A myrosinase-producing Cupriavidus strain and its application in preventing and controlling soil-borne diseases

Abstract

The present invention discloses a myrosinase-producing Cupria virescens strain and its application in preventing and controlling soil-borne diseases. Cupriavidus sp.) is deposited under CGMCC No. 34736. Further provided are uses of the strain for preventing and controlling soil-borne fungal diseases and methods for soil disinfection. The soil disinfection method provided by the present invention significantly increases the amount of isothiocyanates produced during biological fumigation technology and significantly inhibits soil-borne pathogens.

Description

Technical Field

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

[0002] With the advancement of intensive planting and continuous cultivation of high-value-added crops, the occurrence of soil-borne diseases has gradually intensified. Common soil-borne diseases include bacterial wilt, fusarium wilt, root-knot nematode disease, seedling blight and blight, which are caused by pathogens such as Fusarium oxysporum, Rhizoctonia solani, Phytophthora and root-knot nematodes. The accumulation of pathogens, nematodes and fungal toxins seriously affects the growth of crops and the health of the soil, and poses a significant threat to yield. Soil-borne diseases may cause crop yields to decrease by more than 60%, or even result in total 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). In this context, soil disinfection has been widely used as one of the effective means to prevent and control soil-borne diseases. Soil disinfection is a technique that involves applying soil fumigants to the soil using specialized equipment and covering it with plastic film. The technique uses gases released in a closed environment (such as insecticides, fungicides, and herbicides) to prevent and control soil-borne diseases, insect pests, and weeds (Cao Aocheng, Fang Wensheng, et al. A 60-year review of soil fumigation in my country [J]. Acta Phytophylacica Sinica, 2022, 49(01): 325-335). The promotion of this technology not only reduces dependence on other disease control agents during crop growth, but also effectively reduces pesticide residues and pollution, while also improving crop yield and quality.

[0003] Soil biofumigation technology (Biofumigation) is an environmentally friendly soil fumigation method that uses substances with killing activity produced by plants during decomposition, fermentation, metabolism, etc. to inhibit soil-borne pathogens, pests, weeds, etc. (Szczyglowska M, Piekarska A, Konieczka P, et al. Use of brassica plants in the phytoremediation and biofumigation processes[J]. International Journal of Molecular Sciences,2011,12(11):7760-7771.). When biofumigation is applied in the field, the plants with control effects should be chopped and quickly and evenly turned into the soil. The soil should be irrigated to make it completely moist and covered with a film to form an enclosed space to achieve the best fumigation effect. The fumigation time can be appropriately increased when the temperature is low (Matthiessen J, Warton B,Shackleton M. The importance of plant maceration and water addition in achieving high Brassica -derived isothiocyanate levels in soil[J].Agroindustria, 2004, 3(3): 277-281; ​​Gimsing A, Kirkegaard J. Glucosinolate andisothiocyanate concentration in soil following incorporationof Brassica biofumigants[J]. Soil Biology and Biochemistry, 2006, 38(8): 2255-2264.).

[0004] The main mechanisms of action of biological fumigation of cruciferous plants on soil-borne disease prevention and control include: (1) the volatile substances produced by the crushing and decomposition of cruciferous plants inhibit soil-borne pathogens; (2) changes in soil physical and chemical indicators such as ammonium nitrogen and organic matter increase, improve soil quality and thus enhance crop resistance; (3) soil biological fumigation promotes changes in soil microbial community structure and diversity, improves soil microecological environment through biological competition and structural reconstruction, and reduces the risk of soil-borne diseases. The current difficulties in promoting soil biological fumigation technology are: first, the fumigation time is long, usually about 4-8 weeks; second, the effect is unstable, and the effect of biological fumigation on soil-borne diseases also varies depending on the type of plant, soil physical and chemical properties, and the covering film. The disinfection effect of soil biological fumigation technology on soil-borne pathogenic fungi, bacteria, nematodes, and weeds is between 40.7% and 61.7%. Summary of the Invention

[0005] The present invention obtains a myrosinase-producing Cupriphae sp. strain ( Cupriavidus sp.) BC4. The indoor biological fumigation synergy verification showed that after adding the Cupriavidus strain BC4, compared with the blank control and cauliflower treatment, the control effect on the root-knot nematodes in the soil was as high as 100%, and the inhibition rate on the Fusarium genus could reach 98.50%-99.25%. At the same time, it significantly promoted the growth of cucumber plants.

[0006] The present invention first provides a myrosinase-producing Cupribotium strain BC4, which has a deposit number of CGMCC NO.34736. It was deposited on June 3, 2025 in the General Microbiology Center of the China Culture Collection Administration (abbreviated as CGMCC, the depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China), and is classified as: Cupribotium Cupriavidus sp.

[0007] The present invention further provides the use of the Cupriavidus strain BC4 in synergistic biological fumigation.

[0008] Specifically, the soil-borne fungal diseases are one or more of Fusarium, Phytophthora, and Root-knot Nematode.

[0009] The present invention also provides a soil disinfectant for synergistically preventing and controlling soil-borne diseases, which is prepared from the Cupriavidus strain BC4.

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

[0011] 1) adding the Cupriavida strain BC4 to the soil;

[0012] 2) performing soil biological fumigation disinfection on the soil.

[0013] Preferably, the dosage of the Cupriavidus strain BC4 bacterial solution is 0.04*10 6 -0.2*10 6 cfu / g.

[0014] In addition, preferably, the amount of the Brassica plant of the Cruciferae family used in the soil biological fumigation in step 2) is 6.6 g / kg.

[0015] The present invention shows through indoor biological activity assays that the Cupria viride strain BC4 has inhibitory activity against soil-borne pathogens such as Fusarium, Phytophthora, and Root-knot Nematode. Indoor verification experiments have shown that adding the Cupria viride strain BC4 to the biological fumigation treatment quickly starts the fumigation process and reduces the fumigation time, indicating that the Cupria viride strain BC4 plays a key role in the biological fumigation process. A major difficulty in the current promotion of biological fumigation technology is unstable effects. Adding the Cupria viride strain BC4 to the biological fumigation process can effectively improve the stability of the inhibitory effect on soil-borne pathogens. Traditional biotechnology only adds cruciferous plant materials, and the improved biological fumigation technology needs to add strain BC4 before use. The biological fumigation technology after adding the Cupria viride strain BC4 shows higher biological activity against soil-borne pathogens. In addition, after adding the Cupria viride strain BC4, the commonly used biological fumigation treatment time of 4-8 weeks can be shortened to two weeks, achieving a pathogen killing effect significantly higher than the original technology, significantly reducing the time cost of crop planting. It can be seen that the biological fumigation technology with the addition of Cupriavidus strain BC4 has strong scalability.

[0016] Therefore, based on the screening of the Cupriavidus strain BC4, the present invention provides a soil biological fumigation disinfection method. Using Brassica plants from the Cruciferae family as the model fumigation material, the method rapidly initiates the biofumigation process by adding the selected strain BC4 during the biofumigation process, stabilizing the biofumigation effect. Furthermore, the biofumigation method significantly improves the pathogen-killing efficacy, ensuring fumigation effectiveness and shortening fumigation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Colony image of Cupriavidus strain BC4 on a plate.

[0018] Biomaterial deposit information:

[0019] The present invention's Cupricobacterium strain BC4 was deposited on June 3, 2025 in the General Microbiology Center of the China Culture Collection Administration (CGMCC, the depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China), with the deposit number: NO.34736, and the classification name: Cupricobacterium Cupriavidus sp. DETAILED DESCRIPTION

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0021] The present invention is described below by way of examples.

[0022] Example 1 Screening and identification of key functional bacteria in soil after biological fumigation

[0023] In the early stage, indoor and field biological fumigation experiments were carried out using cruciferous cabbage, cauliflower, rapeseed and other plant materials. After the experiment, soil samples were collected for strain screening and identification, and it was found that the bacteria of the genus Cupriavidus in the soil had the strongest ability to produce myrosinase.

[0024] 1.1 Materials and Methods

[0025] 1.1.1 Test soil

[0026] Soil samples were collected from the Pesticide Biology Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, and Yimudi Ecological Park, Miyun District (40°33′37.94″N, 116°82′97.92″E). Surface debris was removed, and soil samples were collected from the depth of 0–20 cm. The samples were sieved with a 2 mm sieve and stored in a refrigerator at 4°C until use. Soil sampling took place on May 24, 2023. No soil disinfection was performed at the sampling site in 2023. Soil samples were collected after the indoor biofumigation test was completed.

[0027] 1.1.2 Isolation and detection of Cupribotium

[0028] Collected soil samples were numbered, and 1 g of fresh soil sample was weighed and mixed with a selective liquid medium (using glucosinolates as the sole carbon source and the same inorganic salt composition as M9 medium) and shaken at 28°C and 180 rpm for 4 days. Next, 1 mL of this solution was aspirated and serially diluted with sterile water. For each serial dilution, 100 μL of the soil suspension was evenly spread onto a selective solid medium (using glucosinolates as the sole carbon source and the same inorganic salt composition as M9 medium). After the surface of the medium dried, the dish was sealed with parafilm and transferred to a 37°C artificial climate incubator for incubation. Strains grown on the medium were subcultured, purified, and stored.

[0029] 1.1.3 Identification of bacterial species

[0030] The soil was selectively cultured on a selective solid medium. A single colony that could grow and produce a precipitation ring on the selective medium was selected. After two purification cultures, a single colony that showed typical copper-grabbing bacteria on the medium was selected for strain identification. The strain was named BC4. The plate colony diagram is shown below. Figure 1As shown. Add 200 μL of pretreatment solution and three glass beads to a 2 mL sterilized centrifuge tube. Pick a single colony and place it in a centrifuge tube. Grind thoroughly in a grinder. Add 20 μL of Proteinase K solution, mix thoroughly, and incubate at 37°C for 30–60 min. Then, add 200 μL of lysis buffer, invert thoroughly to mix, and incubate at 70°C for 10 min. Add 200 μL of anhydrous ethanol, invert thoroughly to mix, and briefly centrifuge to remove any droplets on the inner wall of the tube cap. Add 200 μL of anhydrous ethanol, invert thoroughly to mix, and briefly centrifuge to remove any droplets on the inner wall of the tube cap. Incubate the adsorption column at room temperature for 5–10 minutes to completely dry any remaining rinse solution from the adsorption material. Transfer the adsorption column to a new centrifuge tube and drip 50–100 μL of ddH2O onto the center of the adsorption membrane. Incubate at room temperature for 5–10 min. Centrifuge at 12,000 rpm for 2 min. Collect the solution into the centrifuge tube, which contains the target DNA. 16S rDNA was amplified and 3 μL of the product was subjected to 1% agarose gel electrophoresis for detection. After the electrophoresis, a bright band at about 1050 bp was observed in the gel imaging system. The PCR product was sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The sequence was subjected to BLAST analysis in the NCBI database and was found to be a bacteria of the genus Cupriphae. Cupriavidus sp., and deposited it with the deposit number: CGMCC NO.34736.

[0031] Example 2 Determination of the synergistic effect of Cupriavidus strain BC4 on biological fumigation

[0032] 2.1 Test method

[0033] 2.1.1 Determination of the control effect of strain BC4 on soil-borne pathogens

[0034] This experiment used cauliflower combined with the myrosinase-producing strain Cuprilobium BC4 for plant fumigation. The experimental settings included treatments with only cauliflower, treatments with cauliflower and a low concentration of BC4, treatments with cauliflower and a medium concentration of BC4, treatments with cauliflower and a high concentration of BC4, and a control without cauliflower.

[0035] The concentration in BC4 is the bacterial solution OD 600 = 0.6, the low BC4 concentration was 1 / 5 times the medium BC4 concentration, and the high BC4 concentration was 5 times the medium BC4 concentration. Based on the preliminary test results, the total amount of bacterial solution added to each bottle was 3 mL. Sterile water was used instead for the broccoli-only treatment and the control without broccoli.

[0036] For each fumigation treatment, the plant material was cut into small pieces and applied to cauliflower at a dose of 6.6 g / kg. The mixture was evenly mixed into a wide-mouth bottle containing 300 g of soil with a 20% moisture content and the corresponding solution was added. The additive was thoroughly mixed with the soil, and the bottle cap was tightly closed and sealed with parafilm. Each treatment was repeated six times. The wide-mouth bottle was placed in a 35°C constant temperature incubator and fumigated for 15 days to simulate the field environment conditions commonly encountered in plant fumigation.

[0037] After fumigation, collect soil samples for isolation of soil-borne pathogens and determination of control effectiveness as described in 2.1.2.

[0038] 2.1.2 Isolation of soil-borne pathogens

[0039] The number of Fusarium and Phytophthora colonies and root-knot nematodes in soil samples was determined according to the Komada method (Komada H. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil[J]. Review of plant protection research , 1975, 8:114-124.), Masago (Masago H.Selective Inhibition of Pythium spp. on a Medium for Direct Isolation of Phytophthora spp. from Soils and Plants[J]. Phytopathology , 1977, 77(3), Huang YH, Mao ZC, Xie BY, Chinese leek (Alliumtuberosum Rottler ex Sprengel) reduced disease symptom caused by root-knotnematode. Journal of Integrative Agriculture 15: 364-372 (2016).) method for separation and detection. The colony counts of Fusarium and Phytophthora and the number of root-knot nematodes in soil samples after biological fumigation with strain BC4 were used to evaluate the synergistic effect of strain BC4 on biological fumigation.

[0040] 2.1.3 Data Analysis

[0041] The formula for calculating the control efficiency of pathogenic fungi is as follows:

[0042]

[0043] Y is the control efficiency of Fusarium and Phytophthora (%), X2 is the number of Fusarium and Phytophthora colonies and the number of root-knot nematodes in the blank control group, and X1 is the number of Fusarium and Phytophthora colonies and the number of root-knot nematodes in the treatment group.

[0044] SPSS 26.0 (IBM, USA) software was used to analyze the effects of different treatments on the spores of Fusarium spp. Fusarium spp.), Phytophthora spp. Phytophthora spp.), root-knot nematodes ( Meloidogyne spp.) were used for statistical analysis of the data, and the significance of differences was tested using one-way analysis of variance (ANOVA) and Duncan's new multiple range method. p =0.05).

[0045] 2.2 Effect of strain BC4 on the control of soil pathogens using biofumigation synergistic effect

[0046] Table 1 shows the inhibitory rates of biofumigation combined with strain BC4 against soilborne pathogens. The results showed that compared with the blank control, both the broccoli addition and the broccoli plus BC4 treatments significantly reduced the CFU counts of Fusarium and Phytophthora in the soil, as well as the number of root-knot nematodes. Furthermore, the treatments significantly increased the inhibitory rates against Fusarium, Phytophthora, and root-knot nematodes. The broccoli addition treatment had an inhibitory rate of 50.16% against Fusarium. The combined treatment with low, medium, and high concentrations of BC4 increased the inhibitory rates by 43.64%, 47.82%, and 49.09%, respectively, compared to the broccoli addition alone treatment. The broccoli addition treatment had an inhibitory rate of 40.68% against Phytophthora. The combined treatment with low, medium, and high concentrations of BC4 increased the inhibitory rates by 15.00%, 25.69%, and 35.23%, respectively, compared to the broccoli addition alone treatment. The cauliflower treatment had a 61.70% inhibition rate against root-knot nematodes. The combined application of low, medium, and high concentrations of BC4 increased the inhibition rate against Fusarium by 55.00%, 38.30%, and 38.30%, respectively, compared to the cauliflower-only treatment. The combination of three concentrations of BC4 also significantly increased the inhibition rate against soil-borne pathogens compared to the cauliflower control. The high concentration significantly outperformed the medium and low concentrations in terms of inhibition against Fusarium and Phytophthora. The high concentration was as effective as the medium concentration and significantly higher than the low concentration in terms of inhibition against root-knot nematodes.

[0047] Table 1. Inhibition rate of plant fumigation combined with Cupribotium BC4 against soil-borne pathogens

[0048] deal with Fusarium spp. (CFU / g) Inhibition rate of blank control (%) Cauliflower control inhibition rate (%) Phytophthora spp. (CFU / g) Inhibition rate of blank control (%) Cauliflower control inhibition rate (%) Root-knot nematodes (number / 100g soil) Inhibition rate of blank control (%) Cauliflower control inhibition rate (%) blank 20427±606a 0 d - 23467±672a 0 e - 1000±91a 0 d - brocoli 10180±477b 50.16±2.34 c 0 d 13920±212b 40.68±0.90 d 0 d 383±25 b 61.70±2.50 c 0 c Broccoli + BC4 low concentration 1267±114 c 93.80±0.56 b 87.55±1.12 c 10400±160c 55.68±0.68 c 25.29±1.15 c 133±23 c 86.70±2.30 b 65.27±6.00 b Broccoli + BC4 medium concentration 413±24 cd 97.98±0.12 a 95.94±0.24 b 7893±92 d 66.37±0.39 b 43.30±0.66 b 0±0 d 100±0a 100±0 a Cauliflower + BC4 high concentration 153±12 d 99.25±0.06 a 98.50±0.12 a 5653±92 e 75.91±0.39 a 59.40±0.66 a 0±0 d 100±0a 100±0 a

[0049] 2.3 Effect of strain BC4 on biofumigation synergistic effect on cucumber growth

[0050] Table 2 shows the effects of biofumigation combined with strain BC4 on cucumber growth. Fumigation with strain BC4 promoted cucumber growth to a certain extent. The cauliflower treated with a high concentration of BC4 had the best growth effect, followed by cauliflower treated with a medium concentration of BC4, cauliflower treated with a low concentration of BC4, cauliflower, and the control. Compared with the blank control, BC4 addition had positive effects on various cucumber growth indicators, including significant increases in germination rate by 33.33-49.99%, plant height by 16.72-30.98%, chlorophyll content by 12.82-24.60%, underground length by 11.65-30.33%, fresh weight by 56.99-75.16%, and dry weight by 72.09-90.70%. The high-concentration BC4 treatment significantly increased stem diameter by 28.11% compared to the blank control, while the other treatments had no significant effect.

[0051] Compared to the cauliflower control, addition of different concentrations of BC4 significantly increased the fresh and dry weights of cucumber plants, with increases ranging from 22.28-36.42% and 27.59-41.38%, respectively. High-concentration BC4 also significantly increased plant height, chlorophyll content, and underground length of cucumber plants, with increases of 20.81%, 11.97%, and 21.90%, respectively. Overall, the effects of plant fumigation combined with BC4 on cucumber growth were greater with addition of the bacterial solution than without it, with high-concentration treatments outperforming medium-concentration and low-concentration treatments (Table 2).

[0052] Table 2 Effects of plant fumigation combined with strain BC4 on cucumber growth

[0053] deal with Germination rate (%) Plant height (cm) Stem diameter (mm) Chlorophyll (SAPD) Underground length (cm) Fresh weight (g) Dry weight (g) comparison 66.67±25.00 b 8.91±0.17c 2.81±0.32 b 28.01±1.26 c 8.67±0.19d 4.79±0.11 c 0.43±0.02 d brocoli 77.78±26.35 ab 9.66±0.28bc 3.03±0.27 ab 31.17±0.42 b 9.27±0.18c 6.15±0.12b 0.58±0.01 c Broccoli + BC4 low concentration 88.89±22.05 ab 10.40±0.17 ab 3.20±0.07 ab 31.60±0.44 b 9.68±0.22c 7.52±0.08 a 0.74±0.01 b Broccoli + BC4 medium concentration 100±0 a 10.83±0.20 ab 3.24±0.08 ab 32.50±0.46 b 10.39±0.15 b 7.59±0.11 a 0.78±0.02 ab Cauliflower + BC4 high concentration 100±0 a 11.67±1.15 a 3.60±0.24 a 34.90±0.23 a 11.30±0.28 a 8.39±0.13 a 0.82±0.02 a

Claims

1. A myrosinase-producing Cupriphae strain ( Cupriavidus sp.), characterized in that Its deposit number is CGMCC NO.34736.

2. Use of the Cupriavidus strain according to claim 1 in preventing and controlling diseases caused by soil-borne fungi, wherein the soil-borne fungi are one or more of the genera Fusarium, Phytophthora and Root-knot Nematode.

3. The soil disinfectant prepared by the Cupriavidus strain according to claim 1, characterized in that It contains active bacteria of the Cupriavida strain.

4. The soil disinfectant according to claim 3, characterized in that It exists in the form of bacterial liquid, and the concentration of active bacteria in the bacterial liquid of the genus Cupriavidus is 0.04*10 6 -0.2*10 6 cfu / g.

5. A method for soil disinfection, characterized in that: The process includes the following steps: 1) adding a bacterial solution of the Cupriavidus strain according to claim 1 to the soil; 2) biofumigating the soil; In step 2), Brassica plants of the genus Brassica in the family Cruciferae are used in the biofumigation.

6. The method according to claim 5, wherein The dosage of the bacterial solution of the Cuprobacterium strain is 0.04*10 6 -0.2*10 6 cfu / g.

7. The method according to claim 5, wherein The Brassica plant of the Cruciferae family is one or more of cabbage, cauliflower or mustard, and the dosage is 6-7 g / kg.

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

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