Bacillus agave, fermentation liquor, biological agent and application

Abstract

The invention belongs to the technical field of plant disease control, and particularly relates to bacillus agave, fermentation liquor, a biological agent and application. The invention discloses a strain of bacillus tequilensis, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the bacillus tequilensis is CGMCC NO.34830. The strain of bacillus tequilensis has the advantages that the preservation number is CGMCC NO.34830; the invention also discloses a fermentation broth and a biological agent prepared according to the bacillus agave, and the bacillus agave, the fermentation broth and the biological agent provided by the invention can effectively reduce the harm of root knot nematode disease to plants and promote seed germination or crop growth, and have the advantages of high efficiency, safety, environmental protection and difficult generation of resistance.

Description

Technical Field

[0001] This invention belongs to the field of agave Bacillus, specifically relating to a strain of agave Bacillus, fermentation broth, biological agent, and its application. Background Technology

[0002] Root-knot nematodes ( Meloidogyne Root-knot nematodes (spp.) belong to the phylum Nematoda and are among the most destructive obligate endoparasitic nematodes in the world, with an extremely wide host range covering thousands of economic crops. They invade root tips through their stylets, repeatedly piercing plant cells, ultimately inducing abnormal division and proliferation of parenchyma cells in the root vascular bundles, forming root knots. These root knots severely damage the normal structure and function of the root system, hindering the absorption of water and mineral nutrients. Plants exhibit slow growth, stunted growth, yellowing leaves, premature aging, significantly reduced yields, and even death. This unique parasitic pattern makes them a major biological stressor for crop production, causing enormous economic losses to agriculture.

[0003] According to statistics, 105 species of root-knot nematodes have been reported worldwide, among which four species are the most common: Southern root-knot nematode (… M. incognita Javan root-knot nematodes ( M. javanica ), Northern root-knot nematodes ( M. hapla ) and peanut root-knot nematodes ( M. arenaria These dominant species exhibit significant genetic diversity and adaptive evolutionary capabilities. Root-knot nematode disease typically leads to a 10%–20% reduction in crop yield; in severe cases, losses can reach 80%, or even result in total crop failure.

[0004] Currently, chemical control remains the primary method for controlling plant parasitic nematodes. However, chemical control suffers from serious environmental residues, ecotoxicity, and harm to non-target organisms, and has been strictly limited or banned. The current reliance on single chemical agents is unsustainable; therefore, developing efficient and environmentally friendly methods for controlling root-knot nematodes has become an urgent problem. Endophytic bacteria, as a potential biocontrol resource, are gradually attracting attention. Endophytic bacteria are microorganisms that can colonize within plant tissues and form symbiotic relationships with the host plant; they are ubiquitous in natural ecosystems. These microorganisms play a crucial role in the plant's microecological environment, acting positively in the biocontrol of plant diseases by producing antimicrobial metabolites or inducing systemic resistance in plants. Therefore, finding a safe and efficient endophytic bacterial strain for controlling root-knot nematodes is of great significance for developing environmentally friendly root-knot nematode control strategies. Summary of the Invention

[0005] In order to solve the above problems, the application provides a bacillus agaves, a biological bacterial agent and a preparation method and application thereof. The bacillus agaves with high anti-nematode activity is screened from soil by taking southern root-knot nematode as a target. The fermentation liquor or biological bacterial agent developed according to the strain can effectively reduce the harm of root-knot nematode disease to plants and promote the growth of plants.

[0006] In order to achieve the above-mentioned purpose, the specific technical solutions of the application are as follows: The application provides a bacillus agaves in a first aspect of the application. Bacillus tequilensis The bacillus agaves is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 34830.

[0007] The application provides a fermentation liquor in a second aspect of the application. The bacillus agaves is inoculated into a liquid culture medium, and after being cultured at 25-35 DEG C for 1-3 days, the fermentation liquor is obtained.

[0008] Further, the liquid culture medium takes NB liquid culture medium as a basic culture medium, and further includes 5.2g of corn powder, 5g of urea and 1g of potassium carbonate.

[0009] Further, the culture temperature is 28-32 DEG C.

[0010] Further, the culture temperature is 30.5 DEG C, and the culture time is 27.5h.

[0011] The application provides a biological bacterial agent in a third aspect of the application.

[0012] Further, the metabolic product is the fermentation liquor or the separation of the fermentation liquor.

[0013] Further, the separation is obtained by centrifuging the fermentation liquor.

[0014] Further, the centrifugal speed is 6000-10000r / min, and the centrifugal time is 2-10min.

[0015] Further, the centrifugal speed is 8000r / min, and the centrifugal time is 5min.

[0016] Further, the biological bacterial agent is a liquid agent or a powder agent.

[0017] Furthermore, the viable count of Bacillus agave in each milliliter of the aforementioned biological agent is 1.0 × 10⁻⁶. 8 CFU ~ 1.0 × 10 9 CFU.

[0018] Furthermore, the powder is obtained by adding a protectant to a suspension or fermentation broth of Bacillus agave and then freeze-drying it.

[0019] Furthermore, the protective agent is glucose, lactose, or sucrose.

[0020] Furthermore, the mass concentration of the glucose solution, lactose solution, or sucrose solution is 1% to 10%.

[0021] The volume ratio of the bacterial suspension or fermentation broth of Bacillus agave to the protective agent is 1:1.

[0022] The fourth aspect of the present invention provides the application of the above-described Bacillus agave, fermentation broth, or biological agent in the preparation of biocontrol agents or seed soaking and coating agents.

[0023] Furthermore, the biological control agent is used to control root-knot nematode disease; the seed soaking and coating agent is used to promote crop growth.

[0024] Furthermore, the above-mentioned fermentation liquid or biological agent can be sprayed or irrigated on crops at a dosage of 5 mL to 15 mL per crop to prevent root-knot nematode disease.

[0025] Furthermore, the prevention and control of root-knot nematode disease involves killing root-knot nematodes and / or inhibiting the hatching of root-knot nematode eggs.

[0026] Furthermore, the crop is soaked in the aforementioned fermentation liquid or biological agent for 1 to 5 minutes to promote crop growth.

[0027] Furthermore, promoting crop growth means promoting seed germination, increasing plant height, root length, root fresh weight, or plant fresh weight.

[0028] Furthermore, the crop mentioned is tomato.

[0029] Furthermore, the tomato variety is L402.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a strain of *Bacillus agave*, a fermentation broth, a biological agent, and its applications. This invention involves isolating a strain from soil, which was identified as *Bacillus agave* (…). Bacillus tequilensisThe specimen, with accession number CGMCC NO.34830, is deposited at the China General Microbiological Culture Collection Center and is classified as *Bacillus agave*. Bacillus tequilensis This invention also discloses a fermentation broth and a biological agent prepared based on Bacillus agave. The Bacillus agave, fermentation broth, and biological agent provided by this invention have disease prevention and growth promotion functions.

[0031] (1) The fermentation broth provided by the present invention has certain contact killing activity against second-instar larvae of root-knot nematodes and can inhibit the hatching of root-knot nematode eggs.

[0032] (2) When the fermentation liquid or biological agent is used in various growth stages of plants, it can effectively prevent and control root-knot nematode disease and promote plant growth. When the fermentation liquid or biological agent is used for seed soaking and coating treatment, it can promote seed germination and has no inhibitory effect on plant growth. When the fermentation liquid or biological agent is used in seedlings, it can resist root-knot nematode disease and promote plant growth.

[0033] (3) The strains, fermentation broth and biological agents of the present invention can be used as agents for the prevention and control of root-knot nematode disease. They have the advantages of simple operation, high efficiency, safety, environmental protection and low resistance, effectively reducing the damage of chemical agents to the environment and the threat to humans.

[0034] Instructions for the Preservation of Biological Materials The *Bacillus agave* strain Sneb2623 used in this invention is classified and named *Bacillus agave*. Bacillus tequilensis Its Latin name is: Bacillus tequilensis It was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34830. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 Phylogenetic tree of Bacillus agave Sneb2623 strain constructed based on 16S rDNA sequence.

[0037] Figure 2 Contour plot showing the effect of corn flour and urea additions on the J2s corrected mortality rate.

[0038] Figure 3 The response surface plot shows the effect of corn flour and urea addition on the J2s-corrected mortality rate.

[0039] Figure 4 Contour plot showing the effect of corn flour and potassium carbonate addition on the J2s corrected mortality rate.

[0040] Figure 5 The response surface plot shows the effect of corn flour and potassium carbonate addition on the J2s-corrected mortality rate.

[0041] Figure 6 Contour plot showing the effect of urea and potassium carbonate additions on the J2s corrected mortality rate.

[0042] Figure 7 The response surface plot shows the effect of urea and potassium carbonate addition on the J2s corrected mortality rate.

[0043] Figure 8 Contour plot showing the effects of pH and time on the corrected mortality rate of J2s.

[0044] Figure 9 The response surface plot shows the effect of pH and time on the J2s corrected mortality rate.

[0045] Figure 10 Contour plot showing the effects of pH and temperature on the corrected mortality rate of J2s.

[0046] Figure 11 Contour plot showing the effects of pH and temperature on the corrected mortality rate of J2s.

[0047] Figure 12 Contour plot showing the effects of time and temperature on the J2s-corrected mortality rate.

[0048] Figure 13 Contour plot showing the effects of time and temperature on the J2s-corrected mortality rate.

[0049] Figure 14 The effect of different protective agents on the survival rate of Sneb2623 lyophilized bacterial agents was investigated. Different letters indicate significant differences between groups. Detailed Implementation

[0050] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0051] The NB solid culture medium, NB liquid culture medium, and 30v / v% glycerol used in the embodiments of the present invention were all prepared according to the following method.

[0052] Preparation of NB solid culture medium: Dissolve 10.0g peptone, 5.0g beef extract, 5.0g sodium chloride and 20g agar in 1L distilled water, adjust the pH to 7.0±0.2, and sterilize by moist heat at 121℃ for 30min.

[0053] Preparation of NB liquid culture medium: Dissolve 10.0g peptone, 5.0g beef extract and 5.0g sodium chloride in 1L distilled water, adjust the pH to 7.0±0.2, and sterilize by moist heat at 121℃ for 30min.

[0054] Preparation of 30v / v% glycerol: Mix 60mL of glycerol with 140mL of deionized water in a 200mL blue-mouth bottle and sterilize by moist heat at 121℃ for 30min.

[0055] Root-knot nematodes ( Meloidogyne spp. Root-knot nematodes are among the most serious obligate endoparasitic nematodes globally, with a wide host range covering thousands of economic crops. They invade root tips via their stylets, inducing abnormal cell division and root knot formation, disrupting root function, leading to stunted growth, dwarfism, yellowing, premature aging, significantly reduced yields, and even death, causing enormous economic losses. Currently, chemical control is limited due to environmental residues and ecotoxicity issues, making the development of efficient and environmentally friendly control methods urgent. Endophytic bacteria, as a potential biological control resource, are receiving increasing attention. Finding safe and efficient endophytic bacterial strains is crucial for developing environmentally friendly root-knot nematode control strategies.

[0056] This invention provides an *Agave* Bacillus strain, a fermentation broth, a biological agent, and their applications. This *Agave* Bacillus strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.34830. The *Agave* Bacillus strain, the fermentation broth prepared based on this *Agave* Bacillus strain, and the biological agent provided by this invention can effectively reduce the damage of root-knot nematode disease to plants, promote seed germination or crop growth, and has the advantages of high efficiency, safety, environmental friendliness, and low likelihood of developing resistance.

[0057] Example 1: Obtaining Bacillus agave Sneb2623 strain Soil samples were taken from tomato fields at the Korla Experimental Base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (81°815′E, 41°75′N). 5g of soil sample was weighed and added to a 250mL Erlenmeyer flask containing 45mL of sterile water. The samples were then serially diluted, and samples were taken at dilutions of 10-10. -2 10 -3 10 -410 -5 10 -6 25 μL of each soil suspension was spread onto NB solid medium and cultured at 37 °C for 12 h. The strain was isolated and purified by plate dilution method, and the strain fermentation broth was prepared and mixed with 30% glycerol and frozen to -80 °C.

[0058] Example 2: Identification of Bacillus agave Sneb2623 strain The taxonomic position of the strain was determined by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis.

[0059] Morphological observation: The purified strain from Example 1 was streak-on NB plates and incubated upside down in a 28°C incubator for 12 hours. The colony morphology, color, odor and other characteristics were observed.

[0060] Physiological and biochemical characteristics: In a clean bench, break open the neck of the physiological and biochemical tube (purchased from Qingdao Haibo Biotechnology Co., Ltd.), use a sterilized pipette tip to pick up a fresh single colony and inoculate it into the tube. After completion, seal the tube with sealing film and place it in a 37°C constant temperature incubator. Follow the instructions of the physiological and biochemical reagent kit (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and add the corresponding reagents as required. Observe the final results.

[0061] 16S rDNA sequence analysis: Bacterial DNA was extracted according to the DNA extraction method described by Sutyak et al. (Sutyak KE, Wirawan RE, Aroutcheva AA and Chikindas M L. 2008. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus samyloliquefaciens[J]. Journal of Applied Microbiology, 104, (1067-1074).). Using genomic DNA as a template, 16S rDNA gene amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-ACGGCTACCTTGTTACGACTT-3', SEQ ID NO.2). A 25 μL PCR amplification system consisted of: 1 μL each of upstream and downstream primers, 2 μL DNA template, 12.5 μL 2×Taq Master Mix (Dye olus), and 8.5 μL ddH2O. PCR amplification conditions were: 94℃ pre-denaturation for 1.5 min; 94℃ denaturation for 0.5 min, 58℃ annealing for 0.5 min, 72℃ extension for 1 min, for 30 cycles; 72℃ extension for 5 min, and storage at 4℃. After detection by 1.0 v / v% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared and analyzed using the BLAST database in NCBI. A phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-joining method. The phylogenetic tree is shown below. Figure 1 .

[0062] like Figure 1 The results showed that submitting the strain's sequence to NCBI's GenBank yielded accession number PQ157562, which was most homologous to Bacillus agave. Bacillus tequilensis, The accession number is PP978608. Therefore, this strain has been identified as Bacillus agave.

[0063] Example 3: Preparation of fermentation broth and biological agent 1. Preparation of fermentation broth: The *Bacillus agave* Sneb2623 strain obtained in Example 1 was streaked on NB solid medium and incubated at 28°C for 12 hours until single colonies appeared, thus obtaining the activated strain. One loopful of the activated strain was inoculated into a 250mL Erlenmeyer flask containing 150mL of NB liquid medium and fermented on a shaker for 2 days at 28°C and a shaking speed of 200r / min to obtain a fermentation broth.

[0064] 2. Preparation of biological agent: Centrifuge the above fermentation broth at 8000 r / min for 5 min, and collect the supernatant, which is the biological agent.

[0065] Example 4: Tests on the contact killing effect of biological agents on root-knot nematodes and the inhibition of root-knot nematode egg hatching. Southern root-knot nematodes were obtained by propagating them from tomato roots. Meloidogyne incognita The egg sacs of J2 were used to make an egg suspension, and the rest were hatched using the shallow dish method to produce J2 eggs, which were then prepared into a nematode suspension.

[0066] Take 200 μL of biological agent, use ddH2O and NB medium as the control group, add 200 μL of nematode suspension (about 30 J2 nematodes), confront for 24 h, observe the mortality of nematodes and record the number of dead nematodes, calculate the mortality rate and corrected mortality rate, and the results are shown in Table 1.

[0067] Mortality rate = (Number of dead nematodes / Total number of nematodes) × 100%; Corrected mortality rate = (treatment nematode mortality rate - control nematode mortality rate) / (1 - control nematode mortality rate) × 100%.

[0068] Table 1. Contact killing effect of biological agents on root-knot nematodes Note: - in the table, it indicates that the corresponding indicator is not included in the statistics.

[0069] Take 200 μL of biological agent, with ddH2O and NB medium as controls, add 200 μL of egg suspension (about 10 eggs), and observe the number of hatched nematodes after 24 h and 48 h of culture. Calculate the egg hatching rate and relative inhibition rate. The results are shown in Table 2.

[0070] Egg hatching rate = (Number of hatched nematodes / Number of eggs tested) × 100%; Relative inhibition rate = [(number of control hatched nematodes - number of treatment hatched nematodes) / number of control hatched nematodes] × 100%.

[0071] Table 2. Effects of biological agents on the hatching rate of southern root-knot nematode eggs. Note: - in the table, it indicates that the corresponding indicator is not included in the statistics.

[0072] Tables 1 and 2 show that the corrected mortality rate of second-instar larvae of *Agave chinensis* reached 89.83% after 24 hours using the bio-inoculum agent; the relative inhibition rate of *Agave chinensis* egg hatching by the bio-inoculum agent reached 77.13% after 24 hours and 87.17% after 48 hours. These results indicate that *Bacillus agave* Sneb2623 can effectively kill second-instar larvae of *Agave chinensis* and inhibit egg hatching.

[0073] Example 5: Seed Coating Germination Experiment The fermentation broth of Bacillus agave Sneb2623 prepared according to the method in Example 3 was prepared for later use. Tomato seeds of variety L-402 were selected, and the tomato seeds were soaked in 0.5% sodium hypochlorite solution, shaken and mixed for 5 minutes, rinsed 3 times with ddH2O, and then placed on sterile filter paper to air dry for later use.

[0074] Seeds were soaked in fermentation broth for 3 minutes and then placed in sterile Petri dishes (d=90mm) lined with sterile filter paper. Each treatment was repeated three times, with 10 seeds per replicate. Control groups were treated with equal volumes of sterile water (ddH2O) and NB liquid medium, respectively. The Petri dishes were incubated in a constant temperature and humidity incubator at 25℃. Germination was counted daily after 24 hours. After 7 days of incubation, the germination index and germination rate were calculated, and shoot and root lengths were measured to calculate the vigor index. The results are shown in Table 3.

[0075] Germination rate = (Number of germinated seeds / Total number of seeds tested) × 100%; Germination index = Σ (number of germinated germinations at different times / corresponding number of germination days); Seed vigor index = seedling length × germination index.

[0076] Table 3. Effects of fermentation broth coating on tomato seeds on their germination and growth. Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate results obtained using the Duncan test. P The difference was statistically significant at levels <0.05.

[0077] As shown in Table 3, after treatment with Bacillus agave Sneb2623 fermentation broth, the germination rate and germination index of tomato seeds increased significantly, while the root length and seed vigor showed no significant difference compared with the control group. This indicates that the Bacillus agave Sneb2623 fermentation broth promotes seed germination by soaking and coating tomato seeds, and has no inhibitory effect on the growth of tomato plants.

[0078] Example 6: Experiment on pot control efficacy of Bacillus agave Sneb2623 fermentation broth Take the fermentation broth of Bacillus agave Sneb2623 prepared in Example 3 and the nematode suspension prepared in Example 4, and set them aside. Select the tomato variety L-402, soak the seeds in 0.5% sodium hypochlorite solution, shake well for 5 minutes, rinse 3 times with ddH2O, place the seeds on sterile filter paper, and after the seeds are air-dried, place them in seedling trays containing nutrient soil, and cultivate them in a greenhouse at 25℃ with a photoperiod L / / D=16h / / 8h (L represents the photoperiod and D represents the dark period) until the four-leaf stage, and set them aside.

[0079] The control groups were drenched with ddH2O and inoculated with Southern Root-knot Nematode J2, drenched with NB liquid medium and inoculated with J2, and drenched with a pesticide (1.8% abamectin emulsion, purchased from Yantai Aubes Biochemical Co., Ltd.) and inoculated with J2. The treatment group was drenched with Bacillus agavesii Sneb2623 fermentation broth and inoculated with J2. 2 mL of second-instar larval suspension (approximately 1000 J2 larvae) was added to each seedling, and each seedling was drenched with 10 mL of Bacillus agavesii Sneb2623 fermentation broth (concentration 1×10⁻⁶). 8 The control group received an equal amount of ddH2O or NB liquid culture medium or 1.8% abamectin emulsion per seedling. Each treatment had 5 replicates, with 1 seedling per replicate, and all seedlings were randomly placed. After 30 days of culture, the number of root knots was investigated, and the root knot reduction rate was calculated. The root knot reduction rate was used to represent the relative control efficacy. The results are shown in Table 4.

[0080] Root knot index grading standard: Level 0, no root knots; Grade 1, with some small root knots on the fibrous roots; Grade 2: There are some obvious small root knots on the fibrous roots, but no root knots on the main root; Grade 3: There are some obvious large root knots on the fibrous roots, but no root knots on the main root; Level 4: Most of the fibrous roots are large root knots, while there are no root knots on the main root. Level 5, with root knots on half of the root system and also on the main root; Level 6, with numerous root knots appearing on the main root; Level 7, with root knots on most of the main roots; Level 8, all main roots have root knots; Level 9, all roots have root knots; Level 10, severe root knots, no fibrous roots.

[0081] Root knot reduction rate = [(number of control root knots - number of treated root knots) / number of control root knots] × 100%.

[0082] Table 4. Root irrigation efficacy of Bacillus agave Sneb2623 fermentation broth against tomato root-knot nematode disease. Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate results obtained using the Duncan test. P <0.05 indicates a significant difference. - indicates that the corresponding indicator was not included in the statistics.

[0083] Table 4 shows that in the pot experiment under room temperature conditions, after drenching tomato seedlings with Bacillus agave Sneb2623 fermentation broth, the number of root knots in the tomato roots was significantly lower than that in the NB liquid culture medium control group, but there was no significant difference compared with the abamectin-treated control group. The root knot reduction rate was 66.17%. Therefore, in the pot experiment, Bacillus agave Sneb2623 can effectively control tomato root-knot nematode disease.

[0084] Example 7: Effects of Bacillus agave Sneb2623 fermentation broth on tomato plant growth Take the fermentation broth of Bacillus agave Sneb2623 prepared in Example 3 and set it aside. Select tomato variety L-402, soak the seeds in 0.5% sodium hypochlorite solution, shake well for 5 minutes, rinse 3 times with ddH2O, place the seeds on sterile filter paper, and after the seeds are air-dried, place them in seedling trays containing nutrient soil, and cultivate them in a greenhouse at 25°C with a light cycle of L / / D=16h / / 8h (L represents the light duration and D represents the darkness duration) until the four-leaf stage, and set it aside.

[0085] Control groups were drenched in ddH2O or NB liquid medium, with each seedling receiving 10 mL of either medium. The treatment group consisted of drenching with *Bacillus agave* Sneb2623, with each seedling receiving 10 mL of *Bacillus agave* Sneb2623 fermentation broth (concentration 1×10⁻⁶) per seedling. 8 (CFU / mL). Five replicates were performed for each treatment, with one plantlet per replicate, and the plants were placed completely randomly. After 30 days of culture, the plant height, root length, root fresh weight, and plant fresh weight of the tomato plants were assessed.

[0086] Table 5. Effects of root irrigation with Bacillus agave Sneb2623 fermentation broth on tomato plant growth. Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate results obtained using the Duncan test. P The difference was statistically significant at levels <0.05.

[0087] Table 5 shows that in the pot experiment under room temperature conditions, the root irrigation treatment of tomato seedlings with Bacillus agave Sneb2623 fermentation broth significantly increased plant height and root length compared to the NB liquid culture medium control group, while there was no significant difference in plant fresh weight and root fresh weight between the two groups. Therefore, in the pot experiment, Bacillus agave Sneb2623 can effectively promote the growth of tomato plants.

[0088] Example 8: Field efficacy experiment of Bacillus agave Sneb2623 fermentation broth Take the fermentation broth of Bacillus agave Sneb2623 prepared in Example 3 and set it aside. Select the tomato variety L-402, soak the seeds in 0.5% sodium hypochlorite solution, shake well for 5 minutes, rinse 3 times with ddH2O, place the seeds on sterile filter paper, and after the seeds are air-dried, place them in seedling trays containing nutrient soil. Cultivate them in a greenhouse at 25°C with a photoperiod of L / / D=16h / / 8h (L represents the photoperiod and D represents the photoperiod) until the four-leaf stage, and then transplant them to the field where root-knot nematode disease is present.

[0089] At transplanting, control groups were treated with ddH2O root drenching, NB liquid culture medium root drenching, and chemical root drenching, with each seedling receiving 10 mL of ddH2O, NB liquid culture medium, or chemical (1.8% abamectin emulsion). The treatment group received root drenching with Bacillus agave Sneb2623 fermentation broth, with each seedling receiving 10 mL of the broth (concentration 1×10⁻⁶). 8 (CFU / mL). After 30 days of cultivation, physiological indicators such as plant height, root length, root fresh weight, and plant fresh weight of tomato plants were measured, the root knot index was investigated, and the control efficacy was calculated.

[0090] Root knot index grading standard: Level 0, no root knots; Grade 1, with some small root knots on the fibrous roots; Grade 2: There are some obvious small root knots on the fibrous roots, but no root knots on the main root; Grade 3: There are some obvious large root knots on the fibrous roots, but no root knots on the main root; Level 4: Most of the fibrous roots are large root knots, while there are no root knots on the main root. Level 5, with root knots on half of the root system and also on the main root; Level 6, with numerous root knots appearing on the main root; Level 7, with root knots on most of the main roots; Level 8, all main roots have root knots; Level 9, all roots have root knots; Level 10, severe root knots, no fibrous roots.

[0091] Root knot index = Σ[(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100%; Control efficacy = [(control root knot index - treatment root knot index) / control root knot index] × 100%.

[0092] Table 6. Efficacy of fermentation broth root irrigation treatment on tomato root-knot nematode disease Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate results obtained using the Duncan test. P <0.05 indicates a significant difference. - indicates that the corresponding indicator was not included in the statistics.

[0093] Table 6 shows that in the field trial, the root irrigation treatment of tomato seedlings with Bacillus agave Sneb2623 fermentation broth resulted in a control efficacy of 49.29%, significantly higher than that of the NB medium control group and the abamectin treatment control group. Compared with the NB medium control group and the abamectin treatment control group, root length, root fresh weight, plant fresh weight, and plant height all increased. Therefore, in the field trial, Bacillus agave Sneb2623 fermentation broth can effectively control tomato root-knot nematode disease and promote plant growth.

[0094] Example 9: Optimization of fermentation conditions for Bacillus agave Sneb2623 strain Given the large number of elements involved in nutrient optimization and fermentation condition screening for Sneb2623 fermentation broth, the Plackett-Burman (PB) design method was adopted. The PB design is an experimental design method used to select the importance of optimization factors among different center combination experiments. The goal of the experimental design is to select important nutrients and fermentation conditions in as few experiments as possible.

[0095] The Plackett-Burman method in the Facilitation model of Design-Exper 8.1.1 software was used for design, and the experimental results were analyzed using Design-Exper 8.1.1 software to screen the important factors affecting the fermentation results of strain Sneb518. Next, based on the identified main factors, the steepest fermentation direction was determined, and steepness experiments were conducted. Finally, referring to the results of the steepest fermentation experiment, the Box-Behnken method was used to determine the response center points of important fermentation factors to establish the optimal fermentation results. In this experiment, the Box-Behnken experiments for nutrients and fermentation conditions were conducted separately. The Box-Behnken experiment for fermentation conditions was conducted based on the optimal values ​​for nutrient fermentation.

[0096] Table 7. Experiments and designs for steepest climbing paths of Bacillus agave Sneb2623 culture medium with different combinations of culture medium components. Table 8. Steepest Climbing Path Experiment and Design for Different Combinations of Fermentation Conditions for Bacillus agave Sneb2623 Based on single-factor experiments, Plackett-Burman screening of important factors, and steepest ascent experiments, three important single factors affecting the J2s corrected mortality rate—corn flour (A), urea (B), and potassium carbonate (C)—were identified as the analytical objects for the composition of the fermentation broth of strain Sneb2623. A response surface methodology experiment with three factors and three levels was designed using Design Expert 8.1.1 software (Table 9).

[0097] Table 9. Sneb2623 culture medium composition and Box-Behnken experimental design and results. Coding level, experimental design and results are shown in Figures 2-7 See Table 10.

[0098] Table 10. Evaluation of the simulation coefficients and significance test of the regression equations for Sneb2623 culture medium components. Note: Blank spaces in the table represent indicators that are not included in the statistics.

[0099] Based on the single-factor experiments of fermentation conditions of Sneb2623 strain, three important factors of culture conditions—pH (D), time (E), and temperature (F)—were identified as the subjects of analysis, and a response surface methodology experiment with three factors and three levels was designed. The coding levels, experimental design, and results are detailed below. Figures 8-13 (Tables 11 and 12).

[0100] Table 11 Fermentation conditions for Sneb2623 and Box-Behnken experimental design and results Table 12 Evaluation of the coefficients of the regression equation model for Sneb2623 fermentation conditions and its significance test. Note: Blank spaces in the table represent indicators that are not included in the statistics. * indicates that... p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0101] The optimal nutrient conditions for cultivation were: using NB liquid medium as the basic medium, with the addition of 5.2 g / L corn flour, 5 g / L urea, and 1 g / L potassium carbonate; the optimal fermentation conditions were: pH 6.08, cultivation time 27.46 h, and cultivation temperature 30.65 ℃.

[0102] Example 10: Preparation of freeze-dried powder of Bacillus agave Sneb2623 strain Preparation of protective agents: Prepare glucose aqueous solution, lactose aqueous solution, and sucrose aqueous solution with concentrations of 1 w / v%, 5 w / v%, and 10 w / v, respectively.

[0103] Fermentation broth of strain Sneb2623 was prepared using optimized fermentation conditions. The cultured broth was centrifuged at 4000 rpm for 20 min, and the bacterial cells were collected after centrifugation. The centrifuged bacterial cells were rinsed into petri dishes with 15 mL of sterile water, and the protective agent and bacterial suspension were mixed at a 1:1 volume ratio to prepare a strain mixture. The Sneb2623 strain mixture was pre-frozen overnight at -20°C in a low-temperature freezer. The vacuum freeze dryer was turned on, and the cold trap temperature of the instrument was lowered to below -50°C. The pre-frozen fermentation broth was placed in the vacuum freeze dryer, the vacuum pump was turned on, and the machine was run for 48 h until the internal pressure dropped below 10 Pa to prepare the Sneb2623 lyophilized bacterial agent. The prepared Sneb2623 lyophilized bacterial agent was diluted to the original volume, and J2s were directly contact-killed, and the J2s mortality rate was calculated.

[0104] The survival rates of strains in Sneb2623 lyophilized bacterial agents prepared with different protectants are as follows: Figure 14 As shown, the effects of different concentrations of the three sugars on the survival rate of the bacterial strains varied significantly. With increasing sucrose and lactose concentrations, the survival rate of the strains increased; with increasing glucose concentrations, the survival rate initially decreased and then increased. The highest survival rates were observed for both lactose and sucrose at a concentration of 10 w / v%, while the survival rates for glucose at 1 w / v and 10 w / v concentrations were similar and higher than at 5 w / v. The survival rates of strains with 5 w / v lactose, 10 w / v lactose, 5 w / v sucrose, and 10 w / v sucrose were all above 50%, with the Sneb2623 lyophilized bacterial agent, using 10 w / v glucose as a protectant, exhibiting the highest survival rate of 83.66%, which was considered ideal.

[0105] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0106] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Bacillus agave ( Bacillus tequilensis ), characterized in that, The *Agave* Bacillus is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34830.

2. A fermentation broth, characterized in that, The fermentation broth is obtained by the following preparation method: The Bacillus agave described in claim 1 was inoculated into a liquid culture medium and cultured at 25℃~35℃ for 1 to 3 days to obtain the fermentation broth.

3. A biological agent, characterized in that, The biocontrol agent contains the Bacillus agave as described in claim 1 or a metabolite of the Bacillus agave.

4. The biological agent according to claim 3, characterized in that, The metabolite is the fermentation broth of claim 2 or the isolate of the fermentation broth of claim 2; the isolate does not contain Bacillus agave cells.

5. The biological agent according to claim 4, characterized in that, The biological agent is a liquid or powder; the viable count of Bacillus agave in each milliliter of the biological agent is 1.0 × 10⁻⁶. 8 CFU ~ 1.0 × 10 9 CFU.

6. The biological agent according to claim 5, characterized in that, The powder is obtained by adding a protectant to a suspension or fermentation broth of Bacillus agave and then freeze-drying it; the protectant is glucose, lactose or sucrose.

7. The biological agent according to claim 6, characterized in that, The mass concentration of the glucose solution, lactose solution, or sucrose solution is 1% to 10%; the volume ratio of the Bacillus agave suspension or fermentation broth to the protectant is 1:

1.

8. The use of the Bacillus agave as described in claim 1, the fermentation broth as described in claim 2, or the biological agent as described in claim 3 in the preparation of biological control agents or seed soaking and coating agents.

9. The application according to claim 8, characterized in that, The biological control agent is used to control root-knot nematode disease; the seed soaking and coating agent is used to promote crop growth.

10. The application according to claim 9, characterized in that, The crop in question is tomato.

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