A tropical bacillus NB379 and its application and bacterial agent

Abstract

The present invention relates to the field of microbial technology, and in particular to a tropical Bacillus NB379, its application and bacterial agent. Tropical Bacillus NB379 was deposited in the China Center for Type Culture Collection on March 18, 2024, with a deposit number of CCTCC No: M2024509. Tropical Bacillus NB379 has acaricidal activity and stability under a variety of environmental stresses, and can grow stably in extreme pH ranges, saline-alkali conditions and heavy metal stresses. It has a unique ability to kill spider mites by contact, and can synthesize a variety of biologically active substances such as antibiotics, enzymes and plant hormones. It has a significant antibacterial effect on a variety of pathogens, shows a growth-promoting effect on plants such as tomatoes and Arabidopsis, and can reduce the toxic effects of heavy metals on plants.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a tropical bacillus NB379 and its application and bacterial agent. Background Art

[0002] With the continuous progress of modern agriculture, agricultural production is facing more and more challenges, including plant mites, plant pathogens and heavy metal pollution. Among them, the prevention and control of mites has always been an important and complex task. Mites are an important type of pest in agricultural production. They cause direct damage to plants by sucking plant sap, and also spread viruses and bacteria, further exacerbating the occurrence of plant diseases. Traditional chemical pesticides and fertilizers can control pests and diseases and increase yields to a certain extent, but the side effects of their long-term use, such as increased resistance to pesticides, environmental pollution, soil degradation and reduced crop quality, cannot be ignored. Therefore, seeking environmentally friendly and efficient microbial solutions has become an important direction for the development of modern agriculture.

[0003] Bacillus is a type of Gram-positive bacteria that is widely present in nature. It is widely used in the fields of biological control and plant growth promotion because of its advantages such as being able to produce a variety of antibacterial substances, promote plant growth, and improve plant stress resistance. However, the efficiency and effectiveness of existing Bacillus in adapting to complex environments, inhibiting pathogenic microorganisms, and promoting plant growth still need to be improved. Especially under multiple environmental stress conditions, its growth and metabolic activity may be inhibited, resulting in a significant reduction in its application effect. In addition, among the existing Bacillus resources, strains that have significant control effects on harmful mites are rare. Therefore, the development of Bacillus strains with multiple biological activities such as acaricidal activity and environmental tolerance is of great significance to improving the sustainability and safety of agricultural production. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a tropical Bacillus NB379 and its application and bacterial agent.

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

[0006] The present invention provides a tropical Bacillus (Bacillus tropicus) NB379, which was deposited in the China Center for Type Culture Collection on March 18, 2024, with a deposit number of CCTCCNo:M2024509.

[0007] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in preventing and controlling spider mites, wherein the spider mites include one or more of two-spotted spider mites, citrus mites and cinnabarinus spider mites.

[0008] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in promoting plant growth.

[0009] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in alleviating heavy metal stress of plants, wherein the heavy metals include one or more of copper, manganese, chromium, lead, cadmium and nickel.

[0010] The present invention also provides the use of tropical Bacillus NB379 described in the above technical scheme in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi include one or more of the pathogens of rice sheath blight, wheat fusarium wilt, wheat glumes blight, tomato wilt, tobacco black shank, cotton verticillium wilt, pear black spot and gray mold.

[0011] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing indole-3-acetic acid.

[0012] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in dissolving phosphorus and / or producing ammonium nitrogen.

[0013] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing catalase.

[0014] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing siderophore, wherein the siderophore is a catechol siderophore.

[0015] The present invention also provides a bacterial agent, which contains the tropical Bacillus NB379 described in the above technical solution, with a bacterial content of 1×10 6 CFU / mL.

[0016] The single colony of Bacillus tropicalis NB379 strain growing on LB agar plate is milky white and round, with the edge protruding from the center ( Figure 1 ), and the cell morphology was rod-shaped under microscopic examination. Through genome average nucleotide identity (ANI) comparison analysis, it was found that the ANI similarity between this strain and the model strain (N24) of Bacillus tropicus was 96.72%. Combining ANI analysis and physiological and biochemical identification, it was confirmed that NB379 was Bacillus tropicus. The strain was deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M2024509 and a deposit date of March 18, 2024.

[0017] The median lethal concentration (LC50) of the fermentation liquid of Bacillus tropicalis NB379 against Tetranychus urticae, Panonychus citri and Tetranychus cinnabarinus 50) were 9.8 μL / mL, 22.7 μL / mL and 16.3 μL / mL respectively.

[0018] Bacillus tropicalis NB379 has good environmental tolerance and shows good viability in the pH range of 5.0-10.0, in the heavy metal concentration range of 0.5mM to 64mM, and in saline-alkali environment (NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1, salt concentration range of 40mM-200mM).

[0019] Tropical Bacillus NB379 can effectively inhibit plant pathogens, including: rice sheath blight (Rhizoctonia solani), wheat fusarium wilt (Fusarium graminearum), wheat glumen blight (Phthys graminearum), tomato wilt (Fusarium oxysporum tomato-specific type), tobacco black shank (Phytophthora nicotianae), cotton verticillium wilt (Verticillium dahliae), pome fruit black spot (Alternaria alternata) and Botrytis cinerea.

[0020] Bacillus tropicalis NB379 has the ability to promote plant growth and development, such as producing siderophore, producing IAA, dissolving phosphate, and producing ammonium nitrogen. It has certain growth-promoting potential for tomato and Arabidopsis, and alleviates the effects of Cu 2+ , Mn 2+ 、Zn 2+ Cr 3+ , Fe 2+ , Pb 2 + 、Cd 2+ 、Ni 2+ The stress effects of heavy metals on plants.

[0021] Therefore, the tropical Bacillus NB379 of the present invention provides a new and efficient microbial resource for solving the problems of harmful mites, plant diseases and heavy metal pollution in agricultural production. The application of the NB379 strain is expected to reduce the use of chemical pesticides and fertilizers, increase crop yield and quality, and promote the sustainable development of agriculture, which has important practical application and promotion value.

[0022] Beneficial effects of the present invention:

[0023] The strain NB379 of the present invention is a tropical Bacillus (Bacillus tropicus), which has acaricidal activity and stability under various environmental stresses, and can grow stably in extreme pH ranges, saline-alkali conditions and heavy metal stresses. Compared with existing reports, the strain has a unique ability to kill spider mites, and can synthesize a variety of biologically active substances such as antibiotics, enzymes and plant hormones, has a significant antibacterial effect on a variety of pathogens, shows a growth-promoting effect on plants such as tomatoes and Arabidopsis, and can reduce the toxic effects of heavy metals on plants. This makes the strain have a wider application prospect in agricultural production, not only can it effectively control agricultural pests and diseases, improve crop yield and quality, but also can adapt to the needs of crop growth under different soil and environmental conditions. At the same time, the environmental tolerance of the strain also provides the possibility for its application in the fields of soil remediation and ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The morphological characteristics of strain NB379 include its colony morphology on LB solid plate (A) and SEM image (B);

[0026] Figure 2 This is a schematic diagram of the toxic effect of strain NB379 on two-spotted spider mites;

[0027] Figure 3 Figure 1 shows the experimental results of strain NB379 inhibiting plant pathogens. (A) Strain NB379 showed antibacterial ability against pathogenic fungi. Liquid LB medium was added to the left well of the bacterial block as a control. The supernatant of the fermentation liquid of strain NB379 was added to the right well as a treatment group. From left to right, the pathogens are: Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum, Fusarium oxysporum, and Glehnia littoralis. (B) strain NB379 showed antibacterial ability against pathogenic bacteria; from left to right, the pathogens were: Erwinia carotovora, Aeromonas hydrophila, Aeromonas verticillatus, Pantoea agglomerans, Escherichia coli, Pseudomonas cereus, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, and Xanthomonas nigromaculata;

[0028] Figure 4 The strain NB379 showed tolerance to heavy metals;

[0029] Figure 5 This is a comparison of the effect of strain NB379 in promoting plant growth in pot experiments;

[0030] Figure 6 The VOCs produced by strain NB379 promote plant growth;

[0031] Figure 7 To alleviate the stress of heavy metals on plant seed germination for strain NB379;

[0032] Figure 8 Strain NB379 alleviates the stress of heavy metals on the growth of plant seedlings.

[0033] Biological Deposit Description

[0034] Bacillus tropicus NB379, Latin name Bacillus tropicus, was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on March 18, 2024, with the deposit number CCTCCNo:M2024509. DETAILED DESCRIPTION

[0035] The present invention provides a tropical Bacillus (Bacillus tropicus) NB379, which was deposited in the China Center for Type Culture Collection on March 18, 2024, with a deposit number of CCTCC No: M2024509.

[0036] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in preventing and controlling two-spotted spider mites, wherein the spider mites include one or more of two-spotted spider mites, citrus mites and cinnabarinus spider mites.

[0037] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in promoting plant growth. In the present invention, the plant preferably includes tomato and / or Arabidopsis thaliana.

[0038] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in alleviating heavy metal stress of plants, wherein the heavy metals include one or more of copper, manganese, chromium, lead, cadmium and nickel.

[0039] The present invention also provides the use of tropical Bacillus NB379 described in the above technical scheme in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi include one or more of the pathogens of rice sheath blight, wheat fusarium wilt, wheat glumes blight, tomato wilt, tobacco black shank, cotton verticillium wilt, pear black spot and gray mold.

[0040] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing indole-3-acetic acid.

[0041] The present invention also provides the use of tropical Bacillus NB379 described in the above technical solution in dissolving phosphorus and / or producing ammonium nitrogen. In the present invention, the phosphorus preferably includes organic phosphorus and / or inorganic phosphorus, the inorganic phosphorus preferably includes calcium phosphate, and the organic phosphorus preferably includes lecithin.

[0042] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing catalase.

[0043] The present invention also provides the use of the tropical Bacillus NB379 described in the above technical solution in producing siderophore, wherein the siderophore is a catechol siderophore.

[0044] The present invention also provides a bacterial agent, which contains the tropical Bacillus NB379 described in the above technical solution, with a bacterial content of 1×10 6 CFU / mL. In the present invention, the bacterial agent preferably further comprises a carrier, and the carrier is preferably a solid carrier or a liquid carrier. In the present invention, the dosage form of the bacterial agent is preferably a liquid, an emulsion, a suspension, a powder, a granule, a wettable powder or a water-dispersible granule.

[0045] In the present invention, the method of using the bacterial agent preferably includes: applying the bacterial agent to the roots, leaves or soil of the plant, and the application amount is preferably: after applying to the roots or soil, the bacterial content per liter of soil is 1×10 7 CFU.

[0046] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] The culture medium components involved in the embodiment are:

[0048] (1) LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, pH 7.2, fixed volume to 1 L.

[0049] (2) Fermentation medium No. 1 (mass percentage): 3% soybean meal, 1.5% corn syrup, 1.5% corn starch, pH 7.0-7.5.

[0050] (3) SPA medium: 5.0 g peptone, 20.0 g sucrose, 0.5 g KH2PO4, 0.25 g MgSO4, fixed volume to 1 L.

[0051] (4) PDA medium: Prepare an extract from 200 g of potato, 20.0 g of glucose, and 15.0 g of agar, and adjust the volume to 1 L without adjusting the pH.

[0052] (5) MSA liquid culture medium: sucrose 20.0 g, L-asparagine 2.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, pH 7.0, fixed volume to 1 L.

[0053] (6) NBRIP medium: glucose 10 g, MgCl2 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 1.0 g, pH 7.0, fixed volume to 1 L, agar 15 g.

[0054] (7) Peptone water: 10.0 g peptone, 5.0 g NaCl, pH 7.0, make up to 1 L.

[0055] (8) CAS detection solution: chrome azuro blue 1.0 mM, FeCl3 0.1 mM, hexadecyltrimethylammonium bromide 4.0 mM, phosphate buffer 0.1 M, pH 6.8.

[0056] Example 1

[0057] Screening of Bacillus tropicalis NB379

[0058] (1) Isolation of Bacillus strains:

[0059] The applicant collected samples from the silt of Xiandao Lake and selected about 100g of fertile soil in the 5-10cm area of ​​the silt surface. The collected silt soil sample was transferred to a triangular flask containing 45mL of water, oscillated and mixed, and then heat treated at 85°C for 15min to inactivate non-spore bacteria. After treatment, the suspension was allowed to stand for 5min to precipitate large particles, and then 1mL of supernatant was taken for gradient dilution. 0.1mL of each dilution gradient was applied to the LB agar plate and cultured at 30°C overnight. Single colonies with obvious morphological characteristics were selected from the plate for streaking purification, and confirmed to be pure Bacillus species through colony observation and microscopy. After being transferred to 5mL LB liquid culture medium, 0.5mL of bacterial solution was taken after overnight culture at 30°C and 220rpm, and an equal volume of 60% glycerol was added and stored at -80°C. Through this process, a total of 17 strains of Bacillus were successfully isolated.

[0060] (2) Screening of strains with acaricidal and antibacterial activities:

[0061] ① Determination of acaricidal activity

[0062] The isolated Bacillus strains were inoculated into 5 mL LB liquid medium, activated at 30°C and 220 rpm overnight, and then transferred to fermentation medium No. 1 and cultured at 30°C and 220 rpm for 48 h. The spore concentration in the fermentation broth was determined by the dilution counting method, and the spore concentration in each fermentation broth was adjusted to 1×106 CFU / mL. Subsequently, according to the spider mite slide immersion method in my country's agricultural industry standard (NY / T1154.12-2008), the acaricidal activity of the fermentation liquid of the strain was evaluated, and the harmful mite was the two-spotted spider mite (Tetranychus urticae). The bioassay steps are as follows:

[0063] Cut the double-sided tape into 2cm pieces 2 2. Paste small pieces of about 100 grams of mites on a clean glass slide, select healthy female adult mites, and make their backs adhere to the double-sided tape, with about 30 mites adhered to each glass slide; quickly immerse the glass slide with mites in the fermentation liquid, gently shake for 5 seconds, then take it out, absorb the excess liquid with absorbent paper, place the glass slide on a white disk padded with a wet sponge, and cover it with a plastic film with good light transmittance. Set 4 repetitions for each treatment, and at the same time, use untreated fresh fermentation medium as a blank control; place the container containing the test insects in an environment of 25°C and a light cycle of L:D=16h:8h for 24 hours, and then count the mortality rate of two-spotted spider mites in each treatment group. If the mortality rate in the control group is <5%, there is no need to correct the mortality rate; if the control mortality rate is between 5% and 20%, the mortality rate of the treatment group is corrected according to the formula: corrected mortality rate=(treatment group mortality rate-control group mortality rate) / (1-control group mortality rate)*100; if the control mortality rate is >20%, the test needs to be repeated.

[0064] ② Antibacterial activity determination

[0065] For pathogenic bacteria, the plate diffusion method was used to test the inhibitory effect of Bacillus on specific pathogenic bacteria. Pathogenic bacteria include: Erwinia carotovora, Xanthomonas nigromatis, Pseudomonas, Escherichia coli, Pantoea agglomerans, Aeromonas hydrophila, Aeromonas vermifuga, Bacillus cereus, Staphylococcus aureus, and Micrococcus luteus. The specific method is: the isolated Bacillus strain is cultured in LB liquid medium to the middle of logarithmic growth, and the supernatant is collected by centrifugation; the pathogen is activated overnight using SPA liquid medium, and the bacterial suspension is mixed with SPA agar medium at a temperature of about 50°C to prepare a solid plate, so that the final concentration of the pathogen is 1×10 8 CFU / mL; after the culture medium solidifies, use a 6mm sterile puncher to punch holes, dig out the bacterial cake, add the collected supernatant of the fermentation liquid of the strain to be tested to the hole, place the plate in a 4℃ refrigerator for 2 hours, and then place the inoculated plate in a constant temperature incubator for overnight cultivation according to the optimal growth temperature of the indicator bacteria; after the cultivation, observe whether a clear antibacterial zone is formed around the hole on the plate, and record the experimental results. Sterile water was used as the control group, and each experiment was repeated at least 3 times.

[0066] For pathogenic fungi, the plate confrontation method was used to test the inhibitory ability of Bacillus against specific pathogenic fungi. Pathogenic fungi include: rice sheath blight (Rhizoctonia solani), wheat head blight (Fusarium graminearum), wheat glume blight (Septoria glume), tomato wilt (Fusarium oxysporum tomato-specific type), tobacco black shank (Phytophthora nicotianae), cotton Verticillium wilt (Verticillium dahliae), pear fruit black spot (Alternaria alternata) and Botrytis cinerea. The specific method is as follows: inoculate each pathogenic fungus on the PDA medium and culture until the colony grows vigorously, usually for 2-3 days; use a 6mm sterile puncher to punch holes on the colony of the pathogenic fungus, dig out the bacterial cake and transfer it to the center of a new PDA plate, and gently press to make the bacterial cake in good contact with the culture medium; culture the Bacillus to be tested in the LB medium to the mid-logarithmic growth stage, and collect the supernatant for use; when the pathogenic fungus grows to a certain extent on the new plate (for example, the colony diameter reaches 5-10mm), punch holes at equal distances on both sides of the bacterial cake, add the fermentation supernatant of the strain to be tested and an equal volume of fresh LB liquid culture medium (as a control); place the inoculated plate in a 28℃ constant temperature incubator for 3-7 days; regularly observe and record the growth of the pathogenic fungus and the changes in the confrontation area. Each experiment was repeated at least 3 times.

[0067] Combining the results of the two-spotted spider mite bioassay experiment and the antibacterial experiment, a strain of Bacillus was screened and named NB379. The spore concentration of the fermentation liquid of this strain reached 1×10 6 CFU / mL, the corrected mortality rate against two-spotted spider mites reached 97.4% (±1.7%) ( Figure 2 ) ; NB379 showed good antibacterial activity against a variety of pathogenic fungi and bacteria tested ( Figure 3 ).

[0068] Example 2

[0069] Identification of Bacillus tropicalis NB379:

[0070] (1) Colony morphology detection

[0071] The strain NB379 was cultured on LB solid medium at 30°C for 36 h. The colonies were round, milky white, with edges protruding from the center. Microscopic observation showed that the cells were rod-shaped. The specific colony morphology is shown in Figure 1 Middle A.

[0072] Scanning electron microscope (SEM) observation: A large number of bacterial samples were obtained by centrifugation, and the supernatant was removed. Then, the bacteria were washed three times with 1×PBS buffer (pH 7.2-7.4) to remove impurities; the bacteria were fixed with 2.5% glutaraldehyde, fixed for 3 hours, centrifuged, washed again with PBS buffer and the supernatant was removed; the samples were gradually dehydrated through a series of ethanol aqueous solutions with increasing concentrations (30%, 50%, 70%, 80%, 90%), each step for about 15 minutes; dehydrated twice with 100% ethanol, each for 15 minutes; continued to use a 1:1 mixture of ethanol and tert-butanol for 15 minutes, and then replaced twice with pure tert-butanol, each for 15 minutes; the coverslip containing the bacteria-tert-butanol suspension was placed in a -80℃ refrigerator and then transferred to a freeze dryer for freeze drying; after the sample was dried, it was adhered to a sample stage with conductive tape and gold-plated; the treated sample was placed on the SEM sample stage for observation and imaging. The strain NB379 was rod-shaped under scanning electron microscopy (see Figure 1 Middle B), length is 1.1-3.2 μm and diameter is about 0.6 μm.

[0073] (2) Analysis of physiological and biochemical characteristics

[0074] Its physiological and biochemical characteristics were detected by BIOLOG (Table 1, Table 2)

[0075] Table 1 Physiological and biochemical characteristics of strain NB379 – enzyme activity, carbon source assimilation

[0076]

[0077] +: positive reaction; -: negative reaction; W: weak positive reaction.

[0078] Table 2 Physiological and biochemical characteristics of strain NB379 – Acid production using carbon sources

[0079]

[0080] +: positive reaction; -: negative reaction.

[0081] (3) Strain genome analysis and taxonomic identification

[0082] The genomic DNA of strain NB379 was extracted using the Qiagen genome extraction kit, and the genome of strain NB379 was sequenced and analyzed using the second-generation high-throughput sequencing technology combined with the Oxford Nanopore third-generation sequencing technology. In order to further clarify the taxonomic status of strain NB379, the applicant uploaded the genome data obtained by sequencing to the bacterial identification platform EzBioCloud (https: / / www.ezbiocloud.net / ), and used the genome average nucleotide consistency comparison tool ANI Calculator to compare the genome sequence of strain NB379 with known model strains. The analysis results showed that the ANI of strain NB379 and the model strain N24 of Bacillus tropicus was 96.72%, and the ANI values ​​of the model strains of other similar species were all less than 92%. According to the standards of microbial taxonomy, the ANI value between strains of the same species is ≥95%. Based on this result, strain NB379 was identified as Bacillus tropicus.

[0083] Example 3

[0084] Determination of acaricidal activity of strain NB379

[0085] The fermentation culture method of Example 1 was used to prepare tropical Bacillus NB379 fermentation liquid, and fermentation liquids with different spore concentrations were prepared by mixing different volumes of fresh fermentation liquid. The toxicity of these fermentation liquids to two-spotted spider mites was detected by the spider mite slide immersion method, and the results are summarized in Table 3. SPSS 26.0 data processing software was used to calculate the insecticidal probability of fermentation liquids of different concentrations, and the unit model equation was obtained as PROBIT (P) = -2.631 + 2.652X (the variable X was converted using a logarithm with a base of 10), and the Pearson model goodness of fit test X 2 =0.490, P=0.135, indicating that the model fits well. The experimental results showed that the LC50 of tropical Bacillus NB379 fermentation liquid against two-spotted spider mite 50 It is 9.8μL / mL.

[0086] Table 3 Toxicidal activity of tropical Bacillus NB379 fermentation liquid against Tetranychus urticae

[0087]

[0088] According to the above experimental steps, the bioassay results of different concentrations of tropical Bacillus NB379 fermentation liquid on Panonychus citri and Tetranychus cinnabarinus were statistically analyzed, and the LC values ​​of NB379 fermentation liquid on Panonychus citri and Tetranychus cinnabarinus were calculated using SPSS26.0 data processing software. 50 The values ​​were 22.7 μL / mL and 16.3 μL / mL respectively.

[0089] Example 4

[0090] Determination of growth-promoting ability of strain NB379

[0091] (1) Ability to produce indole-3-acetic acid

[0092] The NB379 strain was inoculated into LB liquid medium containing 5mM tryptophan and cultured in a shaker at 30℃ and 200rpm for 12h; the culture medium was taken out and centrifuged at 10,000rpm for 5min to separate the supernatant; the supernatant was mixed with Salkowski colorimetric solution (50mL 15% HCIO4 mixed with 1mL 0.5M FeCl3) at a ratio of 1:4, and after standing for 30min under dark conditions, the absorbance at 530nm was detected using a spectrophotometer, with the blank culture medium as the control. According to the pre-prepared IAA standard curve, the absorbance value was converted into the actual IAA concentration (g / L). The results showed that the NB379 strain culture solution showed pink after reacting with the Salkowski reagent, and its IAA production was 68.4±5.0mg / L.

[0093] (2) Phosphorus dissolving capacity

[0094] The strain NB379 was inoculated on NBRIP medium plates, which were supplemented with 0.5% Ca3(PO4)2 (inorganic phosphorus source) or lecithin (organic phosphorus source); the culture plates were placed in a 30°C constant temperature incubator for 10 days, and the growth of the colonies was observed. The results showed that a transparent halo formed around the colonies, indicating that the NB379 strain had the ability to dissolve both inorganic and organic phosphorus.

[0095] (3) Ability to produce ammonium nitrogen

[0096] The strain NB379 was inoculated into a test tube containing 10 mL of peptone water and cultured at 30°C for 3 days; 500 μL of Nessler's reagent was added to the culture solution, and the color change was observed. After adding Nessler's reagent, the culture solution of the NB379 strain turned brown-yellow, indicating that the strain has the ability to produce ammonium nitrogen.

[0097] (4) Catalase activity

[0098] Mix a loop of fresh bacterial culture and 50 μL of 3% (v / v) hydrogen peroxide solution on a sterile glass slide; incubate at room temperature for 1 minute and observe the reaction. Bubbles form when the NB379 strain culture is mixed with hydrogen peroxide, indicating that oxygen has been released, which is a positive reaction for catalase, proving that the NB379 strain can produce catalase.

[0099] (5) Siderophore production ability

[0100] A single colony of strain NB379 was inoculated into MSA liquid culture medium and cultured at 30°C and 200 rpm for 48 h. The culture medium was centrifuged at 10,000 r / min for 10 min to separate the supernatant. The supernatant was mixed with CAS detection solution at a volume ratio of 1:1, and the mixture was placed at 30°C in the dark for 1 h before measuring the OD 630 The absorbance value (Ar) of the mixture of uninoculated blank culture medium and CAS test solution was used as a control, and compared with the absorbance value (As) of the mixture of the strain fermentation broth supernatant and CAS test solution to evaluate the strain's ability to produce siderophores: the lower the As / Ar ratio, the higher the ability to produce siderophores. Generally, bacteria with higher siderophore production have an As / Ar value below 0.5. Siderophore activity unit (SU) calculation formula:

[0101]

[0102] The results showed that the SU of the 24h fermentation product of NB379 strain in MSA medium was 0.952±0.027, and the As / Ar value was 0.048±0.012; the SU of the 48h fermentation product was 0.901±0.019, and the As / Ar value was 0.099±0.024. The NB379 strain showed a gradually decreasing ability to produce siderophores with time, but even after 48h, its ability to produce siderophores remained at a high level.

[0103] (6) Siderophore-producing species test:

[0104] ①Shenker's test (detection of carboxylic acid-type siderophores): Take 1 mL of the supernatant of the fermentation broth of strain NB379, add 1 mL of CuSO4 solution (750 μM) and 2 mL of acetate buffer (pH 4.0), mix well, and use a UV spectrophotometer to scan in the wavelength range of 190-280 nm to record whether there is a specific absorption peak.

[0105] ②Arnow's test (detection of catechol-type siderophores): Take 1mL of the fermentation supernatant of strain NB379, add 1mL of 0.5M HCl and 1mL of 10% sodium molybdate-sodium nitrite solution, and observe the color change of the solution. If yellow appears, it indicates the possible presence of catechol structure; then add 1mL of NaOH solution (1M). If the solution turns red and does not fade for at least 1 hour, it indicates the presence of catechol-type siderophores. Use a UV spectrophotometer to detect the characteristic absorption peak at 510nm.

[0106] ③FeCl3 experiment (to distinguish between hydroxamic acid-type and catechol-type siderophores): Take 1 mL of the supernatant of the fermentation broth of strain NB379, add 1-5 mL of FeCl3 solution (2%), and observe the color change of the solution. If it turns red immediately, it may be a hydroxamic acid-type siderophore; if it turns red or purple after adding more than 1 mL of FeCl3 solution, it may be a catechol-type siderophore.

[0107] The results showed that the siderophore produced by strain NB379 was of catechol type.

[0108] (7) Salt and alkali resistance test

[0109] ①Determination of salt tolerance

[0110] LB liquid medium without NaCl was used as the basic medium, and NaCl was added to the final concentrations of 1%, 3%, 5%, 7%, and 9% respectively; OD 600 The bacterial solution with a concentration of 0.6-0.8 was inoculated into LB medium with different salt concentration gradients at a 3% inoculation rate, and 3 parallel samples were set for each salt concentration; after culturing in a shaker at 30°C and 180 rpm for 12 hours, the growth of the bacterial solution was observed and the OD was measured. 600 The results showed that strain NB379 grew well in the NaCl concentration range of 1%-9%, showing strong salt tolerance.

[0111] ② pH tolerance test

[0112] Adjust the pH value of LB liquid medium to 3.0-12.0, and set up 3 parallel samples for each pH value; 600 The bacterial solution with a concentration of 0.6-0.8 was inoculated into LB medium with different pH gradients at a 3% inoculation rate; after culturing in a shaker at 30°C and 180 rpm for 12 h, the OD was measured. 600 The results showed that strain NB379 grew well in the pH range of 5.0-10.0, showing good pH tolerance.

[0113] ③Determination of salt and alkali resistance

[0114] The LB liquid medium without NaCl was used as the basic medium, and a mixed salt solution of NaCl, Na2SO4, NaHCO3 and Na2CO3 was added in a molar mass ratio of 1:9:9:1, and 5 total salt concentration gradients of 40mM-200mM were set; the OD 600 The bacterial solution with a concentration of 0.6-0.8 was inoculated into the culture medium with different total salt concentration gradients at a 3% inoculation rate, and 3 parallel samples were set for each gradient; after culturing in a shaker at 30°C and 180 rpm for 12 hours, the growth of the bacterial solution was observed and the OD was measured. 600 The results showed that the strain NB379 grew well in the saline-alkali concentration range of 40mM-200mM and had good salt-alkali tolerance.

[0115] (8) Heavy metal resistance test

[0116] Different concentrations of metal ions were added to MSA medium, with final concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 mM, respectively. The treatment group without metal ions was used as a blank control. The strain NB379 was inoculated and cultured at 30°C and 180 rpm for 24 h. The culture solution was streaked on a solid plate and cultured at 30°C for 12 h to observe the growth status of the strain. Metal ions include Cu 2+ , Mn 2+ 、Zn 2+ Cr 3+ , Fe 2+ , Pb 2+ 、Cd 2+ 、Ni 2+ wait.

[0117] The experimental results show (such as Figure 4 The strain NB379 showed significant differences in tolerance to different heavy metal ions in MSA medium: 2+ The maximum tolerance concentration of manganese ions (Mn 2+ ) showed extremely high tolerance, with the maximum tolerance concentration reaching 64mM, which was much higher than other tested heavy metal ions; 3+ ) is 2 mM. Chromium is a heavy metal that is toxic to many organisms, but NB379 can still maintain growth at this concentration. 2+ ) and cadmium ions (Cd 2+ ) were all 32 mM, indicating that strain NB379 had a high tolerance to these two heavy metals; 2+ ) had a maximum tolerated concentration of 12 mM, showing good tolerance.

[0118] Example 5

[0119] The growth-promoting effect of strain NB379 on tomato and Arabidopsis

[0120] (1) Growth-promoting effect of strain NB379 on tomatoes

[0121] Select tomato seeds that are uniform in size and full in appearance, soak the seeds in 15% hypochlorous acid solution for 15 minutes for surface disinfection, rinse with distilled water three times, and then soak the seeds in a culture dish containing sterile water; select vermiculite as the cultivation medium, sow the tomato seeds 2 cm below the surface of the sterilized vermiculite, and sow 3 seeds per dish. Use the weighing method to maintain the moisture content of the medium at 80% to ensure suitable moisture conditions to promote seed germination and seedling growth; after about two weeks of seedling cultivation, select the seedlings with uniform growth and transplant them to In new flower pots (the inner diameter and height of the upper and lower bottom of the pot were 18.8cm, 13.5cm, and 18.5cm, respectively), one seedling was retained in each pot; root irrigation treatment began on the 7th day after transplanting, and the sample groups were: 5-fold dilution of NB379 strain MSA fermentation liquid (treatment group 1#), 15-fold dilution (treatment group 2#), 50-fold dilution (treatment group 3#), and an equal amount of deionized water as a control group; 100mL of treatment liquid was irrigated in each pot, and every 6 pots formed a sample group, with an interval of 7 days between each irrigation. After 4 weeks of root irrigation treatment, the growth indicators of tomato seedlings, including plant height, plant fresh weight, and plant dry weight, were measured.

[0122] The results show (such as Figure 5 The height of tomato seedlings in treatment group 1# was significantly higher than that in the control group and other treatment groups, and was 46% higher than that in the control group. The stem diameter of all treatment groups increased, and the stem diameter of treatment group 1# was slightly higher than that of other treatment groups, and was 1.14 times that of the control group. The fresh weight (45.85±0.77g) and dry weight (0.86±0.03g) of tomato seedlings in treatment group 1# were 1.38 times and 2.03 times that of the control group, respectively. In general, the fermentation liquid of strain NB379 at a 5-fold dilution, i.e., 1×10 6 CFU / mL fermentation liquid has a significant promoting effect on the growth of tomato seedlings, which is specifically manifested in the increase of plant height, stem diameter, fresh weight and dry weight.

[0123] (2) Growth-promoting effect of strain NB379 on Arabidopsis thaliana

[0124] Arabidopsis seeds were placed in 15% hypochlorous acid solution for 15 minutes for surface disinfection. After washing with sterile water, the seeds were placed at 4°C for 3 days of vernalization. MS solid culture medium was poured on one side of the two-divided culture dish, and 30 vernalized Arabidopsis seeds were evenly placed on the culture medium after the culture medium was heated. An appropriate amount of strain NB379 bacterial blocks were placed on the other side of the two-divided culture dish to release volatile organic compounds (VOCs). A culture dish without bacterial blocks was set up as a control group to evaluate the effect of VOCs of strain NB379 on the growth of Arabidopsis. The culture dish was sealed with sealing film and placed in a 25°C greenhouse for cultivation. The photoperiod treatment was implemented with 16h light and 8h dark per day to simulate the natural light-dark cycle. There were 3 replicates in each group. After 2 weeks of cultivation, the germination and growth of Arabidopsis seeds were observed and recorded.

[0125] Depend on Figure 6 As shown in the figure, the density of Arabidopsis plants in the treatment group was higher, indicating that the seed germination rate was improved under the action of VOCs of strain NB379. At the same time, the leaf area of ​​Arabidopsis in the treatment group was larger and bright green, indicating that its growth was healthier and more vigorous.

[0126] (3) Strain NB379 alleviates heavy metal stress on Arabidopsis seed germination

[0127] MS solid medium was added to a 24-well plate, and after solidification, one vernalized Arabidopsis seed was planted in each well. The sample groups were: a control group without any treatment substances; a group treated with MSA fermentation broth of NB379 strain at different dilutions to evaluate its direct effect on seed germination; and a group subjected to heavy metal ion stress at different concentrations, including Cr 3 +、Cd 2 +、Ni 2 +, etc., to simulate heavy metal pollution environment; strain NB379 and heavy metal ion co-treatment group to evaluate the mitigation effect of strain NB379. Each group of samples was set up in 4 parallel groups. The treated plates were placed in the greenhouse for further cultivation for 2 weeks, and the germination and growth of Arabidopsis seeds in each sample group were observed and recorded. Figure 7 The results shown show that heavy metal ions inhibit the germination of Arabidopsis seeds in the concentration range of 0.5mM-8.0mM; strain NB379 can alleviate the stress of low concentration heavy metal ions (0.5mM-1.0mM) on seed germination, and the germination rate and growth rate of Arabidopsis seeds are improved with the increase of the concentration of the fermentation supernatant of strain NB379.

[0128] (4) Strain NB379 alleviates heavy metal stress on Arabidopsis seedling growth

[0129] MS solid medium was added to a 24-well plate, and after solidification, 1-2 vernalized Arabidopsis seeds were planted in each well. The 24-well plate was sealed and placed in a 25°C greenhouse for culture, with a photoperiod of 16 h light and 8 h dark per day. After culturing for 1 week, the excess Arabidopsis seedlings were removed to ensure that there was one seedling with similar growth in each well. A variety of treatments were performed, including a control group, a group treated with MSA fermentation broth of NB379 strain at different dilutions, and a group treated with heavy metal ions (Cr 3 +、Cd 2 +、Ni 2 + etc.) stress group, strain NB379 and heavy metal ion co-treatment group, each treatment set 4 groups in parallel. The treated plates were placed in the greenhouse and continued to be cultured for 2 weeks, and the growth of Arabidopsis thaliana in each treatment group was observed and recorded. Figure 8 As shown in the figure, in the control group, the growth of Arabidopsis seedlings weakened with the increase of heavy metal ion concentration, among which the stress effect of 8mM heavy metal ions was the most obvious. After being treated with NB379, the growth of Arabidopsis seedlings under heavy metal stress conditions was significantly improved compared with the untreated stress group, especially the high concentration of NB379 fermentation liquid supernatant, that is, treatment group 1#. Under the stress concentration of heavy metal ions of 1.0mM-4.0mM, the leaf area and color of Arabidopsis were similar to those of seedlings without heavy metal ions, indicating that strain NB379 can effectively alleviate Cr 3 +、Cd 2 + and other negative effects of heavy metals on seedling growth.

[0130] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus tropicus NB379, characterized in that: It was deposited in the China Center for Type Culture Collection on March 18, 2024, with the deposit number being CCTCC No: M2024509.

2. Use of the tropical Bacillus NB379 according to claim 1 in controlling spider mites, wherein the spider mites include one or more of Tetranychus urticae, Panonychus citri and Tetranychus cinnabarinus.

3. Use of the tropical Bacillus NB379 described in claim 1 in promoting plant growth.

4. Use of the tropical Bacillus NB379 according to claim 1 in alleviating heavy metal stress in plants, wherein the heavy metals include one or more of copper, manganese, chromium, lead, cadmium and nickel.

5. Use of the tropical Bacillus NB379 described in claim 1 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi include one or more of the pathogen of rice sheath blight, the pathogen of wheat fusarium wilt, the pathogen of wheat glumen blight, the pathogen of tomato wilt, the pathogen of tobacco black shank, the pathogen of cotton Verticillium wilt, the pathogen of pear black spot and gray mold.

6. Use of the tropical Bacillus NB379 according to claim 1 in producing indole-3-acetic acid.

7. Use of the tropical Bacillus NB379 according to claim 1 in dissolving phosphate and / or producing ammonium nitrogen.

8. Use of the tropical Bacillus NB379 according to claim 1 in producing catalase.

9. Use of the tropical Bacillus NB379 according to claim 1 in producing siderophore, wherein the siderophore is a catechol siderophore.

10. A bacterial agent, characterized in that: The bacterial agent contains the tropical Bacillus NB379 of claim 1, and the bacterial content is 1×10 6 CFU / mL.

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