Bacillus Velez had the best control effect on tomato fruit rot, reaching 81.75%.

[0123] Example 6. Study on the growth-promoting effect of Bacillus Velez NJ157 on tomato

[0124] 1. Effect of Bacillus Velez NJ157 on tomato seed germination

[0125] Tomato seed treatment: Select healthy, plump and uniform-sized tomato seeds, soak them in 30% sodium hypochlorite solution for 2 minutes, then soak them in 75% alcohol solution for 30 seconds, rinse them with sterile water 5 times, absorb 50 μL of the rinse solution and add it to PDA solid culture medium and LB solid culture medium, spread it evenly with a sterile spreading rod, and culture at a constant temperature of 25°C for 24 hours. If no colonies appear, it means that the seed treatment is complete.

[0126] The Bacillus Velezii bacterial solution was washed twice with water to remove the secondary metabolites produced by the strain itself, and then diluted to obtain a final concentration of 10 7CFU / mL of Velez's Bacillus suspension. Place the sterilized tomato seeds in a seed germination bag and inject 25mL of Velez's Bacillus suspension into the seed germination bag. Place the seed germination bag in a 25℃ greenhouse and collect samples after 7 days to measure the root length and shoot length of the tomato seedlings. The results are as follows: Fig.12 shown.

[0127] The results showed that Bacillus Velez NJ157 had a significant growth-promoting effect on tomato seeds. Strain NJ157 had the best effect on promoting tomato root length, with a growth promotion rate of up to 52.64%; the growth promotion rate of tomato sprout length by strain FZB42 was 20.21%, and the growth promotion rate of tomato sprout length by strain NJ157 was 35.19%. The growth promotion rate of Bacillus Velez NJ157 on tomato growth was higher than that of the model strain FZB42, indicating that this strain has a good growth-promoting effect.

[0128] 2. Determination of the growth-promoting effect of Bacillus velez NJ157 on tomato plants

[0129] Disinfection and germination of tomato seeds: Pick complete and full seeds, soak them in 30% sodium hypochlorite solution for 120s, then soak them in 75% ethanol for 30s, and then rinse them repeatedly with ddH2O for 5 times until there is no irritating smell left on the surface of the tomato seeds; spread a sterile filter paper sheet on a 9cm sterilized glass culture dish, add an appropriate amount of sterile water with a pipette, so that the filter paper in the dish is completely soaked with water; use sterile tweezers to pick up tomato seeds and place them on the filter paper, paying attention to the certain distance between the seeds so that they can germinate; after the glass dish is sealed, place it in a 25℃ greenhouse and culture it away from light for 3-5 days. After it grows radicles, it can be transplanted to 1 / 2MS.

[0130] Planting of tomato seedlings: sieve the black soil and mix it with vermiculite in a ratio of 1:1, sterilize it twice at 121℃ for 20min and set aside; use a hole puncher with a diameter of about 2-3cm to punch a hole in the center of a disposable plastic cup, then add 80% of the mixture of black soil and vermiculite, use tweezers to pick up tomato seeds that have grown radicles, plant them in the soil with the roots facing down, cover the top with a layer of soil, place the disposable plastic cup in a tray, add an appropriate amount of water in the tray, and culture it in a greenhouse at 28℃ for about 20 days.

[0131] Root irrigation treatment with fermented liquid of Bacillus Velez NJ157: After the tomato seedlings break through the soil and spread their leaves, select the plants with the same growth trend and dilute the seed liquid of Bacillus Velez NJ157 and the model bacteria FZB42 strain to 10% with sterile water. 7 CFU / mL, 50 mL of a final concentration of 10 7 CFU / mL bacterial suspension, sterile water as blank control, after 20 days of culture, statistical growth data, the results are as follows Fig.13 shown.

[0132] The results showed that NJ157 had a good promoting effect on the growth of tomato plants. Among them, strain NJ157 had the best promoting effect on the aboveground dry weight of tomatoes, underground dry weight and fresh weight, with the promotion rate reaching 43.75%, 81.25% and 172.73%; strain NJ157 had a good promoting effect on the production of chlorophyll and stem diameter of tomatoes, with the promotion rate being 42.43% and 36.00% respectively; the growth promotion rate of this strain on tomato growth was higher than that of the model strain FZB42, indicating that Bacillus Velezii NJ157 had a good growth promoting effect and had certain development potential.

[0133] Example 7. Study on the growth-promoting effect of Bacillus Velez NJ157 on tomato plants

[0134] Tomato seed disinfection and germination: Pick complete and full seeds, disinfect with 30% sodium hypochlorite solution for 3 minutes, disinfect with 75% ethanol for 45 seconds, and then wash with sterile water for 4 times until there is no irritating smell left on the surface of the tomato seeds; spread a sterile filter paper sheet on a 9 cm sterilized glass culture dish, add an appropriate amount of sterile water with a pipette, so that the filter paper in the dish is completely soaked with water; use sterile tweezers to pick up tomato seeds and place them on the filter paper, with a certain distance between the seeds to facilitate germination; after the glass dish is sealed, place it in a 25°C greenhouse and culture it away from light for 3-5 days. After it grows radicles, it can be transplanted into a disposable plastic cup.

[0135] Planting of tomato seedlings: sieve the black soil and mix it with vermiculite in a 1:1 ratio, sterilize it twice at 121℃ for 20mins, and set aside for later use; use a hole puncher with a diameter of about 2-3cm to punch a hole in the center of a disposable plastic cup, then add the sterilized mixture of black soil and vermiculite, use tweezers to pick up tomato seeds that have grown radicles, plant them in the soil with the roots facing down, cover the top with a layer of soil, place the disposable plastic cup in a tray, add an appropriate amount of water in the tray, and culture it in a greenhouse at 25℃ for about 20 days.

[0136] Root irrigation treatment with fermented liquid of Bacillus Velez NJ157: After the tomato seedlings break through the soil and spread their leaves, select the plants with the same growth trend and dilute the seed liquid of Bacillus Velez NJ157 and the model bacteria FZB42 strain to 10% with sterile water. 7 CFU / mL, 50 mL of a final concentration of 10 7 CFU / mL bacterial suspension, sterile water as a blank control. Salt stress treatment began 2 days after the irrigation treatment. A 200mM NaCl solution was added to each tray for treatment. The salt solution was poured every three days. After 30 days of cultivation, the growth promotion data were counted. The results are as follows Fig.14 shown.

[0137] The experimental results showed that Bacillus Velez NJ1507 had a good promoting effect on tomato growth in salt stress environment. Among them, Bacillus Velez NJ157 had the best promoting effect on tomato plant height, chlorophyll content, fresh weight of aboveground part, fresh weight of underground part and dry weight, with the promotion rate as high as 71.21%, 63.63%, 246.62%, 468.75% and 328.57%, and the growth promotion rate of strain NJ157 on tomato growth was not lower than that of model strain FZB42, indicating that Bacillus Velez NJ157 still had a good growth promotion effect under salt stress environment and had high development potential.

[0138] As can be seen from the above embodiments, the present invention provides a strain of Bacillus Velez NJ1507 and its application. The Bacillus Velez NJ1507 of the present invention has the ability of salt-alkali tolerance, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot, has a good growth-promoting effect on tomatoes, and has great application potential in biological pesticides.

[0139] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus Velezii NJ157, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31984, the deposit date is September 14, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing the bacterial solution of Bacillus Velez NJ157 according to claim 1, characterized in that: The Bacillus Velez NJ157 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

3. The preparation method according to claim 2, characterized in that: The fermentation temperature is 28-32° C., the fermentation speed is 180-220 rpm, and the fermentation time is 22-26 h.

4. Use of the Bacillus Velez NJ157 of claim 1 in all or part of the following (1) to (6): (1) Application in inhibiting pathogenic oomycetes, pathogenic bacteria and pathogenic fungi; (2) Application in inhibiting tomato diseases; (3) Application in promoting tomato growth in severe saline-alkali land; (4) Application in decomposing protein or cellulose; (5) Application in the preparation of tomato growth promoter in severe saline-alkali soil; (6) Application in the preparation of bacterial agents for decomposing protein or cellulose.

5. The use according to claim 4, characterized in that: The pathogenic oomycetes include Phytophthora capsici; the pathogenic bacteria include Pseudomonas syringae and Ralstonia solanacearum; the pathogenic fungi include Botrytis cinerea, Fusarium moniliforme, Fusarium solani and Rhizoctonia solani.

6. The use according to claim 4, characterized in that: The tomato diseases include bacterial wilt, root rot and fruit rot.

7. A microbial agent, characterized in that: The microbial agent comprises the bacterial suspension of Bacillus Velez NJ157 according to claim 1.

8. The microbial agent according to claim 7, characterized in that: The spore concentration in the bacterial suspension is 1 to 9×10 7 CFU / mL.

9. A method for promoting tomato growth in severe saline-alkali land, characterized in that: The tomato plants are irrigated with the microbial agent described in claim 7 or 8.

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