Continuous-cropping-resistant bacterial fertilizer based on bacillus velezensis and application of continuous-cropping-resistant bacterial fertilizer

Abstract

The invention provides an anti-continuous cropping bacterial fertilizer based on bacillus velezensis and application of the anti-continuous cropping bacterial fertilizer. According to the invention, firstly, a strain of bacillus velezensis zhanshen-K1 is separated from soil for continuous cropping of hot peppers, and the preservation number of the strain of bacillus velezensis zhanshen-K1 is CGMCC (China General Microbiological Culture Collection Center) No.34459. The strain has the effects of fixing nitrogen, dissolving potassium, decomposing protein, producing exopolysaccharides, antagonizing various pathogenic bacteria such as fusarium fusiforme, fusarium asian, alternaria alternata and phytophthora capsici, degrading various phenolic acid autotoxic substances such as benzoic acid, p-hydroxybenzoic acid and cinnamic acid, and the like; and meanwhile, the continuous cropping-resistant bacterial fertilizer is prepared by adding wormcast and sodium humate into the compound serving as a main effective component, so that continuous cropping obstacles of crops can be reduced.

Description

Technical Field

[0001] The invention relates to a continuous cropping resistance bacterial fertilizer based on Bacillus velezinsis and application thereof, belonging to the field of agricultural biotechnology. Background Art

[0002] Continuous cropping disorder refers to the phenomenon of stunted growth, reduced yield, and decreased quality of the same crop or closely related crops after continuous planting on the same plot of land. Causes include: soil nutrient imbalance and the accumulation of harmful substances, such as excessive consumption of a single nutrient, autotoxic substances produced by root secretions and residual decomposition; an imbalance in the soil microbial community, with an increase in pathogenic microorganisms and a decrease in beneficial bacteria; and a decrease in crop root vitality and a weakened absorption capacity. The main hazards of continuous cropping disorder are low crop emergence rates, slow growth, and increased pests and diseases, leading to significantly reduced yields, poor quality, and even total crop failure in severe cases. It also affects subsequent planting structures and increases production costs.

[0003] Continuous cropping is a widespread problem in my country's agricultural production, particularly in areas where vegetables, fruits, flowers, and grain crops like wheat are grown. This problem poses a serious threat to the sustainable development of agriculture in my country: long-term continuous cropping leads to soil degradation, fertility imbalances, and damage to farmland ecosystems; pest and disease problems are becoming increasingly severe, forcing increased pesticide use, exacerbating environmental pollution and agricultural product safety risks; and crop yields and quality continue to decline, restricting the efficient use of arable land resources, threatening food security and farmers' income, and becoming a major bottleneck to achieving green, low-carbon, and sustainable agricultural development.

[0004] There are currently a variety of methods to alleviate continuous cropping problems, but all have limitations. Crop rotation is a traditional and effective method that can improve soil structure and balance nutrients through the rotation of different crops. However, it is difficult to promote in some areas due to planting habits and market demand. In terms of soil improvement, the application of organic fertilizers, lime, etc. can regulate the physical and chemical properties of the soil, but the cost is high, the effect is slow, and the degradation of accumulated autotoxic substances is limited. Grafting and root replacement can enhance the disease resistance of crops and is widely used in melon and vegetable production. However, the grafting operation is complicated, the technical requirements are high, and there may be problems with the compatibility of the rootstock and scion. Although chemical treatment can quickly sterilize, it will destroy the soil microbial community, increase the resistance of pathogens to drugs, and may also cause pesticide residue problems in agricultural products. The application of microbial fertilizers to improve the physical and chemical properties of the soil and the soil microecology is a relatively safe, cost-effective and effective method for preventing and controlling continuous cropping problems, and is receiving increasing attention. Microbial fertilizers have been widely used on vegetables, fruits, fruit trees, Chinese medicinal herbs, and some grain and cash crops. They have been particularly rapidly adopted in greenhouse vegetable, fruit, peanut, and other cash crop production areas. Microbial fertilizers can improve soil quality, reduce crop disease incidence, promote crop growth, and increase crop yield and quality, which is crucial for alleviating the problem of continuous cropping.

[0005] However, currently available microbial fertilizer products are generally produced using a combination of "functional bacteria + organic matter." The "functional bacteria" themselves are relatively simple, primarily promoting growth. Few possess disease resistance, and even fewer possess the ability to degrade autotoxic substances and increase the production of extracellular polysaccharides. The "organic matter" used is primarily composted agricultural and forestry waste and livestock and poultry manure. There is little emphasis on the role that organic materials with specific functional properties, such as humic acid and earthworm castings, can play in enhancing product effectiveness. Furthermore, there is no research on how to optimize their combination and rationally process them to enhance their effectiveness in addressing continuous cropping problems. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a bacterial fertilizer for resisting continuous cropping based on Bacillus velez and its application. The present invention first isolates a strain of Bacillus velez from the soil of pepper continuous cropping. Bacillus velezensis )zhanshen-K1, this strain has the functions of fixing nitrogen, releasing potassium, decomposing protein, producing extracellular polysaccharides, antagonizing various pathogens and degrading phenolic acid self-toxic substances. At the same time, it is used as the main active ingredient to add earthworm castings and sodium humate to prepare anti-cropping bacterial fertilizer. Experiments have shown that it can alleviate the problems of continuous cropping of crops such as peppers and wheat.

[0007] The present invention firstly isolates a strain of Bacillus velezinis from pepper continuous cropping soil. Bacillus velezensis )zhanshen-K1, which has been deposited in the General Microbial Culture Collection Center of the China Microorganism Culture Collection Committee (CGMCC) on May 8, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 34459. This strain has the ability to fix nitrogen, dissolve potassium, decompose protein, produce extracellular polysaccharides, and antagonize Fusarium fujikura ( Fujikura Fusarium ), Fusarium asiatica ( Asian Fusarium oxysporum ), Alternaria alternata ( Alternaria ) and Phytophthora capsici ( Phytophthora capsici ) and other pathogens, and degrade various phenolic acid self-toxic substances such as benzoic acid, p-hydroxybenzoic acid and cinnamic acid.

[0008] The present invention also provides a fermentation and culture method for the Bacillus velezensis strain zhanshen-K1, which comprises the steps of strain activation, triangular flask seed preparation, liquid seed tank strain preparation and solid-state fermentation and culture.

[0009] During the activation step, culture the culture in LB medium on a slant tube at 33-37°C for 20-24 hours. For flask seed preparation, use LB medium at 33-37°C with shaking at 180-200 rpm for 20-24 hours. For seed tank seed preparation, use LB medium at 33-37°C with stirring at 180-220 rpm for 20-24 hours, with an aeration rate of 0.8-1.0 VVM.

[0010] The solid-state fermentation culture comprises a composite culture medium. After the culture medium is cooled, the culture medium is inoculated into a seed tank with a 5%-10% inoculum. The culture medium is incubated at 37-42°C with ventilation for 48-60 hours. The culture medium is then air-dried at low temperature and ground into 80-100 mesh size, resulting in Bacillus velezensis zhanshen-K1 powder. The composite culture medium comprises: 18-22% crop straw, 28-32% soybean meal, 37-42% bran, 8-12% corn flour, 0.3-0.8% potassium dihydrogen phosphate, 0.1-0.3% magnesium sulfate, and 0.2-0.4% calcium carbonate. Preferably, the composition is: 20% crop straw, 30% soybean meal, 39% bran, 10% corn flour, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and 0.3% calcium carbonate. The crop straw includes, but is not limited to, one or more of wheat, corn, and rice straw.

[0011] The present invention also provides an anti-consecutive-cropping bacterial fertilizer with Bacillus velez zhanshen-K1 as the main active ingredient, which is characterized by comprising, by weight: 20 to 30 parts of the above-mentioned Bacillus velez zhanshen-K1 bacterial powder, 20 to 30 parts of sodium humate, and 40 to 60 parts of earthworm manure, which are uniformly mixed to obtain the anti-consecutive-cropping bacterial fertilizer with Bacillus velez zhanshen-K1 as the main active ingredient, and the effective viable bacterial count is ≥2.0×10 9 cfu / g. Vermicompost must be thoroughly decomposed, dried, and crushed to 80-100 mesh before use. Sodium humate must be crushed to 80-100 mesh before use.

[0012] The application method of the above-mentioned anti-consecutive cropping bacterial fertilizer is: 25~50 kg / mu, used for basal application and topdressing.

[0013] The present invention also discloses the above-mentioned Bacillus Velezii ( Bacillus velezensis ) Application of zhanshen-K1 or anti-cropping bacterial fertilizer in alleviating the obstacles of continuous cropping.

[0014] The above-mentioned reduction of crop continuous cropping obstacles can be manifested in: improving soil enzyme activity, promoting crop growth, enhancing the antioxidant defense capacity of crop roots, reducing oxidative damage, preventing and controlling soil-borne diseases, and increasing crop yields.

[0015] The advantages of the present invention are as follows: 1. The Bacillus velezensis zhanshen-K1 strain has multiple functions, including nitrogen fixation, potassium solubilization, protein decomposition, antagonism against various pathogens such as Fusarium fujikura, Fusarium asiatica, Alternaria alternata and Phytophthora capsici, and degradation of various phenolic acid autotoxic substances such as benzoic acid, p-hydroxybenzoic acid and cinnamic acid. It can play a role in improving soil, increasing nutrient supply, preventing and controlling soil-borne diseases, degrading crop autotoxic substances, etc., and has a better application effect in alleviating continuous cropping obstacles.

[0016] 2. The Bacillus Velez zhanshen-K1 bacterial fertilizer uses functional organic matter such as earthworm castings and sodium humate as carriers. Both earthworm castings and sodium humate have the effects of improving soil, promoting crop growth, and enhancing crop resistance. They work synergistically with the Bacillus Velez zhanshen-K1 strain to achieve better application results.

[0017] 3. Potted pepper trials have demonstrated that Bacillus Velez zhanshen-K1 significantly promotes pepper growth, increases soil enzyme activity, and strengthens the antioxidant defenses of pepper roots, mitigating oxidative damage. Furthermore, the application of Bacillus Velez zhanshen-K1 fertilizer can reduce the incidence of pepper blight in continuously cropped soils, demonstrating a significant control effect. These findings contribute to alleviating the challenges associated with continuous pepper cropping.

[0018] 4. Wheat field trials have shown that the use of Bacillus Velez zhanshen-K1 bacterial fertilizer in continuously cropped wheat fields can alleviate continuous cropping obstacles, significantly promote wheat growth and increase wheat yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Colony morphology of Bacillus velezinsis zhanshen-K1; Figure 2 :Bacterial morphology of Bacillus velezchensishen-K1; Figure 3 : Phylogenetic tree constructed based on partial 16S rDNA sequences; Figure 4 :Bacillus velez zhanshen-K1 produces hydrolysis circles in skim milk powder medium; Figure 5 :Bacillus velezensis zhanshen-K1 was grown in Ashby nitrogen-free medium; Figure 6 :Bacillus velez zhanshen-K1 was grown on silicate bacterial culture medium; Figure 7 :Exopolysaccharide production by Bacillus velez zhanshen-K1; Figure 8 :Effects of Bacillus Velez zhanshen-K1 on plant height and stem diameter of pepper; Figure 9 : Effects of Bacillus velez zhanshen-K1 on fresh weight and dry weight of pepper. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the embodiments.

[0021] Example 1: Screening of Bacillus velezensis zhanshen-K1 (1) Bacillus velezensis zhanshen-K1 was isolated from pepper continuous cropping soil. 10 g of continuous cropping soil was weighed and placed in a pre-sterilized 250 mL conical flask. 90 mL of deionized water and 10-20 glass beads were added and shaken on a shaker at 37°C and 180 rpm for 30 min. The soil suspension was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Concentration. Take 0.1 mL of soil solution of different dilutions, evenly spread it on an LB medium plate, and place it in an incubator at 37°C for 24-48 hours. Select single colonies with different colony morphology, perform streak culture, and obtain single colonies. The formula of the LB medium is: 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar, 1000 mL distilled water, pH 7.0, and sterilize at 121°C for 20 minutes.

[0022] (2) The strains isolated in step (1) were transferred to skim milk powder culture medium, with 4 different strains being inoculated on each plate. The culture was carried out at 37°C for 24 to 48 hours. The strains that produced a large transparent zone were selected as protein-degrading strains. The strains were cultured in pure form and stored for future use. The skim milk powder culture medium was formulated as follows: 3.0 g skim milk powder, 1.6% agar, 200 mL deionized water, pH 7.0, and sterilized at 108°C for 15 minutes.

[0023] (3) The strain screened in step (2) is inoculated on Ashby nitrogen-free medium and cultured at 37°C for 2-3 days to observe whether the strain can grow normally. The strain with better growth is selected and preserved for future use, which is the strain with nitrogen-fixing function. The formula of the Ashby nitrogen-free medium is: mannitol 10.0 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.2 g, calcium sulfate dihydrate 0.1 g, calcium carbonate 5 g, agar 20 g, distilled water 1000 mL, pH 7.0, sterilized at 115°C for 30 min.

[0024] (4) The strain screened in step (3) was transferred to a silicate bacterial culture medium plate and cultured at 37°C for 2-3 days. Strains that can grow normally, produce capsules, and have smooth, transparent, oily colonies were selected and preserved for future use. These strains are strains with potassium solubilization ability. The formula of the silicate bacterial culture medium is: sucrose 5.0 g, magnesium sulfate 0.5 g, calcium carbonate 0.1 g, sodium hydrogen phosphate 2.0 g, ferric chloride 0.005 g, glass powder 1.0 g, agar 15 g, distilled water 1000 mL, pH 7.0, sterilized at 121°C for 15 min.

[0025] Note: The glass powder in silicate bacterial culture media can provide a source of potassium for bacterial strains and is used to screen for strains with potassium-solubilizing abilities. Glass powder contains silicates such as potassium silicate, and the potassium in these substances is insoluble within the glass powder's lattice structure. Strains with potassium-solubilizing abilities secrete organic acids, enzymes, and other substances that disrupt the glass powder's lattice structure, releasing the potassium.

[0026] (5) From the strains screened in step (4), further antagonistic bacteria were screened by plate confrontation test. The pathogenic fungi used in the antagonistic test were Fusarium fujikura ( Fujikura Fusarium ), Fusarium asiatica ( Asian Fusarium oxysporum ), Alternaria alternata ( Alternaria ) and Phytophthora capsici ( Phytophthora capsici Activate the pathogenic fungus and transfer it to a PDA plate. Cultivate until a moss of fungi forms. Use a sterile microporator to punch out circular fungal masses, transfer them to the center of the PDA plate, and incubate at 28°C for 3 days. Use a toothpick to pick the strain to be screened and inoculate it 2 cm from the edge of the pathogen. Continue incubating at 28°C for 3 days and observe for the appearance of an inhibition zone. Select strains with strong antagonism against Fusarium fujikura, Fusarium asiatica, Alternaria alternata, and Phytophthora capsici for future use.

[0027] (6) The strains screened in step (5) were inoculated onto inorganic salt solid culture medium plates containing benzoic acid, p-hydroxybenzoic acid, and cinnamic acid as the sole carbon source (all at a concentration of 30 mg / L), and cultured at 37°C for 3 days to observe whether the strains grew. Strains that can grow on inorganic salt solid culture medium containing benzoic acid, p-hydroxybenzoic acid, and cinnamic acid were selected and preserved for future use. These strains are strains that can degrade benzoic acid, p-hydroxybenzoic acid, and cinnamic acid. The inorganic salt culture medium formula is: 2.0 g ammonium sulfate, 0.50 g sodium dihydrogen phosphate, 0.50 g potassium hydrogen phosphate, 0.20 g magnesium sulfate heptahydrate, 0.10 g calcium chloride dihydrate, 1000 mL distilled water, pH 7.0, and sterilized at 115°C for 30 min.

[0028] (7) The strains screened in step (6) were inoculated into 100 mL conical flasks containing 40 mL LB liquid medium. The culture was carried out in a shaker at 37°C and 180 r / min for 36 h. 5 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 8000 r / min for 10 min. The supernatant was removed and 3 volumes of 95% ethanol were added. The mixture was gently stirred with a glass rod and allowed to stand for 2 min to observe whether flocculent precipitation was produced. If obvious flocculent precipitation was produced, it indicated that the strain had the ability to produce extracellular polysaccharides.

[0029] Through the above multiple screening, taking into account the various beneficial functions of the strain, a strain was obtained with strong ability to decompose protein, fix nitrogen, solubilize potassium, produce extracellular polysaccharides, antagonize multiple pathogens such as Fusarium fujikura, Fusarium asiatica, Alternaria alternata and Phytophthora capsici, and degrade multiple phenolic acid autotoxic substances such as benzoic acid, p-hydroxybenzoic acid and cinnamic acid. It is numbered zhanshen-K1.

[0030] Example 2: Identification of Bacillus velezensis zhanshen-K1 (1) Morphological and physiological and biochemical characteristics The morphological characteristics of the strain are as follows: when cultured on LB medium at 37°C for 24-48 hours, the colonies are milky white, relatively uniform in texture, opaque, and have a diameter of about 1.5-2.5 mm ( Figure 1 The bacteria are short rod-shaped and Gram-positive ( Figure 2 The physiological and biochemical characteristics of the strain are as follows: positive in VP test, positive in nitrate reduction test, positive in starch hydrolysis test, positive in methyl red test, positive in citrate utilization test, and positive in catalase test.

[0031] (2) 16S rDNA sequence analysis The Bacillus velezensis zhanshen-K1 strain was inoculated into LB liquid medium and cultured at 37°C and 180 r / min for 24 h. The bacteria were collected, DNA was extracted, and 16S rRNA gene amplification was performed. Universal primers were used, with the forward sequence being 27-F: 5'-AGAGTTTGATCCTGGCTCAG-3' and the reverse sequence being 1492-R: 5'-GGTTACCTTGTTACGACTT-3'. PCR amplification conditions were as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 60 s, for 30 cycles, and finally 72°C extension for 5 min. The PCR products were sequenced by Qingdao Bioengineering (Shanghai) Co., Ltd., and the sequencing results were Blast-aligned with the Gen Bank nucleic acid database on the NCBI website. Sequences with more than 97% homology were selected to construct a phylogenetic tree using the software MEGA7.0 ( Figure 3 ).

[0032] The morphological, physiological and biochemical characteristics and 16S rDNA sequence (SEQ No. 1) analysis showed that the strain was Bacillus velezinis ( Bacillus velezensis The strain has been deposited in the General Microbiological Culture Collection Center of the China Microorganism Culture Collection Committee (CGMCC) on May 8, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 34459.

[0033] Example 3: Determination of the beneficial functions of Bacillus velezensis zhanshen-K1 (1) Protein degradation ability: Bacillus velezensis zhanshen-K1 was inoculated into skim milk powder medium and cultured at 37℃ for 72 h. The colony diameter d and the transparent zone diameter D were measured, and the D / d ratio was calculated ( Figure 4 The strain D / d was 2.11, indicating strong protein degradation. The skim milk powder culture medium formula was: 3.0 g skim milk powder, 1.6% agar, 200 mL deionized water, pH 7.0, and sterilized at 108°C for 15 min.

[0034] (2) Nitrogen fixation ability: The Bacillus velezensis zhanshen-K1 strain was inoculated on Ashby nitrogen-free medium and cultured at 28°C for 3 days to observe whether the strain can grow normally. The strain grew normally and formed a bacterial lawn, indicating that it has the ability to fix nitrogen ( Figure 5The Ashby nitrogen-free medium formula is: 10.0 g mannitol, 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.2 g sodium chloride, 0.1 g calcium sulfate dihydrate, 5 g calcium carbonate, 20 g agar, 1000 mL distilled water, pH 7.0, sterilized at 115°C for 30 min.

[0035] (3) Potassium-dissolving ability: The strain of Bacillus zeae zhanshen-K1 was inoculated onto a silicate bacterial culture plate and cultured at 37°C for 3 days. The strain was able to grow on the plate and produce capsules in the form of smooth, transparent oil droplets, indicating that it has the ability to dissolve potassium ( Figure 6 The silicate bacterial culture medium is formulated as follows: 5.0 g sucrose, 0.5 g magnesium sulfate, 0.1 g calcium carbonate, 2.0 g disodium hydrogen phosphate, 0.005 g ferric chloride, 1.0 g glass powder, 15 g agar, 1000 mL distilled water, pH 7.0, and sterilized at 121°C for 15 min.

[0036] (4) Antagonistic effect on pathogens: Fusarium fujikura ( Fujikura Fusarium ), Fusarium asiatica ( Asian Fusarium oxysporum ), Alternaria alternata ( Alternaria sp. ) and Phytophthora capsici ( Phytophthora capsici Pathogenic fungi were transferred to PDA plates. Once fully grown, they were punched into circular clumps using a sterilized puncher and transferred to the center of the PDA plates for incubation at 28°C for 3 days. Bacillus Velez zhanshen-K1 was inoculated 2 cm from the edge of the pathogens and incubated at 28°C for another 3 days, observing for the appearance of an inhibition zone. The colony diameters d and D of the pathogens in the antagonistic and non-antagonistic areas were measured, and the inhibition rate was calculated as (Dd) / D × 100%. Bacillus Velez zhanshen-K1 exhibited strong antagonism against Fusarium fujikura, Fusarium asiatica, Alternaria alternata, and Phytophthora capsici. As shown in Table 1, the inhibition rates were 66.2%, 63.4%, 61.3%, and 58.1%, respectively.

[0037] Table 1 Antagonistic ability of Bacillus velezensis zhanshen-K1 against pathogenic fungi

[0038] (5) Polysaccharide production capacity: The Bacillus zehanshen-K1 strain was inoculated into LB liquid culture medium and cultured at 37°C and 180 r / min for 36 h. 5 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 8000 r / min for 10 min. The supernatant was collected and 3 times the volume of 95% ethanol was added. The mixture was gently stirred with a glass rod and allowed to stand for observation. Flocculent precipitation appeared in the solution, indicating that the strain had the ability to produce extracellular polysaccharides ( Figure 7 ).

[0039] (6) Degradation test of benzoic acid, p-hydroxybenzoic acid and cinnamic acid Prepare a benzoic acid-containing culture medium with the following formula: 50 mg of benzoic acid, 5 g of peptone, 0.5 g of ammonium sulfate, 20 g of a complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1), 1000 ml of water, pH 7.0.

[0040] Prepare a culture medium containing p-hydroxybenzoic acid with the following formula: 50 mg p-hydroxybenzoic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1), 1000 ml water, pH 7.0.

[0041] Prepare a cinnamic acid-containing culture medium with the following formula: 50 mg cinnamic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1), 1000 ml water, pH 7.0.

[0042] Bacillus velezensis zhanshen-K1 was inoculated into each of the three aforementioned culture media and cultured at 37°C with shaking at 180 rpm for 7 days. Ten mL of the culture medium was centrifuged at 3500 rpm for 15 minutes, the precipitate discarded, and the supernatant extracted with 10 mL of dichloromethane. The extract was evaporated to dryness using a vacuum rotary evaporator, and the residue was dissolved in 1 mL of methanol and set aside. High-performance liquid chromatography (HPLC) was used to determine the residual amounts of benzoic acid, p-hydroxybenzoic acid, and cinnamic acid in the samples, and the degradation rates were calculated. The mobile phases were acetonitrile and water (pH 2.8 adjusted with acetic acid) at a flow rate of 1.0 mL / min, a C18 reversed-phase column, a column temperature of 30°C, and a detection wavelength of 280 nm. Gradient elution was used: acetonitrile was increased from 5% to 40% over 0-30 minutes, maintained at 40% acetonitrile for 30-40 minutes, and then decreased from 40% to 5% acetonitrile for 40-45 minutes. The results of HPLC showed that the degradation rates of benzoic acid, p-hydroxybenzoic acid and cinnamic acid by Bacillus velezensis zhanshen-K1 were 78.3%, 81.2% and 71.2%, respectively.

[0043] Example 4: Preparation of Bacillus velezensis zhanshen-K1 fermentation broth Activation: Inoculate Bacillus velezensis zhanshen-K1 onto a slant in a test tube containing LB solid medium and incubate at 37°C for 24 hours. The LB solid medium consists of 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 20 g agar powder, and 1000 mL water (pH 7.0).

[0044] Preparation of seed solution in a triangular flask: Inoculate the activated strain into a triangular flask containing LB liquid medium and incubate at 37°C, 200 rpm, and shake for 24 hours to obtain the seed solution in the triangular flask. The LB liquid medium formula is: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, 1000 mL water, pH 7.0.

[0045] Preparation of seed tank strains: Transfer the seed liquid in the triangular flask into a seed tank with a volume of 100 L according to the inoculation volume of 3%. The tank contains 75 L of LB liquid culture medium. Control the temperature at 37°C, the ventilation volume at 0.8~1.0VVM, and the stirring speed at 200r / min for 24 hours.

[0046] Solid-state fermentation: A composite culture medium is used with the following formula (by mass): 20% corn straw meal, 30% soybean meal, 39% bran, 10% corn flour, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and 0.3% calcium carbonate. The ingredients are mixed evenly, the moisture content is adjusted to 55%-60%, and sterilized at 121°C for 25 minutes. After the culture medium cools, the inoculum is inoculated into the seed tank at a 10% inoculum level. The culture is incubated at 37-42°C with ventilation for 48 hours. After low-temperature air drying, the powder is pulverized to 80-100 mesh. This results in Bacillus velezensis zhanshen-K1 powder, with an effective viable count of 2.0 × 10 10 cfu / g.

[0047] Example 5: Preparation of anti-repeatable bacterial fertilizer with Bacillus velezchenshen-K1 as the main active ingredient By weight, 25 parts of the above-mentioned Bacillus Velez zhanshen-K1 powder, 25 parts of sodium humate, and 50 parts of earthworm manure are mixed evenly to obtain the anti-repeatable cropping bacterial fertilizer with Bacillus Velez zhanshen-K1 as the main active ingredient of the present invention, and the effective viable bacterial count is ≥2.0×10 9 cfu / g. Vermicompost must be thoroughly decomposed, dried, and crushed to 80-100 mesh before use. Sodium humate must be crushed to 80-100 mesh before use.

[0048] Application example 1: Pepper potted plant experiment A pot experiment was conducted in Ancheng Town, Pingyin County, Shandong Province. The pots used had an upper inner diameter of 20 cm, a lower inner diameter of 14 cm, and a height of 16 cm. Each pot was filled with 3 kg of soil. The soil was from a continuous pepper cropping system. The test crop was pepper. The pot experiment included one treatment group (T1) and one control group (CK), each with 20 pots. Two pepper seedlings were transplanted into each pot. In the T1 treatment group, 3 grams of the anti-repeating bacterial fertilizer prepared in Example 5 of the present invention, containing Bacillus velezensis zhanshen-K1 as the main active ingredient (hereinafter referred to as "anti-repeating bacterial fertilizer") was applied to the soil of each pot. In the CK control group, 3 grams of the anti-repeating bacterial fertilizer of the present invention, sterilized at 121°C, was applied to the soil of each pot. Seventy days after transplanting, relevant indicators such as plant height, stem diameter, dry weight, fresh weight, soil enzyme activity, root antioxidant enzyme activity, and MDA content were measured. Plant height and stem diameter were measured using a tape measure and a vernier caliper, respectively. Urease activity was determined by the sodium phenolate colorimetric method. Catalase activity was determined by potassium permanganate titration. Sucrase activity was determined by the DNS colorimetric method. Superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) reduction method. Peroxidase (POD) activity was determined by the guaiacol method. Catalase (CAT) activity was determined by UV-visible spectrophotometry. Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) colorimetric method.

[0049] The experimental results showed that compared with the control group CK, the treatment group T1 showed different degrees of increase in plant height, stem diameter and fresh weight ( Figure 8 , Figure 9 ), indicating that the Bacillus Velez zhanshen-K1 strain described herein significantly promotes pepper growth. Compared with the control group CK, the activities of urease, catalase, and sucrase in treatment group T1 were all increased (Table 2), demonstrating that Bacillus Velez zhanshen-K1 can enhance soil enzyme activity. Compared with the control group CK, antioxidant enzyme activities in pepper roots in treatment group T1 were all increased, and MDA content was decreased (Table 3), indicating that Bacillus Velez zhanshen-K1 can enhance the antioxidant defense capacity of pepper roots and reduce oxidative damage. These results demonstrate that the application of Bacillus Velez zhanshen-K1 can help alleviate the problem of continuous cropping of peppers.

[0050] Table 2 Effects on enzyme activities in pepper rhizosphere soil

[0051] * Data in the same column marked with different letters indicate significant differences (P < 0.05).

[0052] Table 3 Effects on antioxidant enzymes and MDA content in pepper roots

[0053] * Data in the same column marked with different letters indicate significant differences (P < 0.05).

[0054] Application Example 2: Control Effect on Pepper Blight A pot experiment was conducted in Ancheng Town, Pingyin County, Shandong Province. The pots used had an upper inner diameter of 20 cm, a lower inner diameter of 14 cm, and a height of 16 cm. Each pot was filled with 3 kg of soil. The soil was from a continuous pepper crop. The test crop was pepper. The pot experiment included two treatment groups (T) and one control group (CK), each with 30 pots. Two pepper seedlings were transplanted into each pot. The control group (CK) received no application of the present invention's anti-repeatable microbial fertilizer. Five days after transplanting the pepper seedlings, 10 mL of a spore suspension of Phytophthora capsici was applied to the roots of each pot. Treatment group T1 received a thorough mixing of the present invention's anti-repeatable microbial fertilizer and soil at a ratio of 1:200, then potted. Five days after transplanting the pepper seedlings, 10 mL of water was applied to the roots of each pot. Bacterial agent treatment group T2: The anti-repeat bacterial fertilizer of the present invention was thoroughly mixed with soil at a ratio of 1:200, and then potted; 5 days after the pepper seedlings were transplanted, 10 mL of the pepper spore suspension was watered at the roots of each pot.

[0055] The preparation method of the spore suspension of Capsici Phytophthora capsici is as follows: the Capsici Phytophthora capsici strain is inoculated into a PDA culture medium, cultured at a constant temperature of 25°C for 7 days, and after the mycelium has fully grown on the plate, 10 mL of sterile water is added, and the surface of the mycelium is gently scraped with a sterile spatula to remove the spores to obtain a spore suspension, and then an appropriate amount of sterile water is added to adjust the spore concentration to 1×10 6 pieces / mL.

[0056] The disease situation was investigated on the 30th day after transplanting. The disease level of each pot of pepper was recorded according to the following grading standards: Level 0, no lesions; Level 1, small water-soaked lesions appeared at the base of the stem, with an area ≤ 1 / 4 of the circumference of the stem base, and no wilting of leaves; Level 3, the area of ​​lesions at the base of the stem accounted for 1 / 4~1 / 2 of the circumference, and the leaves were slightly wilted; Level 5, the area of ​​lesions at the base of the stem accounted for 1 / 2~3 / 4 of the circumference, the leaves were obviously wilted (more than half of the true leaves drooped), and the cotyledons did not fall off; Level 7, the area of ​​lesions at the base of the stem accounted for >3 / 4 of the circumference, almost around the stem, the leaves were severely wilted or partially fell off, the plant fell over but did not die; Level 9: the lesions at the base of the stem circled the stem, the cortex rotted and constricted, and the plant died. The disease index and control effect were calculated according to the disease level, and the calculation formula is as follows:

[0057]

[0058] The test results are shown in Table 5. The application of the anti-repeatable bacterial fertilizer of the present invention can reduce the incidence index of pepper blight in continuously cropped soil, and has a good prevention and control effect, which is beneficial to alleviating the continuous cropping obstacles (Table 5).

[0059] Table 5 The control effect of Bacillus Velez zhanshen-K1 bacterial fertilizer on pepper blight

[0060] Application Example 3: Effect of Bacillus Velezii zhanshen-K1 bacterial fertilizer on promoting growth and increasing yield of wheat A plot experiment was conducted in Ancheng Town, Pingyin County, Shandong Province, using fields that had been continuously cropped with wheat for many years. Two treatment groups were set up, each with six replicates, for a total of 12 plots, each measuring 30 square meters. For the control treatment, conventional fertilization was applied: 40 kg of a conventional compound fertilizer (15-15-15) was applied as a base fertilizer per mu, and 10 kg of a compound fertilizer (20-5-20) was applied as a topdressing fertilizer per mu. For the microbial fertilizer treatment, 10 kg of the present invention's anti-repeatable microbial fertilizer was applied as a base fertilizer per mu, in addition to the CK treatment. The plots were arranged in randomized blocks, with 1-meter-wide isolation rows between plots and 2-meter-wide protective rows surrounding the experimental field to prevent interference between adjacent treatments. Land preparation was carried out according to local wheat cultivation practices to ensure the soil was loose and flat. Sowing was done in rows, with a row spacing of 20 cm, a sowing depth of 3-5 cm, and a sowing rate of 15 kg per mu. All treatment groups were sown on the same day. Aside from the different fertilization treatments, field management measures such as irrigation, weeding, and pest control remained consistent across all treatment groups and were carried out according to local routine management practices. Plant height, tiller number, leaf number, and yield per mu were measured at maturity.

[0061] The test results (Table 6) show that the plant height, tiller number, leaf number and per mu yield of wheat in treatment group T are all higher than those in group CK, which indicates that the application of the anti-continuous cropping bacterial fertilizer of the present invention to wheat planted in continuous cropping fields can alleviate continuous cropping obstacles, significantly promote wheat growth and increase yield.

[0062] Table 6 Effects of Bacillus Velez microbial fertilizer on wheat growth and yield

Claims

1. A strain of Bacillus velezinoffii ( Bacillus velezensis )zhanshen-K1, the deposit number of the strain is: CGMCC No.34459.

2. Use of the Bacillus Velez strain zhanshen-K1 according to claim 1 in nitrogen fixation, potassium solubilization, protein decomposition and exopolysaccharide production.

3. Use of the Bacillus Velez strain zhanshen-K1 according to claim 1 in antagonizing pathogens, wherein the pathogens are Fusarium fujikura, Fusarium asiatica, Alternaria alternata and Phytophthora capsici.

4. Use of the Bacillus Velez strain zhanshen-K1 according to claim 1 in degrading phenolic acid self-toxic substances, wherein the phenolic acid self-toxic substances are benzoic acid, p-hydroxybenzoic acid and cinnamic acid.

5. The fermentation culture method of the Bacillus Velez strain zhanshen-K1 according to claim 1, characterized in that: The method includes strain activation, triangular flask seed preparation, liquid seed tank strain preparation and solid-state fermentation culture. The solid-state fermentation culture comprises the following steps: solid-state fermentation culture adopts a composite culture medium; after the culture medium is cooled, the seed tank strain is inoculated with an inoculation amount of 5% to 10%; the culture medium is ventilated at 37 to 42°C for 48 to 60 hours; the fermentation liquid is air-dried at low temperature and then crushed to 80 to 100 meshes to obtain Bacillus velezensis zhanshen-K1 bacterial powder; and the composite culture medium is composed of the following mass ratios: 18 to 22% crop straw, 28 to 32% soybean meal, 37 to 42% bran, 8 to 12% corn flour, 0.3 to 0.8% potassium dihydrogen phosphate, 0.1 to 0.3% magnesium sulfate, and 0.2 to 0.4% calcium carbonate.

6. A bacterial fertilizer for resisting repeated cropping with Bacillus velezensis zhanshen-K1 as the main active ingredient, characterized in that: In parts by weight: 20-30 parts of Bacillus velezensis zhanshen-K1 powder prepared by the method according to claim 5, 20-30 parts of sodium humate, and 40-60 parts of earthworm castings.

7. The anti-repeatable bacterial fertilizer according to claim 6, wherein The earthworm castings must be thoroughly decomposed, dried and crushed to 80-100 meshes before use; the sodium humate must be crushed to 80-100 meshes before use.

8. The method for applying the anti-repeatable fertilizer according to claim 6 or 7, wherein: For crops with continuous cropping problems, 25~50 kg / mu, used for basal application and topdressing.

9. Use of the Bacillus velezensis zhanshen-K1 according to claim 1 or the anti-continuous cropping bacterial fertilizer according to claim 6 in alleviating obstacles to continuous cropping.

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