A kind of Bacillus coagulans and its application in preventing and controlling soil-borne diseases

Abstract

The present invention discloses a Bacillus coagulans and its application in preventing and controlling soil-borne diseases. A method for soil disinfection is provided, comprising: 1) adding Bacillus coagulans NJYB-1 to the soil; and 2) performing anaerobic soil disinfection (ASD) on the soil. The soil disinfection method provided by the present invention greatly reduces the amount of organic carbon source used in the ASD technology and can significantly increase crop yields.

Description

Technical Field

[0001] The invention relates to the fields of microorganisms and plant protection, and in particular to a bacillus coagulans and application thereof in preventing and controlling soil-borne diseases. Background Art

[0002] Tomatoes are nutritious and delicious, and they play an important role in people's daily diet. They are an important fruit and vegetable species with a long history of cultivation and a wide range of cultivation areas in the world. Due to the huge economic benefits and the continuous development of scale and specialization of facility crops, my country's tomato planting area ranked first in Asia as early as 2011. With the intensive planting and continuous planting of tomatoes, the occurrence of soil-borne diseases has become increasingly serious. Common soil-borne diseases of tomatoes include bacterial wilt, wilt, root-knot nematode disease, seedling blight, and blight, which are caused by Fusarium oxysporum, Rhizoctonia solani, Phytophthora, and root-knot nematodes. The accumulation of pathogens, nematodes, fungal toxins, etc. seriously affects the healthy growth of tomatoes and has a serious impact on yield. Soil-borne diseases can reduce crop yields by more than 60% or even cause crop failure, causing huge economic losses to farmers (Ghoname AA, Riad GS, et al. Finding natural alternatives to methyl bromide in greenhouse cantaloupe for yield, quality and disease control[J]. International Journal of ChemTech Research , 2015, 8(9):84-92.). At present, soil disinfection before planting crops is one of the most effective and stable ways to prevent and control soil-borne diseases. Soil fumigation refers to the application of soil fumigants into the soil through professional pesticide application equipment and covering with special plastic film to produce gases with insecticidal, bactericidal, herbicidal or nematicidal effects in an artificially enclosed space, so as to prevent and control soil-borne diseases, insects, weeds and other hazards. (Cao Aocheng, Fang Wensheng, et al. A review of 60 years of soil fumigation in my country [J]. Journal of Plant Protection, 2022, 49(01): 325-335.). The emergence of soil fumigation technology has reduced the use of other disease prevention agents during crop growth, reduced pesticide residues and pollution, and improved crop yield and quality.

[0003] Anaerobic soil disinfestation (ASD), also known as reductive soil disinfection (RSD) or biological soil disinfection (BSD), is widely used in the production of most high-value-added crops to control soil-borne fungi, bacteria, nematodes, weeds and other pathogens (Lee, CG et al. (2020) “Soil prokaryotes are associated with decreasing Fusarium oxysporum density during anaerobic soildisinfestation in the tomato field,” Applied Soil Ecology . Elsevier, 155(November 2019), p. 103632. doi: 10.1016 / j.apsoil.2020.103632.; Song, Z. et al. (2020) “Maltose and totally impermeable film enhanced suppression ofanaerobic soil disinfestation on soilborne pathogens and increased strawberryyield,” Sustainability (Switzerland) , 12(13), pp. 1–21. doi: 10.3390 / su12135456.). In Japan, the Netherlands, the United States, China and other regions, ASD can effectively control soil-borne diseases in various economic crop production. The main steps of ASD technology include adding a certain amount of organic carbon source to the soil, irrigating with sufficient water, and using plastic film to keep it sealed for more than 3 weeks (Zhu, W. et al. (2022) “Influence of reductivesoil disinfestation on the chemical and microbial characteristics of agreenhouse soil infested with Fusarium oxysporum,” Physiological and Molecular Plant Pathology. Elsevier Ltd, 118, p. 101805. doi: 10.1016 / j.pmpp.2022.101805.). The main mechanisms of ASD in controlling soil-borne diseases include: (1) the volatile organic compounds produced by the decomposition of organic carbon sources inhibit soil-borne pathogens; (2) changes in soil physical and chemical indicators such as reduced pH and increased organic matter improve soil quality and thus enhance crop stress resistance; (3) anaerobic soil conditions promote changes in the structure and diversity of soil microbial communities, improve soil microecological environment through biological competition and structural reconstruction, and reduce the risk of soil-borne diseases. Difficulties in promoting ASD technology at this stage: 1. Disinfection time. ASD disinfection usually takes about 3-11 weeks, and the disinfection time requirement is relatively long; 2. High cost. ASD treatment requires the addition of organic carbon sources to the soil. In the early stage, we have determined the ASD technology with one or more of maltose, sucrose, glucose, and lactose as organic carbon sources. In traditional ASD technology, the application amount of one or more of maltose, sucrose, glucose, and lactose is about 900g / ㎡, which is large in dosage and has high labor costs; 3. Unstable effect. The prevention and control effect of ASD on soil-borne diseases also varies depending on the type of organic carbon source, soil physical and chemical properties, and covering film. The effective range of ASD disinfection for solid carbon sources such as wheat bran and organic fertilizers, and liquid carbon sources such as syrup and alcohol added to the soil is usually within 20-60 cm of the soil surface. The disinfection effect of ASD technology on soil-borne pathogenic fungi, bacteria, nematodes, and weeds is between 50-85%. Summary of the invention

[0004] The present invention first provides a coagulant Bacillus NJYB-1, which has a preservation number of CGMCC NO. 31570 (hereinafter referred to as strain W23), which is isolated from soil after ASD. Indoor biological activity assay shows that strain W23 has an antibacterial activity of more than 50% against soil-borne pathogenic fungi such as Fusarium oxysporum and Rhizoctonia solani.

[0005] The present invention first provides a Bacillus coagulans ( Bacillus coagulans ), its deposit number is CGMCCNO. 31570.

[0006] The present invention further provides the use of the condensed spore bacilli in preventing and controlling soil-borne fungal diseases.

[0007] Specifically, the soil-borne fungal diseases are one or more of Fusarium, Phytophthora, Root-knot Nematode, and Colletotrichum.

[0008] Preferably, the soil-borne fungal disease is Fusarium oxysporum ( Fusarium oxysporum )、Verticillium spp.( Fusarium spp.), Neodiscus hirsutus ( P. neoformans), Verticillium dahliae ( V. dahlia ) R. solani )

[0009] The invention also provides a strain for preventing and controlling soil-borne diseases, which is prepared from the coagulated spore bacilli.

[0010] The present invention provides a method for soil disinfection, which comprises the following steps:

[0011] 1) adding the Bacillus coagulans to the soil; and

[0012] 2) performing anaerobic soil disinfection on the soil.

[0013] Preferably, the dosage of the Bacillus coagulans liquid is 5-30 L / mu.

[0014] In addition, preferably, the organic carbon source used in the anaerobic soil disinfection in step 2) is maltose, and the amount used is 200-600 kg / mu.

[0015] The present invention further provides a method for planting crops, wherein the soil is disinfected by the method before planting the crops. Preferably, the crops are tomatoes.

[0016] The present invention shows that strain W23 has an antibacterial activity of more than 50% against soil-borne pathogenic fungi such as Fusarium oxysporum and Rhizoctonia solani through indoor biological activity determination. Field experiments show that adding strain W23 to ASD treatment quickly starts the disinfection process, and can significantly promote the growth of tomato plants after treatment. In particular, the addition of strain W23 significantly increases the plant height and stem thickness of tomatoes, and can significantly increase tomato yield, indicating that strain W23 plays a key role in the ASD disinfection process. A major difficulty in the current promotion of ASD technology is that the cost is too high. Adding strain W23 during the ASD process can effectively reduce the cost of ASD use. In traditional ASD technology, the application amount of maltose is about 900g / ㎡, and the addition amount of strain W23 in the field test of bacterial solution is about 7.5mL / ㎡. The amount of organic carbon source maltose used after adding strain W23 to the improved ASD technology is only 450g / ㎡. The ASD technology after adding strain W23 shows higher biological activity against soil-borne pathogens and can significantly increase tomato yield. The ASD technology with the addition of strain W23 reduces the amount of carbon source used by 50%, greatly reducing its use cost, ensuring high disinfection efficiency, and significantly increasing tomato yields. In addition, after adding strain W23, the commonly used ASD three-week treatment time can be shortened to two weeks, while achieving basically the same microbial killing effect, significantly reducing the time cost of crop planting. It can be seen that the ASD technology with the addition of strain W23 has strong scalability.

[0017] Therefore, based on the screening of strain W23, the present invention provides a soil disinfection method, using maltose as a model carbon source, and quickly starting the ASD disinfection process by adding the screened strain 23 in the ASD disinfection process, stabilizing the ASD disinfection effect, and increasing crop yields. The amount of organic carbon source used in the ASD technology is greatly reduced, ensuring the disinfection effect, shortening the disinfection time, and significantly increasing crop yields.

[0018] Biomaterial Deposit Information

[0019] The strain NJYB-1 of the present invention was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC) on August 12, 2024, with the deposit number: CGMCC NO. 31570, and the classification name: Bacillus coagulans ( Bacillus coagulans ). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the antibacterial activity of the lactic acid bacteria culture liquid and supernatant after 1d and 7d of culture against Fusarium oxysporum (A, C) and Verticillium dahliae (B, D) are shown. The horizontal axis shows the number of the isolated lactic acid bacteria; the vertical axis shows the antibacterial rate.

[0021] Figure 2 The results of the antibacterial activity of lactic acid against pathogenic fungi are shown. a, Fusarium oxysporum; b, Rhizoctonia solani; c, Neodiscus hirsutum; d, Colletotrichum acanthosporium.

[0022] Figure 3 The results of the 16S rDNA BLSAT homology alignment of strain W23 are shown.

[0023] Figure 4 The agarose gel electrophoresis of 16S rDNA identification of strain W23 is shown. Note: M is 5000, 3000, 2000, 1000, 750, 500, 250, 100 bp from top to bottom, and the other lanes are duplicates of the same strain samples.

[0024] Figure 5 The results of the antibacterial activity of the bacterial solution of strain W23 are shown. a, Neodiscusspora; b, Fusarium oxysporum; c, Verticillium dahliae.

[0025] Figure 6 The inhibitory effect of strain W23 on cucumber leaves inoculated with Rhizoctonia solani is shown. a, front of leaves of the water control group; b, front of leaves of the 10X diluted bacterial solution of strain W23; c, back of leaves of the water control group; d, underside of leaves of the 10X diluted bacterial solution of strain W23.

[0026] Figure 7The control effect of ASD disinfection with strain W23 on soil microorganisms (after film removal) is shown. A, Fusarium; B, Phytophthora; C, Root-knot nematodes; D, Lactobacillus. The test site on the left is Shunyi District, and the test site on the right is Fangshan District.

[0027] Figure 8 The control effect of ASD disinfection with strain W23 on soil microorganisms (100 days) is shown. A, Fusarium; B, Phytophthora; C, Root-knot nematodes; D, Lactobacillus. The left test site is Shunyi District, and the right test site is Fangshan District.

[0028] Fig. 9 The effect of ASD disinfection with strain W23 on tomato growth and yield is shown. A, plant height; B, stem diameter; C, leaf chlorophyll; D, yield. The left test site is Shunyi District, and the right test site is Fangshan District. DETAILED DESCRIPTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0030] The present invention is described below by way of examples.

[0031] Example 1: Screening and identification of key functional microorganisms in the ASD process

[0032] In the early stage, an ASD indoor test was conducted using maltose as an organic carbon source. After the test, soil samples were collected for analysis of microbial taxonomic changes. It was found that after ASD treatment, the relative abundance of Sporolactobacillus in the soil exceeded 50%, becoming the absolute dominant genus in the disinfected soil.

[0033] 1.1 Materials and methods

[0034] 1.1.1 Test materials are shown in Table 1.

[0035] Table 1. Test materials

[0036]

[0037] 1.1.2 Test soil

[0038] The soil samples used in the experiment were from Tianyi Strawberry Garden in Changping District, Beijing (40°12′27.74″N, 116°24′25.02″E). After the strawberry vines were pulled, the surface debris of the soil was removed and soil samples from 0-20 cm were collected. The soil samples were sieved with a 2 mm sieve and stored in a refrigerator at 4°C for future use. The park has been planting strawberries continuously for 20 years. The soil samples were taken on January 24, 2022. The soil at the sampling point was not disinfected in 2021. Due to continuous planting of strawberries, strawberry wilt and verticillium wilt occurred seriously at this site. After completing the ASD test indoors, the dryer was opened and placed in a fume hood for 8 days. Soil samples were collected after the airing was completed.

[0039] 1.1.3 Isolation and detection of lactic acid bacteria from soil microorganisms

[0040] The collected soil samples were numbered, and 5 g of fresh soil samples were weighed and mixed with 95 mL of 0.7‰ sterilized agar water, and shaken at 25°C and 200 r / min for 20 min. Then, 1 mL of the above solution was taken and gradient diluted with sterile water. 50 μL of soil suspension was evenly spread on MRS solid medium for each dilution gradient. After the surface of the medium was dry, the culture dish was sealed with a sealing film and transferred to a 37°C anaerobic artificial climate incubator for 24 h. The strains that met the lactic acid bacteria were subcultured, purified and preserved. Lactic acid bacteria culture medium: MRS (Lactobacilli MRS Broth) medium (1L): 20 g glucose, 10 g tryptone, 5 g yeast extract, 10 g beef extract, 0.25 g manganese sulfate tetrahydrate, 0.58 g magnesium sulfate heptahydrate, 1 mL Tween-80, 2.6 g dipotassium hydrogen phosphate, 5 g sodium acetate, and 2 g ammonium citrate. MRS solid medium: Add 1.2% to 1.5% agar powder to the MRS liquid medium.

[0041] 1.1.4 Identification of lactic acid bacteria

[0042] After the single colony of lactic acid bacteria selectively cultured from the soil with MRS solid medium was purified and cultured twice, the typical single colony of lactic acid bacteria on the medium was selected for strain identification, and the strain was named W23. Take a 2 mL sterilized centrifuge tube, add 200 μL of pretreatment solution and three glass beads, pick a single colony in the centrifuge tube, put it in a grinder and grind it thoroughly, add 20 μL of Proteinase K solution, mix it, and place it at 37℃ for 30~60 min, then add 200 μL of lysis solution, mix it thoroughly by inversion, and place it at 70℃ for 10 min; add 200 μL of anhydrous ethanol, mix it thoroughly by inversion, and centrifuge it briefly to remove the droplets on the inner wall of the tube cap. Add 200 μL of anhydrous ethanol, mix it thoroughly by inversion, and centrifuge it briefly to remove the droplets on the inner wall of the tube cap. The adsorption column is placed at room temperature for 5~10 minutes to completely dry the residual rinsing liquid in the adsorption material. Transfer the adsorption column to a new centrifuge tube, add 50~100 μL ddH2O to the middle of the adsorption membrane, place it at room temperature for 5~10 minutes, centrifuge it at 12,000 rpm for 2 minutes, and collect the solution into the centrifuge tube, which is the target DNA. Amplify 16S rDNA. After amplification, take 3 μL of the product and perform 1% agarose gel electrophoresis for detection. After the electrophoresis, a bright band was observed at about 1050 bp in the gel imaging system ( Figure 4 ). The PCR products were sent to Beijing Liuhe BGI Technology Co., Ltd. for sequencing. The sequences were analyzed by BLAST in the NCBI database, and a phylogenetic tree was constructed using Mega7.0 software, with 16S rDNA gene sequence homology A>98% as the identification standard.

[0043] 1.1.5 Indoor activity test of lactic acid bacteria against soil-borne pathogenic fungi

[0044] The lactic acid bacteria isolated from ASD soil were activated using MRS medium for standby use. The pathogenic bacteria block used for confrontation culture was inoculated into the center of the PDA medium. On the PDA plate, vertical lines were drawn at a diagonal distance of 2 cm from the center point, starting from the center point. The activated lactic acid bacteria on the solid MRS medium were dipped with a coating rod, and a 1 cm inoculation line was evenly applied at the line. After sealing with a sealing film, the culture dish was placed in a 28°C constant temperature incubator for light shielding. When the mycelium of the pathogenic bacteria inoculated on the PDA medium of the control group grew to 2 / 3, the colony diameter was measured by the cross method. For the supernatant of lactic acid bacteria, four sterile filter paper pieces with a diameter of 1 cm were placed at a diagonal distance of 2 cm from the center point of the PDA. 25 μL of the supernatant after centrifugation was aspirated and inoculated onto the filter paper pieces for confrontation experiment.

[0045] 1.1.6 Indoor activity test of lactic acid against soil-borne pathogenic fungi

[0046] The mycelium growth rate method was used to determine the sensitivity of the four pathogenic fungi to lactic acid. This experiment was performed under a sterile environment. According to the results of the preliminary experiment, the lactic acid concentration gradient was set as shown in Figure 2. Figure 2 . Take the activated fungal strain PDA plate, prepare the test agent into a series of drug-containing PDA plates with different concentrations, use a hole puncher to punch a 5.0 mm diameter fungus cake on the same circumference of the edge of the strain colony, insert the mycelium side down into the center of the plate, and inoculate one fungus cake per dish. Use the PDA plate without the agent but containing the same amount of distilled water as the control, and set up 3 replicates for each treatment. Invert and culture in a 28℃ incubator under dark conditions for 4-7 days. When the colony of the control group fills 2 / 3 of the PDA plate, the colony diameter is measured by the cross method (Duan Xuelan, Qin Qinglian, Song Chunlin, et al. Indoor toxicity of 10 fungicides and their combinations against wheat sheath blight [J]. Plant Doctor, 2019, 32(06):18-23.DOI:10.13718 / j.cnki.zwys.2019.06.003.).

[0047] 1.1.7 Data Analysis

[0048] Calculation method:

[0049] Calculation method of growth inhibition rate of two pathogenic fungi

[0050] Y = (1 – ( D1-D0 ) × 100 (Formula 1-1)

[0051] Y represents the growth inhibition rate of fungal hyphae (%); D0 represents the diameter of the fungal cake; D1 represents the diameter of the colony in the lactic acid or strain W23 treatment group; D2 represents the diameter of the colony in the blank control treatment group.

[0052] Data processing:

[0053] The experimental data were processed using Excel 2016, IBM SPSS 26.0 and DPS 19.0 data processing systems to calculate the EC values ​​of each agent for the tested strains. 50 value.

[0054] 1.2 Results of indoor activity tests of lactic acid bacteria and lactic acid against soil-borne pathogenic fungi

[0055] 1.2.1 Antibacterial activity of lactic acid bacteria

[0056] Several single colonies were selected from the purified culture W23 strain plate, cultured in MRS medium for 1 day, and named W042, W043, W044, W045, and W046. The bacterial solution and the supernatant after centrifugation had antibacterial activity against Fusarium oxysporum and Verticillium dahliae; the average antibacterial activity of the bacterial solution and supernatant against Fusarium oxysporum was 32.86% and 24.58%, respectively, and the average antibacterial activity of the bacterial solution and supernatant against Verticillium dahliae was 41.65% and 44.73%, respectively. There was no significant difference in the protective effect of the lactic acid bacteria solution and supernatant cultured for 1 day and 7 days against Fusarium oxysporum and Verticillium dahliae. It can be seen that the isolated lactic acid bacteria and its metabolites have antibacterial activity against Fusarium oxysporum and Verticillium dahliae ( Figure 1 ).

[0057] 1.2.2 Antibacterial activity of lactic acid

[0058] Lactic acid against Fusarium oxysporum (EC 50 =1138 mg / kg), Rhizoctonia solani (EC 50 =509 mg / kg), Neodiscus hirsutus (EC 50 =2500 mg / kg), Colletotrichum spinulosum (EC 50 =876 mg / kg) and others have a good inhibitory effect ( Figure 2 ).

[0059] 1.3 Identification of strain W23

[0060] The culture characteristics of strain W23 are as follows: it is rod-shaped with blunt ends, Gram-positive, catalase-positive, with terminal spores and no flagella, and can decompose sugars to produce L-lactic acid.

[0061] After 16S rDNA sequencing, strain W23 was accessed to Genbank with accession number 9GS145SM01R. Homology comparison was performed with related species within the genus Bacillus using MEGA7.0 software. If the 16S rDNA sequence homology was greater than 99%, it was considered to belong to the same species. Figure 3 As shown, strain W23 and Bacillus coagulans were compared by BLSAT homology. Bacilluscoagulans The strain has the closest relationship with the homology of 100.00%, so it belongs to Bacillus coagulans and has been biologically preserved with the preservation number: CGMCC No:31570.

[0062] Example 2: Effect of strain W23 on ASD

[0063] 2.1 Materials and methods

[0064] 2.1.1 Test materials

[0065] See Table 1.

[0066] 2.1.2 Indoor activity test of strain W23 against soil-borne pathogenic fungi

[0067] The specific method is the same as 1.1.5.

[0068] 2.1.3 Determination of the antibacterial activity of strain W23 against cucumber leaves inoculated with Rhizoctonia solani

[0069] Collect fresh and healthy cucumber leaves, pierce 5 holes on the front and back of the cucumber leaves with a sterile needle, try not to pierce through the leaves, and then inoculate 1 cm diameter Rhizoctonia solani fungus blocks at the pierced positions. Spread sterile filter paper in a 20 cm diameter petri dish, then add 5 ml sterile water, and place the inoculated fresh cucumber leaves on the filter paper. Then evenly spray the 100-fold diluted strain W23 bacterial solution on the leaf surface. After the leaf surface is dry, cover the petri dish and place it in a 28°C constant temperature incubator to observe the changes on the leaf surface.

[0070] 2.1.4 ASD field trials

[0071] In 2022, field trials were conducted at Hongke Farm in Fangshan, Beijing (39°74′91.44″N, 116°14′32.67″E) and Shiwaiyuan Farm in Shunyi (40°13′03.47″N, 116°65′45.61″E) to control soil-borne diseases such as tomato wilt and root-knot nematodes. Specific information on the test sites is shown in (Table 2).

[0072] Table 2 Field test schedule

[0073]

[0074] The tomato solar greenhouse did not undergo soil disinfection in 2021. In the experiment, the amount of maltose used for ASD treatment was 300 kg / mu, which was dissolved in water and directly applied to the ground, and then irrigated with clean water, with a water consumption of 4 tons / mu, covered with PE film, and the closed disinfection time was 3 weeks. The amount of the supplemented probiotic strain W23 was 5 L / mu, which was evenly sprinkled on the soil surface and tilled evenly with a rotary tiller, and then operated according to the ASD steps. The amount of Weibaimu was 53.37 kg / mu, and drip irrigation was used for drug application, with a water consumption of 4 tons / mu and covered with PE film. The specific experimental plan is shown in Table 3.

[0075] Table 3. ASD field trial design for controlling soil-borne diseases of tomatoes - Beijing

[0076]

[0077] 2.1.5 Soil sample collection

[0078] At the end of fumigation, soil samples were collected twice after film removal and 100 days after tomato planting for isolation and detection of soil-borne pathogens.

[0079] 2.1.6 Isolation of soil-borne pathogens

[0080] The colony counts of Fusarium and Phytophthora in soil samples were calculated according to Komada (Komada H. Development of a selective medium for quantitative isolation of Fusarium oxysporum fromnatural soil[J]. Review of plant protection research , 1975, 8: 114-124..) and Masago (Masago H.Selective Inhibition of Pythium spp. on a Medium for DirectIsolation of Phytophthora spp. from Soils and Plants[J]. Phytopathology , 1977,77(3)) method for separation and detection. The colony counts of Fusarium and Phytophthora in soil samples after ASD disinfection were used to evaluate the disinfection effect of ASD.

[0081] 2.1.7 Tomato yield and growth indicators

[0082] 100 days after transplanting, 20 tomatoes were randomly selected from each plot to measure plant height, stem diameter, and chlorophyll. The measurements were made using a tape measure, a vernier caliper, a chlorophyll meter, and a balance. During the entire tomato growth period, 2 beds were fixed in each plot to record tomato yield data, and the total tomato yield of each treatment plot was calculated after the experiment.

[0083] 2.1.8 Data Analysis

[0084] Calculation formula

[0085] The calculation method of the mycelial growth inhibition rate of pathogenic fungi is as follows:

[0086] (Formula 2)

[0087] Y is the growth inhibition rate of pathogenic fungal mycelium (%), D1 is the diameter of the test fungus cake (7 mm), D2 is the colony diameter of the pathogenic fungus after fumigation treatment, and D3 is the colony diameter of the pathogenic fungus in the blank control group.

[0088] The formula for calculating the control efficiency of pathogenic fungi is as follows:

[0089] (Formula 3)

[0090] Y is the control efficiency of Fusarium and Phytophthora (%), X2 is the colony count of Fusarium and Phytophthora in the blank control group, and X1 is the colony count of Fusarium and Phytophthora in the treatment group.

[0091] SPSS 26.0 (IBM, USA) was used to analyze the effects of different treatments on the spores of Fusarium spp. Fusarium spp.), Phytophthora spp. Phytophthora spp.) and tomato plant height, stem diameter and other experimental data were statistically analyzed, and one-way analysis of variance (ANOVA) and Duncan's new multiple range method were used for significant difference test ( p =0.05), and Origin 2018 was used to draw the table.

[0092] Data processing

[0093] The experimental data were processed using Excel 2016, IBM SPSS 26.0 and DPS 19.0 data processing systems to calculate the EC values ​​of each agent for the tested strains. 50 value.

[0094] 2.2 Strain W23 for ASD enhancement

[0095] 2.2.1 Antibacterial activity of strain W23

[0096] The inhibition rates of strain W23 isolated from ASD soil against pathogenic bacteria Fusarium oxysporum, Fusarium verticillium and Neodiscus polytrichosporium were 61.64%, 52.67% and 68.06%, respectively. Figure 5 ).

[0097] 2.2.2 Antibacterial activity of strain W23 against cucumber leaves inoculated with Rhizoctonia solani

[0098] The results of the indoor experiment showed that after the cucumber leaves were inoculated with the pathogenic bacteria of Rhizoctonia solani and cultured at 28℃ for 3 days, it was found that compared with the water control, the cucumber leaves sprayed with 10X diluted strain W23 solution were dark green in color, and no lesions were seen at the inoculated site; while the cucumber leaves treated with the water control turned yellow overall, and the lesions were obviously expanded. It can be seen that strain W23 has an inhibitory effect on Rhizoctonia solani by foliar spraying ( Figure 6 ).

[0099] 2.2.3 Effect of ASD disinfection with beneficial bacteria on soil microorganisms

[0100] After ASD disinfection and film removal (on August 7 and August 6, 2022, respectively, in Fangshan and Shunyi), soil samples were taken during tomato growth (100 days) for analysis and detection of Fusarium, Phytophthora and root-knot nematodes in the soil. The test results showed that compared with the blank control, the inhibition rates of Fusarium, Phytophthora and root-knot nematodes in the soil after ASD treatment and ASD+B treatment at Fangshan and Shunyi test points after film removal exceeded 97.99%, 95.98% and 91.60% respectively; the number of lactic acid bacteria in the soil samples treated with ASD+B after film removal was significantly higher than that in the blank control and ASD treatment (Table 4 and Figure 7 ).

[0101] Table 4. Data on the control of soil microorganisms after ASD disinfection with beneficial bacteria added (after film removal)

[0102]

[0103] After 100 days of disinfection, the inhibition rates of Fusarium, Phytophthora and root-knot nematodes in the soil of Fangshan and Shunyi after ASD treatment and ASD+B treatment were as high as 80.13%, 74.86% and 67.29%, respectively, during the tomato growth period. The inhibition effect of ASD and ASD+B disinfection treatments on Phytophthora was significantly higher than that of MS treatment. The colony count of lactic acid bacteria in the soil samples treated with ASD+B disinfection was significantly higher than that of other treatments. It can be seen that lactic acid bacteria have a good colonization ability in the soil (Table 5 and Figure 8 ).

[0104] Table 5. Data on the control effect of ASD disinfection with strain W23 on soil-borne pathogenic microorganisms (100 days after tomato planting)

[0105]

[0106] 2.2.4 Effects of ASD disinfection with beneficial bacteria on tomato growth and yield

[0107] ASD and ASD+B treatments can significantly promote the growth of tomato plants, especially after ASD+B treatment, the height and stem diameter of tomatoes increased by 44.26% and 24.02%, respectively. It can be seen that the disinfection technology of ASD and the combination of ASD and strain W23 can effectively prevent and control soil-borne diseases caused by Fusarium, Phytophthora and root-knot nematodes in tomato crops, and can significantly promote the growth of tomatoes (Table 6 and Fig. 9 ).

[0108] Table 6. Data on growth and yield of tomatoes after ASD disinfection with strain W23

[0109]

[0110] Field trials showed that the (ASD+B) treatment supplemented with strain W23 (6.17 kg / m 2 ) and blank treatment (4.60kg / m 2 ) yield, the ASD+B treatment significantly increased tomato yield by 34.12%, and was superior to the yield of the ASD treatment alone (5.48 kg / m 2 )(Table 6 and Fig. 9 ).

Claims

1. A Bacillus coagulans ( Bacillus coagulans ), characterized in that, Its deposit number is CGMCC NO.31570.

2. Use of Bacillus coagulans as claimed in claim 1 in preventing and controlling soil-borne fungal diseases, wherein the soil-borne fungal diseases are one or more of Fusarium, Phytophthora, Root-knot Nematode, and Colletotrichum.

3. The use according to claim 2, characterized in that The soil-borne fungal diseases are Fusarium oxysporum, Fusarium verticillium, Neodiscus polytrichosporium, and Verticillium dahliae.

4. A microbial agent for preventing and controlling soil-borne diseases, characterized in that: The bacterial liquid or supernatant is prepared from the Bacillus coagulans as claimed in claim 1.

5. A method for soil disinfection, characterized in that: The steps include: 1) adding the Bacillus coagulans according to claim 1 to the soil; and 2) performing anaerobic soil disinfection on the soil.

6. The method according to claim 5, characterized in that The dosage of the Bacillus coagulans liquid is 5-30 L / mu.

7. The method according to claim 5, characterized in that The organic carbon source used in the anaerobic soil disinfection in step 2) is maltose, and the amount used is 200-600 kg / mu.

8. A method for planting crops, characterized in that: Before planting crops, the soil is disinfected using the method according to any one of claims 5 to 7.

9. The method according to claim 8, characterized in that The crop is tomato.

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