Bacillus paralicheniformis NJ102 and application thereof

Abstract

The invention provides bacillus paralicheniformis NJ102 and application thereof, and belongs to the technical field of microorganisms. The bacillus paralicheniformis NJ102 disclosed by the invention has the capabilities of resisting low temperature and saline and alkaline, and can endure the low temperature of 10 DEG C, the salt stress with the salt content of 17% and the strong alkali stress with the pH value of 12.5. Meanwhile, the bactericidal composition has a relatively good inhibition effect on ralstonia solanacearum, rhizoctonia solanacearum and phytophthora capsici, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot. The bacillus paralicheniformis disclosed by the invention also has relatively strong capability of producing protease, cellulase, amylase and IAA (Indoleacetic Acid), has a relatively good growth promoting effect on tomatoes, and has great application potential in the aspect of biopesticide.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus paralicheniformis NJ102 and an application thereof. Background Art

[0002] As we all know, traditional chemical pesticides have problems such as low safety, large residue, and difficult toxin degradation. Irrational use often causes water, soil, air pollution and toxic residues in agricultural products, seriously endangering the ecosystem and human health, and has become an important factor restricting human environmental governance, healthy survival, steady agricultural growth and sustainable development. At present, countries around the world attach importance to the development of biological pesticides with high safety, low residue, no pollution, high biological activity and strong selectivity to replace the currently widely used chemical pesticides or reduce the use of chemical pesticides.

[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 is 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 multifunctional biocontrol strains that are resistant to low temperatures and salt and alkali isolated from Antarctica. Summary of the invention

[0004] The object of the present invention is to provide a strain of Bacillus paralicheniformis NJ102 and an application thereof. The Bacillus paralicheniformis NJ102 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, thereby 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 paralicheniformis NJ102, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC No. 31983, a deposit date of September 14, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a method for preparing the bacterial solution of the Bacillus paralicheniformis NJ102, wherein the Bacillus paralicheniformis NJ102 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial solution.

[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 paralicheniformis NJ102 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, cellulose or starch;

[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, cellulose or starch.

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

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

[0018] Preferably, the microbial agent comprises a bacterial suspension of the strain 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 severe saline-alkali land, wherein the tomato plants are irrigated with the bacterial agent.

[0021] By adopting the above technical scheme, the present invention has the following beneficial effects: the Bacillus paralicheniformis NJ102 of the present invention has the ability to resist low temperature and salt and alkali, can tolerate low temperature of 10°C, salt stress with a salt content of 17% and strong alkali stress with a pH of 12.5. At the same time, it has a good inhibitory effect on Ralstonia solanacearum, Rhizoctonia solani and Phytophthora capsici, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot. The Bacillus paralicheniformis of the present invention also has a strong ability to produce protease, cellulase and amylase, and can also produce IAA, which has a good growth-promoting effect on tomatoes. The Bacillus paralicheniformis NJ102 of the present invention has the ability to resist low temperature, salt and alkali, prevent and treat a variety of pathogens, produce a variety of enzymes, and has a good growth-promoting effect on tomatoes, and has great application potential in biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of Bacillus paralicheniformis NJ102.

[0023] Figure 2 This is a diagram showing the inhibitory effect of Bacillus paralicheniformis NJ102 on pathogens, wherein A is Ralstonia solanacearum, B is Pseudomonas syringae, C is Botrytis cinerea, D is Phytophthora capsici, and E is Rhizoctonia solani.

[0024] Figure 3 This is a graph showing the results of the cold stress tolerance test of Bacillus paralicheniformis NJ102, where the left side shows the growth of the strain at 10°C; the right side shows the growth of the strain at 7°C.

[0025] Figure 4 The graphs are the results of the salt stress tolerance test of Bacillus paralicheniformis NJ102, wherein AC are the plate phenotype results of the salt tolerance (11%, 15%, 17%) of Bacillus paralicheniformis NJ102 and the model strains FZB42, 168, and DMW1, respectively.

[0026] Figure 5 This is the test for the alkali stress resistance of Bacillus paralicheniformis NJ102, where AF: alkali resistance (pH = 8, 9, 10, 11, 12, 12.5) plate phenotype results of Bacillus paralicheniformis NJ102, FZB42, 168, and DMW1.

[0027] Figure 6 This is the HPLC-MS identification result of the secondary metabolites of Bacillus paralicheniformis NJ102.

[0028] Figure 7 This is the antibacterial effect diagram of the crude extract of secondary metabolites of Bacillus paralicheniformis NJ102, where A is Ralstonia solanacearum, B is Pseudomonas syringae, C is Botrytis cinerea, D is Phytophthora capsici, and E is Rhizoctonia solani.

[0029] Figure 8 This is a graph showing the detection results of protease, cellulase, amylase and indoleacetic acid (IAA) produced by Bacillus paralicheniformis NJ102, wherein A is protease, B is cellulase, C is amylase, and D is indoleacetic acid.

[0030] Fig. 9 This is a diagram showing the disease prevention effect of Bacillus paralicheniformis NJ102 on bacterial wilt infection on tomato plants.

[0031] Fig.10 This is a diagram showing the disease prevention effect of Bacillus paralicheniformis NJ102 against pepper phytophthora (P. capsici) infection on tomato plants.

[0032] Fig.11 This is a diagram showing the disease prevention effect of Bacillus paralicheniformis NJ102 against pepper phytophthora (P. capsici) infection of tomato fruits.

[0033] Fig.12 This is a diagram showing the disease prevention effect of Bacillus paralicheniformis NJ102 against Fusarium solani infection in tomato fruit.

[0034] Fig.13 This is a diagram showing the effect of Bacillus paralicheniformis NJ102 on promoting tomato seed germination.

[0035] Fig.14 Figure 1 is the effect of Bacillus paralicheniformis NJ102 on the growth of tomato plants, where A is the effect of Bacillus paralicheniformis NJ102 on tomato plant height and root length; and B1 is the statistical chart of the effects of Bacillus paralicheniformis NJ102 on tomato plant height, root length, chlorophyll content, stem diameter, aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight.

[0036] Fig.15Figure 3 shows the effect of Bacillus paralicheniformis NJ102 on the growth of tomato plants under salt stress, where A is the effect of Bacillus paralicheniformis NJ102 on tomato plant height and root length under salt stress; BI is a statistical chart of the effect of Bacillus paralicheniformis NJ102 on 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.

[0037] Biological Deposit Description

[0038] The Bacillus paralicheniformis NJ102 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.31983, a deposit date of September 14, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1. Analysis of the taxonomic status of Bacillus paralicheniformis NJ102 based on the core genome

[0041] The NJ102 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 amplified by PCR using the forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No.3) and the 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 the amplification of the 16SrRNA fragment was observed by gel electrophoresis, the PCR stock solution was sent for sequencing. The 16SrDNA gene sequence is shown in SEQ ID No.1.

[0042] 16SrDNA gene sequence:

[0043]

[0044] PCR amplification and sequence determination of gyrB gene

[0045] Forward primer gyrB-F:

[0046] 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'

[0047] (SEQ ID No.5);

[0048] Reverse primer gyrB-R:

[0049] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'

[0050] (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 1186 bp, and the result is shown in the sequence table SEQ ID No.2.

[0051] gyrB gene sequence:

[0052]

[0053] The results of 16S rDNA and gyrB identification showed that strain NJ102 belonged to Bacillus paralicheniformis. Secondly, according to the genome sequence of NJ102, 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 NJ102 and Bacillus paralicheniformis was the closest. The above results show that the Antarctic strain NJ102 belongs to Bacillus paralicheniformis.

[0054] Example 2 Determination of antibacterial ability of Bacillus paralicheniformis NJ102

[0055] The antibacterial ability of Bacillus paralicheniformis NJ102 was preliminarily determined, and several representative pathogenic bacteria and pathogenic fungi were selected. Among them, the pathogenic bacteria included Ralstonia solanacearum and Pseudomonas syringae, and the pathogenic fungi included Botrytis cinerea, Phytophthora capsici, and Rhizoctonia solani.

[0056] Determination of the antibacterial ability of Bacillus paralicheniformis NJ102 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 rate of 1%, mix thoroughly and pour into the bacteria-containing plate. Add 5 μL of the bacterial solution of Bacillus paralicheniformis NJ102 (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.

[0057] Determination of the antibacterial ability of Bacillus paralicheniformis NJ102 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, and 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. When 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 cake, and drop 5 μL of the bacterial solution of Bacillus paralicheniformis NJ102 (OD 600=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 paralicheniformis NJ102 had good antagonistic effects on a variety of plant pathogenic fungi and bacteria, among which the best inhibitory effects were on Ralstonia solanacearum, Rhizoctonia solani and Phytophthora capsici.

[0058] Example 3 Determination of low temperature resistance, salt resistance and alkali resistance of Bacillus paralicheniformis NJ102

[0059] 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 paralicheniformis NJ102 was tested.

[0060] 1. Determination of low temperature stress tolerance

[0061] Different low temperature stresses (temperature gradient: 10℃, 7℃) were simulated to determine the ability of Bacillus paralicheniformis NJ102, model strain Bacillus subtilis 168, Bacillus velezensis FZB42 and Bacillus velezensis DMW1 to resist low temperature stress. Bacillus paralicheniformis NJ102 and model strains 168, FZB42 and DMW1 were inoculated into LB liquid medium, respectively, and cultured at 200r / min and 37℃ for 24h. When the bacterial solution concentration was OD 600 =2.0, transfer 5 μL of bacterial solution and drop it on LB solid medium, grow it in 10℃ and 7℃ incubators for 5 days, and observe the growth of the test strain at different temperatures. Repeat biologically for 3 times. The results are as follows Figure 3 As shown. The experimental results show that Bacillus paralicheniformis NJ102 can grow at a minimum temperature of 10°C, while the model strains FZB42, 168 and DMW1 can hardly grow. Bacillus that can grow at 4°C or 10°C are called low-temperature adaptable Bacillus. The results show that Bacillus paralicheniformis NJ102 has good low-temperature resistance and belongs to low-temperature adaptable Bacillus.

[0062] 2. Determination of salt stress tolerance

[0063] Different salt stresses (salt gradient: 11%, 15%, 17% NaCl concentration) were simulated to determine the ability of Bacillus paralicheniformis NJ102, model strain 168, FZB42 and DMW1 to resist salt stress. Single colonies of the test strains were picked into LB liquid culture 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%, 15%, 17%), and then put the medium into a 37°C incubator. After 5 days, observe the colony growth of each strain under different salt gradients. Biologically repeat 3 times. The results are as follows Figure 4 The results showed that Bacillus paralicheniformis NJ102 could grow under salt stress conditions with a salt content of 17%, while the model strain FZB42 could only tolerate salt stress of 11% NaCl. Its salt stress tolerance was higher than that of the model strain FZB42.

[0064] 3. Determination of alkaline stress tolerance

[0065] Different alkaline stresses (pH gradient: 8, 9, 10, 11, 12, 12.5) were simulated to test the ability of Bacillus paralicheniformis NJ102, 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 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 alkaline gradients (pH 8, 9, 10, 11, 12, 12.5), 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 5 The results showed that Bacillus paralicheniformis NJ102 could grow under strong alkaline stress conditions of pH 12.5, and its ability to resist alkaline stress was higher than that of the model strain FZB42.

[0066] Example 4 Extraction, identification and activity determination of secondary metabolites of Bacillus paralicheniformis NJ102

[0067] 1. Extraction and identification of secondary metabolites of Bacillus paralicheniformis NJ102

[0068] Activate Bacillus paralicheniformis NJ102 on LB solid medium and place in a 37°C incubator until a single colony grows; pick a single colony of NJ102 to 20 mL of LB liquid medium and culture overnight at 37°C and 200 rpm; the next day, transfer five Bacillus paralicheniformis NJ102 bacterial liquids to 200 mL of LB liquid medium at a ratio of 1%, and culture at 37°C and 200 rpm for 24 hours; add 5 mL of resin (XAD-16N) to each NJ102 bacterial liquid, and continue to culture for 24 hours. The next day, the bacterial solution was centrifuged at 8000 rpm for 10 min to collect the bacteria; 30 mL of methanol was used to dissolve the bacteria, and then the bacterial suspension was continued to be placed at 37°C and shaken at 200 rpm for 4 h, and the bacterial suspension was centrifuged at 8000 rpm for 10 min, the supernatant was collected and filtered with filter paper, and the supernatant was concentrated to 2 mL using a rotary evaporator, and then the crude extract was filtered with a 0.22 μm organic filter, and the production of lipopeptides was identified and detected using a high performance liquid chromatography-mass spectrometry (HPLC-MS). The results are as follows Figure 6 As shown in the results of identification, it can be seen that the secondary metabolites of NJ102 contain iturin and surfactin.

[0069] 2. Determination of antibacterial ability of secondary metabolites of Bacillus paralicheniformis NJ102

[0070] A plate confrontation experiment was conducted using the crude extract of secondary metabolites produced by Bacillus paralicheniformis NJ102 against (A) Ralstonia solanacearum, (B) Pseudomonas syringae, (C) Botrytis cinerea, (D) Phytophthora capsici and (E) Rhizoctonia solani. A certain number of bacterial cakes were punched from the outermost edge of the colony on the original culture plate of the pathogenic fungi using a sterile puncher with a diameter of about 0.5 cm. The bacterial cakes (the side containing hyphae) were then transferred to the center of a new PDA culture plate and cultured in an incubator at a suitable temperature for 10-18 hours. The cakes were ready for use after new hyphae grew around them. Then, a 0.5 cm diameter sterile punch was used to punch a hole at the same distance from the bacterial cake, and 50 μL of the secondary metabolites of Bacillus paralicheniformis NJ102 was dripped into the center of the hole. Methanol solution was used as a control. After drying, the hole was returned to the incubator. The antibacterial effect was observed after 48 hours. The results are as follows: Figure 7 shown.

[0071] The results of the plate confrontation experiment showed that the secondary metabolites of Bacillus paralicheniformis NJ102 had a good antagonistic effect on a variety of plant pathogenic fungi and bacteria, among which the inhibitory effect on Ralstonia solanacearum, R. solani and B. cinerea was the best.

[0072] 3. Determination of the ability of Bacillus paralicheniformis NJ102 to produce extracellular hydrolases

[0073] The ability of Bacillus paralicheniformis NJ102 to produce extracellular hydrolases was detected by plate assay, and the qualitative detection of protease production by Bacillus paralicheniformis NJ102 was performed on skim milk agar plates: the ability of Bacillus paralicheniformis NJ102 to produce protease was determined in agar medium containing skim milk powder (20g skim milk powder, 20g agar, pH=7.0, sterilized at 115℃ for 10min). A single colony of Bacillus paralicheniformis NJ102 was picked up and placed in LB liquid medium, and cultured overnight at 37℃, 200r / min for 12h; 5μL (OD 600 =1.0) was placed in a 37°C constant temperature incubator for culture, and after 24-96 hours, whether a transparent circle was formed around the colonies was observed.

[0074] The cellulase production capacity of Bacillus paralicheniformis NJ102 was determined using CMC-Na medium: a single colony of Bacillus paralicheniformis NJ102 was picked up and placed in LB liquid medium, and cultured overnight at 37°C and 200 r / min for 12 hours. On the CMC-Na medium plate, sterile filter paper discs with a diameter of 4 mm were placed in a "cross" shape; 5 μL of bacterial suspension (OD 600 =1.0), inoculated in the center of the filter paper, sealed the plate, and placed it in a 37°C constant temperature incubator for culture; after 2 days, took out the culture dish, injected Gram iodine staining solution until the surface of the plate was submerged, let it stand for 4 minutes, then poured out the staining solution, and observed whether a transparent circle was formed around the colony. If a transparent hydrolysis circle appeared around the bacteria, it means that the strain can produce the corresponding enzyme. If no transparent hydrolysis circle appeared, it means that the strain cannot produce such enzyme.

[0075] Starch agar medium was used to detect the amylase activity of Bacillus paralicheniformis NJ102: colonies of Bacillus paralicheniformis NJ102 were picked into 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 8 shown.

[0076] The results showed that Bacillus paralicheniformis NJ102 could produce a large transparent hydrolysis zone in CMC-Na medium, which indicated that it had a strong ability to produce cellulase.

[0077] 4. Determination of IAA production capacity of Bacillus paralicheniformis NJ102

[0078] 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.

[0079] Determination of the ability of Bacillus paralicheniformis NJ102 to produce indoleacetic acid (IAA): 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 100 mg / L tryptophan at a content of 1%. After culturing at 37°C and 180rpm for 24 hours, 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. An equal volume of LB liquid containing 100 mg / L tryptophan was used as a blank control. The results are as follows Figure 8 As shown in D.

[0080] The results showed that Bacillus paralicheniformis NJ102 was able to produce IAA, and the IAA production capacity of strain NJ102 was stronger than 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.

[0081] Example 5 Determination of the ability of Bacillus paralicheniformis NJ102 to control tomato diseases

[0082] 1. Determination of the control ability of Bacillus paralicheniformis NJ102 against tomato bacterial wilt

[0083] 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 paralicheniformis NJ102 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 paralicheniformis NJ102 solution per treatment. Sterile water irrigated roots 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 Fig. 9 And as shown in Table 1.

[0084] Incidence rate (%) = (number of diseased plants / total number of plants) × 100%

[0085] Disease classification survey standards:

[0086] 0: no wilting;

[0087] 1: 1%-25% leaf area wilting;

[0088] 2: 26%-50% leaf area wilting;

[0089] 3: 51%-75% leaf area wilting;

[0090] 4: 76%-100% leaf area wilts or dies.

[0091] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0092] Control effect = (blank control disease index - treatment disease index) / blank control disease index x 100%.

[0093] Table 1 Control effect of Bacillus paralicheniformis NJ102 on tomato bacterial wilt

[0094]

[0095] 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).

[0096] The experimental results showed that the incidence of tomato bacterial wilt was reduced after the tomato plants were infected with Bacillus paralicheniformis NJ102. Among them, the incidence of tomatoes treated with strain NJ102 was significantly reduced to 66.67%, which was 16.66% lower than the blank control group; the disease index of tomatoes treated with strain NJ102 was significantly reduced, and the prevention and control effect of NJ102 on tomato bacterial wilt was 48.32%.

[0097] 2. Determination of the ability of Bacillus paralicheniformis NJ102 to control tomato root rot

[0098] 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 paralicheniformis NJ102 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 10 mL of Bacillus paralicheniformis NJ102 bacterial 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 inoculated at the rhizosphere 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.10 And as shown in Table 2.

[0099] Disease classification survey standards:

[0100] 0: no wilting;

[0101] 1: 1%-25% leaf area wilting;

[0102] 2: 26%-50% leaf area wilting;

[0103] 3: 51%-75% leaf area wilting;

[0104] 4: 76%-100% leaf area wilts or dies.

[0105] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0106] Control effect = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0107] Table 2 Control effect of Bacillus paralicheniformis NJ102 on tomato root rot

[0108]

[0109] 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).

[0110] The experimental results showed that the incidence of tomato root rot was reduced after the tomato plants were treated with Bacillus paralicheniformis NJ102. The incidence of tomato treated with Bacillus paralicheniformis NJ102 was the lowest, at 66.67%, which was 13.88% lower than the blank control group. The disease index of tomato root rot treated with Bacillus paralicheniformis NJ102 was significantly reduced, and the control effect on tomato root rot was the best, reaching 40.75%.

[0111] 3. Determination of the disease-preventive ability of Bacillus paralicheniformis NJ102 against Phytophthora capsici on tomato fruits

[0112] Activate Bacillus paralicheniformis NJ102 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. Cultivate overnight at 37°C and 200 rpm. The next day, transfer to 1% and culture 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 results are as follows. Fig.11And as shown in Table 4.

[0113] Table 3 Pepper Phytophthora disease classification survey standards

[0114]

[0115] Disease index (%) = [∑ (disease level value × number of plants with that disease level) / (highest level number × number of plants surveyed)] × 100.

[0116] Control effect = (blank control disease index - treatment disease index) / blank control disease index × 100%.

[0117] Table 4 Control effect of Bacillus paralicheniformis NJ102 on Phytophthora capsici on tomato fruits

[0118]

[0119] 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).

[0120] The experimental results showed that treatment of tomato plants with Bacillus paralicheniformis NJ102 inhibited the occurrence of P. capsici in tomato fruits, and the disease index of tomato fruits treated with strain NJ102 was significantly reduced. Among them, the control effect of NJ102 on tomato bacterial wilt reached 52.94%, which was higher than the control effect of the model strain FZB42.

[0121] 4. Determination of the disease protection ability of Bacillus paralicheniformis NJ102 against Fusarium solani (fruit rot) on tomato fruits

[0122] Activate Bacillus paralicheniformis NJ102 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. Cultivate overnight at 37°C and 200 rpm. The next day, transfer to 1% and culture 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.12 And as shown in Table 5.

[0123] Table 5 Control effect of Bacillus paralicheniformis NJ102 on fruit rot on tomato fruit

[0124]

[0125] 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).

[0126] The experimental results showed that treatment of tomato plants with Bacillus paralicheniformis NJ102 inhibited the occurrence of F. solani on tomato fruits, and the disease index of tomato fruits treated with strain NJ102 was significantly reduced. Among them, the control effect of NJ102 on tomato fruit rot reached 51.63%, which was higher than the control effect of the model strain FZB42.

[0127] Example 6 Study on the Growth-Promoting Effect of Bacillus paralicheniformis NJ102 on Tomato

[0128] 1. Effect of Bacillus paralicheniformis NJ102 on tomato seed germination

[0129] 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.

[0130] The bacterial solution of Bacillus paralicheniformis NJ102 was washed twice to remove the secondary metabolites produced by the strain itself, and then diluted to obtain a final concentration of 10 7CFU / mL of Bacillus paralicheniformis NJ102 suspension. Place the sterilized tomato seeds in a seed germination bag and inject 25mL of Bacillus paralicheniformis NJ102 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 bud length of the tomato seedlings. The results are as follows: Fig.13 The results showed that Bacillus paralicheniformis NJ102 had a certain growth-promoting effect on tomato seed root length and sprout length, among which the growth-promoting effect on tomato root length was 32.27%; the growth-promoting effect on tomato sprout length was 27.55%; and the growth-promoting effect on tomato growth was higher than that of the model strain FZB42, indicating that Bacillus paralicheniformis NJ102 had a good effect on promoting tomato seed germination.

[0131] 2. Determination of the growth-promoting effect of Bacillus paralicheniformis NJ102 on tomato plants

[0132] Disinfection and germination of tomato seeds: Pick up complete and full seeds, soak them in 30% sodium hypochlorite solution for 120 seconds, then soak them in 75% ethanol for 30 seconds, 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 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. During this process, pay attention to the certain distance between the seeds so that they can germinate; after the glass dish is sealed, place it in a 25°C greenhouse and culture it in the dark for 3-5 days. After it grows radicles, it can be transplanted to 1 / 2MS.

[0133] 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.

[0134] Root irrigation treatment with Bacillus paralicheniformis NJ102 fermentation liquid: After the tomato seedlings break through the soil and unfold their leaves, pick the plants with the same growth trend, dilute the seed liquid of Bacillus paralicheniformis NJ102 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.14As shown. The results showed that Bacillus paralicheniformis NJ102 had a good promoting effect on the growth of tomato plants. Among them, strain NJ102 had the best promoting effect on tomato root length, fresh weight of aboveground part and dry weight, with the promotion rate reaching 50.84%, 40.08% and 43.75%; strain NJ102 had a good promoting effect on tomato chlorophyll production, root fresh weight and dry weight, with the promotion rate being 28.86%, 43.75% and 72.73% respectively; and the growth promotion rate of this strain on tomato growth was similar to that of the model strain FZB42, indicating that Bacillus paralicheniformis NJ102 had a good growth promoting effect and had certain development potential.

[0135] Example 7. Study on the growth-promoting effect of Bacillus paralicheniformis NJ102 on tomato plants under salt stress

[0136] 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 the tomato seeds and place them on the filter paper. During this process, pay 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 into a disposable plastic cup.

[0137] 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.

[0138] Root irrigation treatment with Bacillus paralicheniformis NJ102 fermentation liquid: After the tomato seedlings break through the soil and unfold their leaves, pick the plants with the same growth trend, dilute the seed liquid of Bacillus paralicheniformis NJ102 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.15As shown. The experimental results show that Bacillus paralicheniformis NJ102 has a good promoting effect on the growth of tomatoes in salt stress environment. Among them, NJ102 has a good promoting effect on tomato root length, chlorophyll content, fresh weight, dry weight of aboveground parts and fresh weight and dry weight of underground parts, with promotion rates of 90.41%, 54.23%, 191.73%, 257.14% and 191.25%, 85.27% respectively. The growth promotion rate of Bacillus paralicheniformis NJ102 on tomato growth is not lower than that of the model strain FZB42, indicating that this strain still has a good growth-promoting effect under salt stress environment and has certain development potential.

[0139] From the above examples, it can be seen that the present invention provides a strain of Bacillus paralicheniformis NJ102 and its application. The Bacillus paralicheniformis NJ102 of the present invention has the ability of low temperature resistance and salt-alkali resistance, 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.

[0140] 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 paralicheniformis NJ102, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31983, 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 paralicheniformis NJ102 according to claim 1, characterized in that: The Bacillus paralicheniformis NJ102 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 paralicheniformis NJ102 according to 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, cellulose or starch; (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, cellulose or starch.

5. The use according to claim 4, characterized in that: The pathogenic oomycetes include Phytophthora capsici; the pathogenic bacteria include Ralstonia solanacearum and Pseudomonas syringae; the pathogenic fungi include Botrytis cinerea, Rhizoctonia solani and Fusarium 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 paralicheniformis NJ102 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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