A Bacillus subtilis 04 bactericide for preventing and controlling root rot of traditional Chinese medicinal materials and its application

Abstract

The present invention provides a Bacillus subtilis strain 04 bactericide for preventing and controlling root rot of Chinese medicinal materials and its application. The preservation number of Bacillus subtilis strain 04 of the present invention is CGMCC No. 30084. Bacillus subtilis strain 04 of the present invention has good antibacterial effects on various Fusarium species, produces biocontrol active substances such as protease, amylase, cellulase, glucanase and pullulanase, has strong salt tolerance and no hemolytic property; the control effect of the Bacillus subtilis strain 04 bactericide on the root rot of Lycium barbarum and Astragalus membranaceus reaches 68.01% - 80.44%, and can promote the root length and plant height of Astragalus membranaceus and Scutellaria baicalensis. It is a biocontrol strain with excellent effects and has important significance for the biological control of plant diseases.

Description

Technical Field

[0001] The present invention belongs to the field of microbial pesticides, and mainly relates to a Bacillus subtilis strain, a bacterial agent for preventing and controlling root rot of Chinese medicinal materials, and an application method thereof. Background Art

[0002] Root rot is a common soil-borne disease occurring in crop planting areas, mainly caused by the combined infection of pathogenic bacteria such as Fusarium and Alternaria, which severely restricts the sustainable development of modern agriculture and is known as the "plant cancer". Root rot has been found in medicinal plants in many parts of the world. The root rot of Lycium barbarum causes 5% of the Lycium barbarum trees to wither every year, the diseased plant rate is 15%, and in severely affected areas, the diseased plant rate is 37.6% and the withered plant rate reaches 26.5%; in low-lying and poorly drained plots, the incidence of Astragalus membranaceus root rot is 32% - 41%, and in severely diseased fields, it reaches 55%; for Isatis indigotica Fort. with mild root rot, the yield reduction in the medicinal material area is 11.2% - 21.6%, and for those with severe root rot, the yield reduction is more than 35%, and in severe years, the yield reduction is 50%; the field incidence of Stellaria dichotoma root rot is 15% - 30%, and in severely affected plots, it is as high as more than 60%. Planting disease-resistant varieties and chemical pesticides are the main measures for preventing and controlling crop diseases and pests. Since the breeding cycle of disease-resistant varieties is relatively long, the residues of chemical pesticides pose a threat to the ecological environment and human health. Therefore, exploring environmentally friendly and highly efficient biological control technologies for root rot of Chinese medicinal materials has important scientific value for protecting the ecological environment and the high-quality development of the Chinese medicinal materials industry.

[0003] Biocontrol bacteria in the rhizosphere soil can colonize on the surface of the roots. As the roots grow, the colonies gradually increase and finally connect to surround the roots to form a mucilage layer or mucilage. Microorganisms are all embedded in the mucilage, and the mucilage will prevent the colonization of later microorganisms. Protected by this physical barrier, it is difficult for some plant pathogens to cross this barrier and invade the plant roots. Therefore, the plant roots can grow in a relatively stable environment. The bacteria used for preventing and controlling diseases and pests of Chinese medicinal materials mainly include Bacillus, among which Bacillus has the advantages of good environmental compatibility, high disease and insect control activity, strong stress resistance, harmless to humans and animals, etc., and is the most widely used biocontrol bacteria. Some Bacillus subtilis can effectively colonize on plant roots and regulate the physiological functions of host plants to promote plant growth.

[0004] Bacillus can produce chitinase, sugar degrading enzyme, cell wall degrading enzyme, etc., which can dissolve the cell walls of plant pathogenic hyphae. Especially those senescent or even nearly dead hyphae are more likely to be dissolved. Bacillus subtilis XG-7 has good antibacterial activity against Fusarium head blight. D1 / 2 and DZSG23 can inhibit the mycelial growth of Fusarium graminearum, slow down spore germination, and make spores deformed; Z54, AF0907, B-30210, RC218, etc. can reduce the incidence of wheat Fusarium head blight; SG6 inhibits mycelial growth, sporulation amount and DON production. Wang Tingting et al. (2024) isolated Bacillus subtilis XH-1, which inhibits the germination of conidia of Fusarium graminearum, and also makes the spores deformed and enlarged, significantly reducing the damage of wheat Fusarium head blight. Qin Nan et al. (2023) isolated the Bacillus subtilis LF17 agent, and the control effect on downy mildew was 59.59%, significantly higher than that of agents such as dimethomorph and propamocarb hydrochloride. After treatment, the fresh weight of Chenopodium album seedlings and stem length increased by 54.5% and 24.2% respectively compared with the control, and the spike length and 1000-grain weight increased by 20.4% and 29.1% respectively compared with the control. In the pot experiment of the fermentation broth of Bacillus subtilis GXHZ16 isolated by Fan Qiling (2023), the relative pot control effect of the GXHZ16 fermentation broth (1×10 8 CFU / mL) against tobacco black shank reached 51.49±2.69%. However, at present, there are few Bacillus subtilis agents that can effectively control the diseases of traditional Chinese medicinal materials. It is necessary to carry out research on Bacillus subtilis suitable for the prevention and control of diseases of traditional Chinese medicinal materials. SUMMARY OF THE INVENTION

[0005] Aiming at the problems existing in agricultural production and the deficiencies of the prior art, the present invention provides a Bacillus subtilis that has good antibacterial effects against various pathogenic bacteria such as Fusarium and has a high control effect on root rot of traditional Chinese medicine (such as wolfberry root rot, astragalus root rot, etc.). Another object of the present invention is to provide the application of this Bacillus subtilis.

[0006] The Bacillus subtilis of the present invention is classified and named as Bacillus subtilis, collected from the rhizosphere soil of traditional Chinese medicinal materials in Longde County, Ningxia Hui Autonomous Region, and named Bacillus subtilis 04. It was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) on March 21, 2024, and the deposit number is CGMCC No. 30084.

[0007] The morphological characteristics of the Bacillus subtilis 04 of the present invention are as follows: Gram-positive, the colony is rough, irregularly round, the outer edge of the colony shows radial ripples, white, opaque, without luster. The spores are rod-shaped, arranged singly or in chains, evenly colored, without capsules, and motile.

[0008] The application of Bacillus subtilis 04 in inhibiting plant pathogenic bacteria and / or preventing plant diseases caused by plant pathogenic bacteria also falls within the protection scope of the present invention.

[0009] The application of the described Bacillus subtilis 04 in preparing a biocontrol agent or microbial fertilizer for inhibiting plant pathogenic bacteria also falls within the protection scope of the present invention.

[0010] Among them, the plant pathogenic bacteria are one or any combination of two or more of Fusarium oxysporum, Fusarium verticiliodes, Fusarium moniliforme, and Fusarium solani.

[0011] The application of Bacillus subtilis 04 in promoting the growth of traditional Chinese medicine plants also falls within the protection scope of the present invention. Preferably, the traditional Chinese medicine plants are Scutellaria baicalensis and / or Astragalus membranaceus.

[0012] Advantages of the present invention:

[0013] The Bacillus subtilis 04 of the present invention is collected from the rhizosphere soil of Lycium barbarum in Hongsibao District, Wuzhong City, Ningxia Hui Autonomous Region, and has outstanding control effects on Lycium barbarum root rot or Astragalus membranaceus root rot, and also has antibacterial activity against various other plant pathogenic bacteria, such as Fusarium oxysporum, Fusarium verticiliodes, Fusarium moniliforme, and Fusarium solani. This strain has very good application prospects in the development of biological agents for plant diseases.

[0014] Bacillus subtilis 04 also has the effect of promoting the growth of traditional Chinese medicine plants, such as traditional Chinese medicine plants like Scutellaria baicalensis and Astragalus membranaceus

[0015] Bacillus subtilis 04 has good salt tolerance, and the content of nitrogen, phosphorus, and potassium in the fermentation broth is relatively high.

[0016] The Bacillus subtilis 04 of the present invention has strong enzyme-producing ability and can produce protease, amylase, cellulase, dextranase, pullulanase, and volatile substances.

[0017] The Bacillus subtilis 04 of the present invention does not have hemolytic property and complies with the biosafety regulations. Description of the drawings

[0018] Figure 1 It is the colony morphology of Bacillus subtilis 04 on the NA plate.

[0019] Figure 2Phylogenetic evolution tree of Bacillus subtilis 04 based on 16SrDNA sequencing.

[0020] Figure 3 Phylogenetic evolution tree of Bacillus subtilis 04 based on gyrA sequencing.

[0021] Figure 4 Disease prevention effect of Bacillus subtilis 04 on wolfberry root rot under greenhouse conditions.

[0022] Figure 5 Disease prevention effect of Bacillus subtilis 04 on astragalus root rot under greenhouse conditions.

[0023] Figure 6 Enzyme production ability of Bacillus subtilis 04. In the figure, protease, amylase, cellulase, and pullulanase are shown from left to right.

[0024] Figure 7 Hemolytic property of Bacillus subtilis 04.

[0025] Biological material preservation

[0026] Preservation number: CGMCC No.30084

[0027] Name: Bacillus subtilis 04

[0028] Taxonomic naming: Bacillus subtilis

[0029] Whether alive: Alive

[0030] Preservation time: March 21, 2024

[0031] Preservation institution: China General Microbiological Culture Collection Center

[0032] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Detailed implementation method

[0033] Example 1. Obtaining and identification of Bacillus subtilis 04

[0034] (1) Isolation and purification of rhizosphere soil bacillus from traditional Chinese medicine

[0035] Collect the rhizosphere soil of wolfberry in Hongsibao District, Wuzhong City, Ningxia Hui Autonomous Region. Weigh 10 g of soil and put it into a triangular flask containing 90 mL of sterile water. Shake and mix evenly at room temperature for 0.5 h, and according to 10, 10 2 and 10 3Gradient dilution was carried out, and then it was spread on the Nutrient Agar (NA) medium plate and cultured in an inverted position at 30 °C for 48 h. Single colonies were picked, gradient streaking was performed on the NA plate, and the single colonies obtained again were stored in a -80 °C refrigerator with 40% glycerol.

[0036] Nutrient Agar (NA) medium: 10.0 g of peptone, 3.0 g of beef powder, 5.0 g of sodium chloride, 15.0 g of agar, pH 7.3 ± 0.1, sterilized at 121 °C for 20 min.

[0037] The above separation method and antibacterial screening experiment obtained the Bacillus subtilis of the present invention, named Bacillus subtilis 04.

[0038] The morphological characteristics of Bacillus subtilis 04 of the present invention are as follows: Gram-positive, rough colonies, irregular circular shape, with radial ripples on the outer edge of the colonies, white, opaque, without luster. Spores are rod-shaped, arranged singly or in chains, uniformly colored, without capsules, motile ( Figure 1 ).

[0039] (2) Antibacterial test of Bacillus subtilis 04 against Fusarium

[0040] Antibacterial activity of Bacillus subtilis 04 strain: Using a sterile punch, pathogen discs (1 cm) of Fusarium oxysporum 295, Fusarium verticiliodes 173, Fusarium moniliforme N19-2-2, Fusarium solani N18-1-2, etc. were placed in the center of the PDA medium plate. At a distance of 2 cm from the edge of the pathogen colony, single colonies of the test strain were picked with a sterile toothpick and streaked (2 cm). The petri dish without streaking was used as a blank control. All petri dishes were cultured in the dark at 28 °C with 4 replicates. After 5 days, the diameter of the pathogen colony and the antibacterial zone were measured, and the antibacterial effect of Bacillus subtilis 04 against plant pathogens was calculated.

[0041] Antagonistic effect of the fermentation broth: On the potato agar plate medium, pathogen discs (1 cm) of Fusarium oxysporum 295, Fusarium verticiliodes 173, Fusarium moniliforme N19-2-2, and Fusarium solani were placed in the center. Sterile Oxford cups were placed at a distance of 2 cm on both sides of the discs, and the fermentation broth of Bacillus subtilis 04 was added to the cups for confrontation culture. Sterile LB liquid medium was used as a control and placed in an incubator at 28 °C for dark culture. Each treatment was repeated with 10 petri dishes. After 5 days, the diameter of the pathogen colony and the antibacterial zone were measured, and the results are shown in Table 1.

[0042] Antagonistic effect of the sterile supernatant: The sterile supernatant of Bacillus subtilis 04 was mixed with the sterilized PDA medium cooled to 50 °C at a ratio of 1:30 to prepare a plate with the sterile supernatant. After the petri dish had solidified, a pathogen bacterial cake (1 cm) was placed in the center of the plate with the sterile supernatant, and then the petri dish was placed in an incubator and cultured in the dark at 28 °C. Each treatment was replicated in 10 plates. After 5 days, the diameter of the pathogen colony was measured, and the results are shown in Table 1.

[0043] Preparation of the sterile supernatant: Single colonies of Bacillus subtilis 04 that had been activated for 24 h were inoculated into LB liquid medium respectively. After shaking culture at 37 °C and 200 rpm / min for 72 h, the fermentation broth was centrifuged at 4 °C and 10,000 rpm / min for 20 min to remove the bacteria. The supernatant was sterilized by filtration (0.22 μm) to obtain the sterile supernatant.

[0044] Relative mycelium inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0045] Table 1 Antagonistic effect of Bacillus subtilis 04 against various pathogens such as Fusarium

[0046]

[0047] The screening experiment showed that, among them, Bacillus subtilis 04 of the present invention has an antagonistic effect against various pathogens such as Fusarium, and the inhibition rate is very high. It is an excellent antagonistic strain.

[0048] (3) Identification of the strain Bacillus subtilis 04

[0049] The reference for the physiological and biochemical identification method is "Manual for the Systematic Identification of Common Bacteria". The other physiological and biochemical indicators of Bacillus were tested using the HBI Bacillus biochemical identification reagent strip (HBIG14, Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park), and the results are shown in Table 1. The total DNA of Bacillus strain 04 was extracted. Using the DNA sample with qualified concentration and quality as the template, 16S universal primers (27F: AGAGTTTGATCMTGGCTCAG; 1492R: TACGGYTACCTTGTTACGACTT) and gyrA specific primers were used for sequencing amplification. PCR reaction system: 12.8 μL ddH2O; 3 μL Buffer; 2 μL dNTP; 3 μL Primer1; 3 μL Primer2; 1 μL DNA template; 0.2 μL enzyme, with a total volume of 30 μL. Amplification program: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; store at 12°C. The amplified PCR products were sequenced, and each sequencing result was subjected to BlastN alignment analysis on NCBI. The 16S rDNA sequence length was 1257 bp, with 100% similarity to Bacillus subtilis strain MB9-B6 (Sequence 1); the gyrA specific sequence length was 875 bp, with 100% similarity to Bacillus subtilis strain MJ-4 (Sequence 2). Combining the morphological and physiological and biochemical characteristics (Table 2), it was identified as Bacillus subtilis. The phylogenetic evolution tree of Bacillus subtilis 04 based on 16S rDNA sequencing is as Figure 2 shown, and the phylogenetic evolution tree of Bacillus subtilis 04 based on gyrA sequencing is as Figure 3 shown. Bacillus subtilis 04 has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit date of March 21, 2024, and the deposit number CGMCC NO. 30084.

[0050] Table 2. Physiological and biochemical indicators of Bacillus subtilis 04

[0051] Treatment V-P Citrate Propionate D-Xylose L-Arabinose Bacillus subtilis 04 - + - + + Treatment D-Mannitol Gelatin liquefaction Growth at pH5.7 Nitrate reduction Starch hydrolysis Bacillus subtilis 04 - + + + +

[0052] Note: "+" indicates positive; "-" indicates negative

[0053] Cultivation of Bacillus subtilis 04: Inoculate the single colony of strain 04 that has been activated for 24 h into LB liquid medium, and cultivate it at 37°C and 200 rpm / min for 72 h.

[0054] Example 2. Identification of the salt tolerance of Bacillus subtilis 04

[0055] Generally, microorganisms are prone to plasmolysis under high osmotic pressure and excessive water absorption under low osmotic pressure. Therefore, an appropriate salt concentration is a necessary condition for the growth of microorganisms. Add NaCl to the culture medium to prepare plates with concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and 11% for standby. Use the plate without NaCl as the positive control. Incubate at 37°C for 3 - 5 days, observe and record the results as shown in Table 3. The results show that Bacillus subtilis 04 has strong salt tolerance.

[0056] Table 3 Salt tolerance of Bacillus subtilis 04

[0057] Treatment 1％ 2％ 3％ 4％ 5％ 6％ 7％ 8％ 9％ 10％ / 04 + + + + + + + + + -

[0058] Example 3. Control efficacy test of Bacillus subtilis 04 bacterial agent on root rot of Chinese medicinal materials under greenhouse conditions

[0059] Determine the control effect of the antagonistic bacterium on the pathogen through pot experiments with Lycium barbarum and Astragalus membranaceus seedlings: (1) Preparation of mixed spore suspension of multiple Fusarium spp. such as Fusarium oxysporum, Fusarium verticillioides, Fusarium moniliforme, and Fusarium solani: Scrape the pathogens cultured on PDA plates at 28°C for 10 d respectively, disperse them into a 6 g / L CMC (carboxymethyl cellulose sodium) solution to make a spore suspension, and adjust the spore concentration to 2.0×10 7 CFU / mL. (2) Pot experiment design: Select healthy Astragalus membranaceus and Lycium barbarum seedlings with consistent growth from the Astragalus membranaceus and Lycium barbarum cultivation bases and divide them into 3 groups, namely the blank group (without inoculating bacteria), the control group (inoculating the pathogen), and the experimental group (inoculating the pathogen and the antagonistic bacterium). There are 12 pots for each treatment, with 3 plants in each pot. Transplant the seedlings into small plastic flower pots filled with mixed nutrient soil (nutrient soil∶vermiculite∶perlite = 3∶1∶1). After 14 d, inoculate the above pathogen spore suspension (1×10 6 CFU / mL) to the control group and the experimental group by the method of root injury perfusion, with an inoculation amount of 5 mL per pot, and use the inoculation of an equal amount of sterile water as the control. 14 d after inoculating the pathogen, inoculate 5 mL of the fermentation filtrate of Bacillus subtilis 04 (1×10 8 CFU / mL) to each pot of the experimental group, and use the inoculation of an equal amount of sterile water as the blank control. (3) Control efficacy determination: Observe and count the disease incidence after culturing for 28 d, calculate the control effect according to the plant disease grading standard, and the results are shown in Table 4 and Table 5.

[0060] There are 2 groups in the greenhouse experiment design: CK is the water control; B is Bacillus subtilis 04.

[0061] Disease index = [∑(number of diseased plants at each level × representative value of relative disease level)] / [total number of investigated plants × representative value of the highest disease level] × 100; Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0062] Table 4 Control effect of Bacillus subtilis 04 agent on wolfberry root rot

[0063]

[0064] Table 5 Preventive effect of Bacillus subtilis 04 agent on astragalus root rot

[0065]

[0066]

[0067] The test results showed that Bacillus subtilis 04 had good preventive effects on wolfberry and astragalus root rot under pot conditions ( Figure 4 and Figure 5 ), and had a certain degree of treatment effect on wolfberry root rot under the condition of serious occurrence of wolfberry root rot in the field.

[0068] Example 4 Growth promotion test of Bacillus subtilis 04 agent on Chinese medicinal materials under greenhouse conditions

[0069] Seed treatment: Place the seeds of astragalus and scutellaria baicalensis in a 1% sodium hypochlorite solution for surface disinfection for 30 min, then rinse the residual sodium hypochlorite on the seed surface with sterile water, and dry the surface moisture on the ultra-clean workbench.

[0070] Greenhouse growth promotion test: Coat the surface-disinfected Chinese medicinal material seeds with the fermentation broth of Bacillus subtilis 04 (1×10 8 CFU / mL). The seeds in the blank control group were not coated with any agent after surface disinfection. Plant astragalus and scutellaria baicalensis in the greenhouse, with 50 g of seeds for each treatment, and the planting area is about 20 m 2 . The treatment methods of microbial agent CK and hymexazol wettable powder are the same as those of the tested strains. No agent is applied in the blank control. The test design includes: ⑴ Blank control CK: ⑵ Bacillus subtilis 04; ⑶ Hymexazol wettable powder YCK; ⑷ Microbial agent control MCK (Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd.). Investigate the growth of astragalus and scutellaria baicalensis 30 days after planting, measure the plant height and root length, and the results are shown in Table 6 and Table 7.

[0071] Table 6 Growth promotion effect of Bacillus subtilis 04 agent on astragalus under greenhouse conditions

[0072]

[0073] Table 7 Growth promotion effect of Bacillus subtilis 04 agent on scutellaria baicalensis under greenhouse conditions

[0074]

[0075]

[0076] The greenhouse pot experiment confirmed that the Bacillus subtilis 04 microbial agent could significantly promote the plant height and root length of Astragalus membranaceus and Scutellaria baicalensis, showing good growth-promoting effects.

[0077] Example 5: Detection of biocontrol active substances of Bacillus subtilis 04

[0078] Detection of xylanase: The activated strain was inoculated onto the xylanase-producing fermentation medium with a sterilized toothpick and cultured with shaking at 30 °C and 170 r / min for 48 h. Take 2 mL of the culture solution, centrifuge at 6000 r and 4 °C for 10 min, and the supernatant is the crude xylanase solution for standby. Use a punch with a diameter of 5 mm to punch three holes in the xylan solid medium. Add 90 μL of the crude enzyme solution to each hole, culture at 37 °C for 5 h, then add absolute ethanol to the solid medium to cover the entire medium surface, and let it stand for 2 - 3 h; absolute ethanol can cause a color reaction of polysaccharides. If the strain can produce xylanase, the xylan around the colony will be decomposed into monosaccharides and a transparent circle will appear. The results are shown in Table 8.

[0079] Detection of pullulanase: The activated strain was spotted onto the pullulanase-producing selective medium with a sterilized toothpick and cultured in an inverted position at 30 °C for 48 h. Add Lugol's iodine solution to the medium and let it stand for 10 min. If the strain can produce pullulanase, the pullulan around the colony will be decomposed into monosaccharides and a transparent circle will appear. The results are shown in Table 8.

[0080] Detection of chitinase: The activated strain was inoculated onto the chitinase-producing fermentation broth medium with a sterilized toothpick and cultured with shaking at 30 °C and 70 r / min for 48 h. Take 2 ml of the fermentation broth, centrifuge at 6000 r and 4 °C for 10 min, and the supernatant is the crude chitinase solution for standby. Use a punch with a diameter of 5 mm to punch three holes in the xylan solid medium. Add 90 μL of the crude enzyme solution to each hole; place it in an incubator at 37 °C and culture for 5 h. Drop Congo red dye onto the medium. Congo red dye can cause a color reaction with polysaccharides. If the strain can produce chitinase, the chitin around the colony will be decomposed into monosaccharides and a transparent circle will appear. The results are shown in Table 8.

[0081] Detection of phytase: The activated strain was spotted onto the phytase-producing screening medium with a sterilized toothpick and cultured in an inverted position at 37 °C for 48 h. Observe whether the calcium phytate around the colony is decomposed to form a transparent circle. The results are shown in Table 8.

[0082] Detection of amylase: Inoculate a newly activated single colony onto an LB plate containing 0.2% soluble starch and culture for 48 h. After obvious colonies are formed, add Lugol's iodine solution to stain on the plate for 10 min, and wash the plate with 70% ethanol. For the strains that can produce amylase, a colorless transparent circle can be formed around the colony growth area against the black background. If there is a transparent circle, it indicates that the strain can produce amylase. There are 3 replicates for each treatment, and the results are shown in Table 8.

[0083] Detection of protease: Inoculate the activated tested strain by stabbing onto a 1% skim milk agar plate and observe the appearance of a transparent circle around it after culturing at 30 °C for 24, 48, and 72 h. The appearance of a transparent circle indicates the production of protease. There are 3 replicates for each treatment, and the results are shown in Table 8.

[0084] Detection of glucanase: Inoculate the tested bacteria onto a plate containing ABP medium and observe whether a digestion circle appears in the plate after culturing at 30 °C for 48 and 72 h. If a digestion circle appears, it indicates the production of glucanase. There are 3 replicates for each treatment, and the results are shown in Table 8.

[0085] Detection of siderophore: Siderophore is detected using CAS medium. Inoculate the activated tested bacteria onto a CAS detection medium plate and observe whether an orange halo appears in the plate after culturing at 30 °C for 72 h. If an orange color appears, it indicates the production of siderophore. Each treatment is repeated 3 times, and the results are shown in Table 8.

[0086] Detection of cellulase: After activating the isolated strain, inoculate it onto a cellulose screening medium plate and culture it in an inverted position at 28 °C for 2 d. Stain it with 0.1% Congo red staining solution for 10 min, and then decolorize it with 1 mol / L NaCl solution for 5 min. If the strain produces cellulase, a clear transparent circle will appear around the colony. Each treatment is repeated 3 times, and the results are shown in Table 8.

[0087]

[0088] Preparation of ABP medium: Grind the massive Poria cocos into powder with a mortar so that it can pass through a 120-mesh sieve. KH2PO4 6.8 g, K2PHO4·12H2O 17.9 g, yeast extract 6.7 g, Poria cocos powder (or β-1.3-glucan) 5.0 g, aniline blue 120 mg, agar powder 12 g, H2O 1 L, pH value is 6.8, water 1 L.

[0089] Preparation of CAS medium: Casein peptone 9 g, yeast extract 5 g, trisodium citrate 10 g, disodium hydrogen phosphate 1 g, sodium dihydrogen phosphate 1 g, glucose 10 g, agar 20 g, dissolved in 1 L of water.

[0090] Cellulose-enriched medium (1 L): 6 g of NaCl, 0.1 g of MgSO₄, 0.5 g of KH₂PO₄, 0.1 g of CaCl₂, 2.0 g of (NH₄)₂SO₄, 2.0 g of K₂HPO₄, 15 g of agar, 5 g of CMC-Na, pH adjusted to 7.0; Cellulose screening medium (1 L): Add 1 g of yeast powder to the above celluose-enriched medium.

[0091] Congo red staining solution: Dissolve Congo red in distilled water to a final concentration of 0.1% (w / v).

[0092] Congo red decolorizing solution: NaCl solution with a final concentration of 1 mol / L.

[0093] Table 8 Detection of biocontrol active substances of Bacillus subtilis strain 04

[0094]

[0095]

[0096] Note: The data are the detection results of three replicates; "+" indicates that the result was detected, and "-" indicates that the result was not detected.

[0097] The detection results of biocontrol-related substances showed that Bacillus subtilis strain 04 could produce protease, amylase, cellulase, glucanase, pullulanase and volatile substances ( Figure 6 ).

[0098] Example 6, Hemolytic test of Bacillus subtilis strain 04

[0099] Pick a single colony of Bacillus activated on the LB plate and streak it on a Columbia blood agar (Beijing Solarbio Science & Technology Co., Ltd.) plate containing 8% sheep blood (Beijing Land Bridge Technology Co., Ltd.). Incubate at 37 °C for 14 - 18 h, observe the morphology around the colony, and judge the type of hemolysis. Each strain has 3 replicates. If a well-defined and completely transparent hemolytic ring is formed around the colony, indicating hemolysis, the hemolytic type is β-hemolysis; if there is no change in the medium around the colony, indicating no hemolysis, it is γ-hemolysis. The results are shown in Table 9 and Figure 7 as shown.

[0100] Table 9 Hemolysis of Bacillus subtilis strain 04

[0101] Strain name Transparent zone Hemolysis zone Hemolytic property Bacillus subtilis 04 \ γ-Hemolysis None

[0102] The detection results showed that Bacillus subtilis strain 04 did not have hemolysis, which complied with the safety use regulations of biocontrol strains in the "Biological Safety Law of the People's Republic of China".

[0103] Example 7, Test of nitrogen, phosphorus and potassium contents of Bacillus subtilis strain 04 bacterium agent

[0104] After Bacillus subtilis 04 was activated on an LB plate, it was inoculated into a liquid LB medium (liquid loading: 50 mL / 150 mL Erlenmeyer flask). After culturing on a shaker at 30 °C and 150 r / min for 36 - 48 h, 1 mL of the bacterial solution with a concentration of 10 8 CFU / mL was inoculated into a liquid NFM culture medium. There were 3 replicates for each strain, and the non-inoculated culture medium was used as a control. After culturing at 30 °C and 160 r / min for 7 d, the nitrogen fixation rate of each culture medium was determined by the Kjeldahl method, and the results are shown in Table 10.

[0105] After Bacillus subtilis 04 was activated on an LB plate, it was inoculated into a liquid LB medium (liquid loading: 50 mL / 150 mL Erlenmeyer flask). After culturing on a shaker at 30 °C and 150 r / min for 4 d, 1 mL of the bacterial solution with a concentration of 10 8 CFU / mL was inoculated into liquid PKO inorganic phosphorus and Mengjina organic phosphorus culture media. There were 3 replicates for each strain, and the non-inoculated culture medium was used as a control. After culturing on a shaker at 30 °C and 150 r / min for 10 d, the phosphorus solubilization rate was determined by the molybdenum antimony anti-colorimetric method, and the results are shown in Table 10.

[0106] After Bacillus subtilis 04 was activated on an LB plate, it was inoculated into a liquid LB medium (liquid loading: 50 mL / 150 mL Erlenmeyer flask). After culturing on a shaker at 30 °C and 150 r / min for 36 - 48 h, 1 mL of the bacterial solution with a concentration of 10 8 CFU / mL was inoculated into a liquid potassium feldspar culture medium. There were 3 replicates for each strain, and the non-inoculated culture medium was used as a control. After culturing at 30 °C and 160 r / min for 7 d, the potassium release rate of each culture medium was determined by flame atomic absorption spectrometry, and the results are shown in Table 10.

[0107] Table 6 Nitrogen, phosphorus and potassium contents of the Bacillus subtilis 04 bacterial agent

[0108] Strain number Nitrogen content rate / % Phosphorus content rate / % Potassium content rate / % Bacillus 04 0.197 0.023 0.038

[0109] The test results show that the contents of nitrogen, phosphorus and potassium in the Bacillus subtilis 04 bacterial agent are relatively high, and can provide absorbable nitrogen, phosphorus and potassium elements for the host plants.

Claims

1. A strain of Bacillus subtilis ( Bacilus subtilis ), named Bacillus subtilis 04, characterized in that, The Bacillus subtilis 04 has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 30084.

2. Use of the Bacillus subtilis 04 according to claim 1 in inhibiting plant pathogens and / or preventing and controlling plant diseases caused by plant pathogens; the plant pathogen is Fusarium oxysporum ( Fusarium oxysporum ), Fusarium verticillium ( Fusarium Verticiliodes ), Fusarium moniliforme ( Fusarium Moniliforme ) and Fusarium solani ( Fusarium Solani ) or any combination of two or more thereof; the plant disease is wolfberry root rot and / or astragalus root rot.

3. The use of the Bacillus subtilis 04 according to claim 1 in the preparation of a biocontrol agent or microbial fertilizer for inhibiting plant pathogens; the plant pathogen is Fusarium oxysporum ( Fusarium oxysporum ), Fusarium verticillium ( Fusarium Verticiliodes ), Fusarium moniliforme ( Fusarium Moniliforme ) and Fusarium solani ( Fusarium Solani ) or any combination of two or more thereof; the plant disease is wolfberry root rot and / or astragalus root rot.

4. A plant disease biocontrol agent, characterized in that: The active ingredient is the Bacillus subtilis described in claim 1; the plant pathogen is Fusarium oxysporum ( Fusarium oxysporum ), Fusarium verticillium ( Fusarium Verticiliodes ), Fusarium moniliforme ( Fusarium Moniliforme ) and Fusarium solani ( Fusarium Solani ) or any combination of two or more.

5. Use of the Bacillus subtilis according to claim 1 in promoting the growth of Chinese medicinal plants; the Chinese medicinal plants are Scutellaria baicalensis and / or Astragalus membranaceus.

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