A Bacillus megaterium LDSR-6 bacterial agent producing lipopeptide antibiotics and its application

Abstract

The present invention provides a Bacillus megaterium LDSR-6 bacterium agent for producing lipopeptide antibiotics and its application. The Bacillus megaterium LDSR-6 of the present invention has a preservation number of CGMCC No. 30083. The Bacillus megaterium of the present invention has antibacterial activity against various plant pathogenic bacteria such as Fusarium, has strong salt tolerance and no hemolysis; the Bacillus megaterium LDSR-6 bacterium agent has a preventive effect of 62.31% on Astragalus root rot under greenhouse conditions, and the lipopeptide antibiotics produced have a broad-spectrum and high-efficiency antibacterial effect on various pathogenic bacteria such as Fusarium and Phytophthora, and can cause the infection hyphae of various Fusarium to be deformed. The Bacillus megaterium of the present invention 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 megaterium LDSR-6 strain that produces lipopeptide antibiotics, a bacterial agent and an application method thereof. Background Art

[0002] Root rot is a common soil-borne disease that occurs in crop planting areas. It is 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 die every year, with a diseased plant rate of 15%. In severely affected areas, the diseased plant rate is 37.6%, and the dead 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%. The reduction in the yield of Isatis indigotica root rot in lightly affected medicinal material areas is 11.2% - 21.6%, in moderately affected areas, the reduction is more than 35%, and in severely affected years, the 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%. Underground pests gnaw on the underground parts of Chinese medicinal materials, seriously affecting the yield and quality of Chinese medicinal materials. Planting disease-resistant varieties and chemical pesticides are the main measures for controlling crop pests and diseases. 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 efficient biological control technologies for Chinese medicinal material root rot has important scientific value for protecting the ecological environment and the high-quality development of the Chinese medicinal material 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 pathogenic bacteria to cross this barrier and invade the plant roots. Therefore, the plant roots can grow in a relatively stable environment. The bacteria used for controlling pests and diseases 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, harmlessness to humans and livestock, etc., and is the most widely used biocontrol bacteria. During the metabolic process, Bacillus megaterium can produce substances such as organic acids, auxins, and antibiotics, which can promote the dissolution of phosphorus; promote the formation of soil aggregate structures; promote crop growth; inhibit the growth of pathogenic bacteria; and have the effects of promoting growth and preventing diseases. Since it is harmless to the environment, it can improve the micro-ecological environment of the soil. The application prospect of Bacillus megaterium is very broad. At present, it has been widely used in agricultural biological pesticides, agricultural biological fertilizers, etc., showing good ecological and social benefits.

[0004] Bacillus can produce chitinase, sugar degrading enzyme, cell wall degrading enzyme, etc., which can dissolve the cell wall of the hyphae of plant pathogens. In particular, those senescent or even nearly dead hyphae are more likely to be dissolved. Wang Qian et al. (2023) screened out that the fermentation broth of Bacillus megaterium YB-3 had a significant effect on controlling ginger bacterial wilt. Xie Qiang et al. (2022) found that the inhibition rates of the fermentation broth of Bacillus megaterium Bm isolated on tobacco mosaic virus were 88.4% and 74.3% respectively. The inhibition rate of the sterile supernatant of Bm on the mycelial growth of Phytophthora parasitica var. nicotianae was 67.5%, and the diameter of the inhibition zone on Ralstonia solanacearum was 10.03 mm. Yang Jingyan (2018) found that the fermentation broth of Bacillus megaterium CP3 had good contact toxicity and fumigation toxicity against Meloidogyne incognita. After treating Meloidogyne incognita with volatile compounds of Bacillus megaterium CP3 at a concentration of 1000 mg / L for 72 h, 2-tetradecanone, 2-tetradecanol and 2-pentadecanone all showed certain nematicidal activities, and the mortality rates of Meloidogyne incognita were 57.18%, 89.04% and 52.16% respectively. At present, there is no research report on the control of Chinese medicinal materials root rot by Bacillus megaterium. However, there are few Bacillus agents that can effectively control the diseases of Chinese medicinal materials at present. It is necessary to carry out research on Bacillus megaterium suitable for the control of Chinese medicinal materials diseases. Summary of the Invention

[0005] In view of the problems existing in agricultural production and the deficiencies of the prior art, the present invention provides a Bacillus megaterium with good antibacterial effects against various pathogens such as Fusarium and high control efficacy against Astragalus membranaceus root rot. Another object of the present invention is to provide the application of the Bacillus megaterium.

[0006] The Bacillus megaterium of the present invention is classified and named as Bacillus megaterium subsp. thuringiensis, collected from the rhizosphere soil of Astragalus membranaceus in Longde County, Ningxia Hui Autonomous Region, named Bacillus megaterium LDSR-6, and was deposited in the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on March 21, 2024, with the deposit number of CGMCC No. 30083.

[0007] The morphological characteristics of the Bacillus megaterium LDSR-6 of the present invention are as follows: Gram-positive, aerobic, usually motile, rod-shaped, single, paired or arranged in chains, oval spores, colonies are white, round to irregular, with neat or irregular edges.

[0008] The application of the Bacillus megaterium LDSR-6 in inhibiting plant pathogens and / or controlling plant diseases caused by plant pathogens also belongs to the protection scope of the present invention.

[0009] The application of the Bacillus megaterium LDSR-6 in preparing a biocontrol agent or microbial fertilizer for inhibiting plant pathogens also belongs to the protection scope of the present invention.

[0010] Among them, the plant pathogen is one or more arbitrary combinations of Fusarium oxysporum, F. verticilliodes, F. moniliforme, F. solani, Phytophthora parasitica var. nicotianae, F. oxysporum f. sp. cucumerinum, F. oxyspirum f. sp. niveum, Colletotrichum gloeospoioides, Alternaria alternata, and A. alternata.

[0011] The present invention also provides a lipopeptide extract, which is obtained by culturing the above-mentioned Bacillus megaterium, collecting the bacterial cells, and extracting with methanol to obtain a crude lipopeptide extract.

[0012] The application of the above lipopeptide extract in inhibiting plant pathogens and / or preventing and controlling plant diseases caused by plant pathogens, and the application of the lipopeptide extract in preparing a biocontrol agent or a microbial fertilizer for inhibiting plant pathogens also belong to the protection scope of the present invention.

[0013] Furthermore, the present invention also protects a plant disease inhibitor, the active ingredient of which is the above-mentioned Bacillus megaterium or the above lipopeptide extract. As a microbial agent, it may also contain acceptable auxiliaries.

[0014] Advantages of the present invention:

[0015] The Bacillus megaterium LDSR-6 of the present invention is collected from the rhizosphere soil of traditional Chinese medicinal materials in Longde County, Ningxia Hui Autonomous Region, and has outstanding control effect on Astragalus root rot and antibacterial activity against many other plant pathogens, such as Fusarium oxysporum, F. verticilliodes, F. moniliforme, F. solani, Phytophthora parasitica var. nicotianae Phytophthora nicotianae , F. oxysporum f. sp. cucumerinum, F. oxyspirum f. sp. niveum, Colletotrichum gloeospoioides, Alternaria alternata, and A. alternata. This strain has very good application prospects in the development of plant disease biological agents.

[0016] Bacillus megaterium LDSR-6 has good salt tolerance, no hemolytic property, and the liquid bacterial agent has relatively high nitrogen, phosphorus, and potassium contents. Description of the Drawings

[0017] Figure 1 Colony morphology of Bacillus megaterium LDSR-6 on the NA plate.

[0018] Figure 2 Morphology of the cells and spores of Bacillus megaterium LDSR-6.

[0019] Figure 3 Phylogenetic tree of Bacillus megaterium LDSR-6.

[0020] Figure 4 Disease prevention effect of Bacillus megaterium LDSR-6 on Astragalus root rot under greenhouse conditions.

[0021] Figure 5 Inhibitory effect of Bacillus megaterium LDSR-6 on the mycelial morphology of Fusarium oxysporum.

[0022] Figure 6 Inhibitory effect of Bacillus megaterium LDSR-6 on the mycelial morphology of Fusarium moniliforme.

[0023] Figure 7 Inhibitory effect of Bacillus megaterium LDSR-6 on the mycelial morphology of Fusarium solani.

[0024] Figure 8 Inhibitory effect of Bacillus megaterium LDSR-6 on the mycelial morphology of Verticillium fusarium.

[0025] Figure 9 Hemolytic property of Bacillus megaterium LDSR-6.

[0026] Biological Material Deposit

[0027] Deposit Number: CGMCC No.30083

[0028] Name: Bacillus megaterium LDSR-6

[0029] Taxonomic Nomenclature: Bacillus megaterium

[0030] Survival Status: Alive

[0031] Deposit Date: March 21, 2024

[0032] Deposit Institution: China General Microbiological Culture Collection Center

[0033] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Detailed implementation manners

[0034] Example 1. Isolation and identification of Bacillus megaterium LDSR-6

[0035] (1) Isolation and purification of rhizospheric soil bacillus of Chinese medicinal materials

[0036] Collect the rhizospheric soil of Astragalus membranaceus in Longde County, 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 dilute it according to the gradients of 10, 10 2 and 10 3 Then coat it on the Nutrient Agar (NA) medium plate and incubate it upside down at 30 °C for 48 h. Pick out single colonies, streak them on the NA plate in gradients, and store the single colonies obtained again in a -80 °C refrigerator with 40% glycerol.

[0037] Nutrient Agar (NA) medium: peptone 10.0 g, beef powder 3.0 g, sodium chloride 5.0 g, agar 15.0 g, pH 7.3 ± 0.1, sterilize at 121 °C for 20 min.

[0038] The above isolation method and antibacterial screening experiment obtained the Bacillus megaterium of the present invention, named Bacillus megaterium LDSR-6.

[0039] The morphological characteristics of the Bacillus megaterium LDSR-6 of the present invention are as follows: Gram-positive, aerobic, usually motile, rod-shaped, single, paired or arranged in chains, oval spores, colonies are white, round to irregular, with neat or irregular edges (see Figure 1 and Figure 2 ).

[0040] (2) Antibacterial test of Bacillus megaterium LDSR-6 against various pathogenic bacteria such as Fusarium

[0041] Mycelial inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%. The mycelial inhibition rates of Bacillus megaterium LDSR-6 against various pathogenic fungi such as Fusarium oxysporum 295, F. verticilliodes 173, F. moniliforme N19-2-2, F. solani N18-1-2, Phytophthora parasitica var. nicotianae, Magnaporthe oryzae, F. oxysporum f. sp. cucumerinum, and F. oxyspirum f. sp. niveum were measured, and the results are shown in Table 1. Agar discs (1 cm) of the above-mentioned pathogenic fungi were placed in the center of PDA medium plates. Single colonies of the test strains were streaked (2 cm) with sterile toothpicks 2 cm away from the edges of the pathogenic fungal colonies. Plates without streaking were used as blank controls. All plates were cultured in the dark at 28 °C with 4 replicates. After 5 days, the diameters of the pathogenic fungal colonies and the inhibition zones were measured to screen for strains with obvious antagonistic effects. The screening experiment showed that Bacillus megaterium LDSR-6 of the present invention has antagonistic effects against various pathogenic fungi such as Fusarium, and is an excellent antagonistic strain.

[0042] Relative mycelial inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0043] Table 1 Antagonistic effects of Bacillus megaterium LDSR-6 against various pathogenic fungi such as Fusarium

[0044] pathogenic bacterium Relative mycelium inhibition rate (%) Fusarium oxysporum 60.90 Fusarium oxysporum f. sp. niveum 44.95 Fusarium moniliforme 42.03 Fusarium solani 42.00 Fusarium verticillioides 53.33 Phytophthora parasitica var. nicotianae 51.61 Fusarium oxysporum f. sp. cucumerinum 42.43 Magnaporthe oryzae 24.32

[0045] (3) Strain identification of Bacillus megaterium LDSR-6

[0046] For physiological and biochemical identification methods, refer to the "Handbook for Systematic Identification of Common Bacteria". 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). The total DNA of Bacillus megaterium LDSR-6 strain was extracted. Using the DNA sample with qualified concentration and quality as the template, amplification was carried out using 16S universal primers (27F: AGAGTTTGATCMTGGCTCAG; 1492R: TACGGYTACCTTGTTACGACTT). PCR reaction system: 12.8 μL ddH 2O; 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, for 30 cycles; 72°C for 10 min; store at 12°C. Sequence the amplified PCR product and perform BlastN alignment analysis on each sequencing result on NCBI. The sequence length is 1320 bp, and the similarity with Bacillus megaterium strain cqs V16 reaches 98% (Sequence 1). Combining the morphological, physiological, and biochemical characteristics (Table 2), it was identified as Bacillus megaterium. The phylogenetic tree of Bacillus megaterium LDSR-6 is as Figure 3 shown.

[0047] Bacillus megaterium LDSR-6 has been deposited in the China General Microbiological Culture Collection Center (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. 30083.

[0048] Table 2 Physiological and biochemical indicators of Bacillus LDSR-6

[0049] treatment V-P citrate propionate D-xylose L-arabinose LDSR-6 - + - - - treatment D-mannitol gelatin liquefaction growth at pH 5.7 nitrate reduction starch hydrolysis LDSR-6 + + - + +

[0050] Note: "+" indicates positive; "-" indicates negative

[0051] Cultivation of Bacillus megaterium LDSR-6: Inoculate a single colony of strain LDSR-6 that has been activated for 24 h into LB liquid medium and culture it with shaking at 37°C and 200 rpm / min for 72 h.

[0052] Example 2. Identification of the salt tolerance of Bacillus megaterium LDSR-6

[0053] Generally, microorganisms are prone to plasmolysis under high osmotic pressure and prone to 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 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, culture at 37°C for 3 - 5 days, observe and record the results in Table 3. The results show that Bacillus megaterium LDSR-6 has strong salt tolerance.

[0054] Table 3 Salt tolerance of Bacillus megaterium LDSR-6

[0055] treatment 1％ 2％ 3％ 4％ 5％ 6％ 7％ 8％ 9％ 10％ / LDSR-6 + + + + + + + + + -

[0056] Example 3: Efficacy Test of Bacillus megaterium LDSR-6 against Root Rot of Astragalus membranaceus under Greenhouse Conditions

[0057] Seed treatment: The Astragalus membranaceus seeds were surface-sterilized in 1% sodium hypochlorite solution for 30 min, then the residual sodium hypochlorite on the seed surface was rinsed off with sterile water, and the surface moisture was dried on a superclean workbench.

[0058] The control effect of the antagonistic bacterium against the pathogen was determined through pot experiments with Astragalus membranaceus seedlings: (1) Preparation of a mixed spore suspension of the pathogens causing root rot of Astragalus membranaceus (Fusarium oxysporum, Fusarium verticillioides, Fusarium moniliforme, and Fusarium solani spores in a weight ratio of 1:1:1:1): The pathogens cultured on PDA plates at 28 °C for 10 d were scraped off and dispersed into a 6 g / L CMC (carboxymethylcellulose sodium) solution to make a spore suspension, and the spore concentration was adjusted to 2.0×10 7 CFU / mL. (2) Pot experiment design: Healthy Astragalus membranaceus seedlings with consistent growth were selected from the Astragalus membranaceus cultivation base and divided 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 were 12 pots for each treatment, with 3 plants in each pot. The seedlings were transplanted into small plastic flower pots filled with a mixed nutrient soil (nutrient soil: vermiculite: perlite = 3:1:1). After 14 d, the above-mentioned pathogen spore suspension (1×10 6 CFU / mL) was inoculated into the control group and the experimental group by the method of root injury perfusion, and the inoculation amount per pot was 5 mL, with an equal amount of sterile water inoculated as the control. 14 d after inoculating the pathogen, 5 mL of the liquid bacterium agent of Bacillus megaterium LDSR-6 (1×10 8 CFU / mL) was inoculated into each pot of the experimental group, with an equal amount of sterile water inoculated as the blank control. (3) Efficacy determination: After culturing for 28 d, the disease incidence was observed and counted, and the control effect was calculated according to the plant disease grading standard.

[0059] Two groups were designed for the greenhouse experiment: CK was the water control; B was the strain LDSR-6.

[0060] Disease index = [∑(number of diseased plants at each level × representative value of relative disease level)] / [total number of plants surveyed × representative value of the highest disease level] × 100; Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0061] Table 4 Control Effect of Bacillus megaterium LDSR-6 against Root Rot of Astragalus membranaceus under Greenhouse Conditions

[0062] treatment disease index Control effect (%) CK 56.92a / LDSR-6 21.32b 62.31

[0063] The test results are shown in Table 4, and the results indicate that Bacillus megaterium LDSR-6 has a good preventive effect against root rot of Astragalus membranaceus under greenhouse conditions( Figure 4 ).

[0064] Example 4: Antibacterial Effect of the Crude Lipopeptide Extract from Bacillus megaterium LDSR-6

[0065] The crude lipopeptide extract from Bacillus megaterium was extracted by the acid precipitation method: First, Bacillus megaterium LDSR-6 was activated and then inoculated into 50 mL of Landy medium (20 g of glucose, 5 g of L-sodium glutamate, MgSO 4 0.5 g, KCl 0.5 g, KH 2 PO 4 1 g, FeSO 4 0.15 mg, MnSO 4 5 mg, CuSO 4 0.16 mg, H 2 O 1000 mL) respectively. After culturing at 28 °C and 150 r / min for 48 h, the fermentation broth was centrifuged at 4 °C and 10,000 r / min for 15 min. The supernatant I was adjusted to pH 2.0 with 6 mol / L HCl, precipitated at 4 °C for 12 h, and then centrifuged at 10,000 r / min for 20 min. The supernatant II was discarded, and the precipitate was collected. The obtained precipitate was added with 2 mL of methanol (analytical pure), and then the pH was adjusted to 7.0 with 1 mol / L NaOH. The extracts were combined and filtered through a bacterial filter with a diameter of 0.22 μm to obtain the crude lipopeptide extract, which was stored in a -20 °C refrigerator for later use.

[0066] Test on the antibacterial effect of the crude lipopeptide extract: 200 μL of the crude lipopeptide extract was evenly spread on the PDA plate. The bacterial cakes (1 cm) of 10 plant pathogenic bacteria such as Fusarium oxysporum 295, Fusarium verticillioides 173, Fusarium moniliforme N19-2-2, Fusarium solani N18-1-2, Phytophthora parasitica var. nicotianae, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. niveum, Colletotrichum gloeosporioides, Alternaria alternata, and Alternaria mali were taken with a punch and placed in the center of the PDA medium plate. The PDA plate without spreading the crude lipopeptide extract was used as the blank control. All the petri dishes were cultured in the dark at 28 °C with 4 replicates. After 5 d, the diameter of the pathogenic bacteria colonies was measured, and the antibacterial effect of the crude lipopeptide extract on 10 plant pathogenic bacteria such as Fusarium was calculated.

[0067] Relative mycelium antibacterial rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0068] The results are shown in Table 5. The results indicate that the above-mentioned crude lipopeptide extract from Bacillus megaterium has a good antibacterial effect on various bacteria shown in Table 5.

[0069] Table 5 Antibacterial Effect of the Crude Lipopeptide Extract from Bacillus megaterium LDSR-6

[0070]

[0071]

[0072] Example 5, Inhibitory Effect of Bacillus megaterium LDSR-6 on the Mycelial Morphology of Fusarium

[0073] Place pathogen mycelial discs (1 cm) such as Fusarium oxysporum 295, Fusarium verticillioides 173, Fusarium moniliforme N19-2-2, and Fusarium solani N18-1-2 in the center of a PDA medium plate. At a distance of 2 cm from the edge of the pathogen colony, place a single colony mycelial disc (5 mm) of Bacillus megaterium LDSR-6 for control. Petri dishes without the single colony mycelial disc of Bacillus megaterium LDSR-6 serve as blank controls. All Petri dishes are cultured in the dark at 28 °C with 4 replicates. After 5 days, use an electron microscope to observe the mycelial morphology of the pathogens in the inhibition zone of each Petri dish. Observe the mycelial morphology of the pathogens at the edge of the colony in the blank control to evaluate the inhibitory effect of Bacillus megaterium LDSR-6 on the mycelial morphology of the 4 species of Fusarium

[0074] Microscopic observation found that Bacillus megaterium LDSR-6 caused the mycelia of Fusarium oxysporum 295 to be deformed, shrunk, and hollow ( Figure 5 ); caused the mycelia of Fusarium moniliforme N19-2-2 to break into small segments and show small protrusions ( Figure 6 ); caused the mycelia of Fusarium solani N18-1-2 to show many beaded vesicular morphologies ( Figure 7 ); caused the mycelia of Fusarium verticillioides 173 to produce deformed swollen protrusions ( Figure 8 ). The results showed that Bacillus megaterium LDSR-6 could cause the infection mycelia of various Fusarium to be deformed, thus reducing or losing the pathogenicity of the infection mycelia

[0075] Example 6, Hemolytic Test of Bacillus megaterium LDSR-6

[0076] Pick a single colony of Bacillus activated on an 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, with 3 replicates for each strain. If a well-defined and completely transparent hemolysis ring is formed around the colony, indicating hemolysis, the hemolysis type is β-hemolysis; if there is no change in the medium around the colony, indicating no hemolysis, it is γ-hemolysis

[0077] Table 6 Hemolysis of Bacillus LDSR-6

[0078] strain name clear zone hemolytic zone hemolytic property Bacillus megaterium LDSR-6 \ γ-hemolysis none

[0079] The detection results are shown in Table 6, indicating that Bacillus megaterium LDSR-6 does not have hemolytic activity and complies with the safety regulations for the use of biocontrol strains in the "Biological Safety Law of the People's Republic of China"( Figure 9 ).

[0080] Example 7. Determination of the nitrogen, phosphorus, and potassium contents of the Bacillus megaterium LDSR-6 bacterial agent

[0081] After activation of Bacillus megaterium LDSR-6 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 solution. There were 3 replicates for each strain, and the non-inoculated culture solution was used as a control. After culturing at 30 °C and 160 r / min for 7 d, the nitrogen fixation rate of each culture solution was measured by the Kjeldahl method. The results are shown in Table 6.

[0082] After activation of Bacillus megaterium LDSR-6 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 solutions. There were 3 replicates for each strain, and the non-inoculated culture solution 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 measured by the molybdenum-antimony anti-colorimetric method. The results are shown in Table 6.

[0083] After activation of Bacillus megaterium LDSR-6 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 solution. There were 3 replicates for each strain, and the non-inoculated culture solution was used as a control. After culturing at 30 °C and 160 r / min for 7 d, the potassium release rate of each culture solution was measured by flame atomic absorption spectrometry. The results are shown in Table 7.

[0084] Table 7. Determination of the nitrogen, phosphorus, and potassium contents of Bacillus LDSR-6

[0085] strain number Nitrogen content / % Phosphorus content / % Potassium content / % Bacillus LDSR-6 0.200 0.044 0.010

[0086] The test results show that the contents of nitrogen, phosphorus, and potassium in the Bacillus megaterium LDSR-6 bacterial agent are relatively high, which can provide absorbable nitrogen, phosphorus, and potassium elements for host plants.

Claims

1. A strain of Bacillus megaterium ( Bacillus megaterium ), named Bacillus megaterium LDSR-6, characterized in that, The Bacillus megaterium LDSR-6 has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number being CGMCC No. 30083.

2. Use of the Bacillus megaterium 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 F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

3. Use of the Bacillus megaterium 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 F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora parasitica var.nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

4. Use of the Bacillus megaterium according to claim 1 in producing lipopeptide antibiotics having an inhibitory effect on plant pathogens; the plant pathogen is Fusarium oxysporum Fusarium oxysporum Fusarium verticillium F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora parasitica var.nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

5. A plant disease inhibitor, characterized in that: The active ingredient is the Bacillus megaterium or its lipopeptide extract as described in claim 1; the pathogen of the plant disease is Fusarium oxysporum Fusarium oxysporum Fusarium verticillium F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

6. A lipopeptide extract, which is prepared by culturing the Bacillus megaterium as claimed in claim 1, collecting the bacterial cells, and extracting the cells with methanol to obtain a crude lipopeptide extract.

7. Use of the lipopeptide extract according to claim 6 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 F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

8. Use of the lipopeptide extract according to claim 7 in the preparation of a biocontrol agent or microbial fertilizer for inhibiting plant pathogens; the plant pathogen is Fusarium oxysporum Fusarium oxysporum Fusarium verticillium F. verticilliodes Fusarium moniliforme F. moniliforme Fusarium solani F. solani , Phytophthora nicotianae Phytophthora nicotianae , Fusarium wilt Fusarium oxysporum f. sp. cucumerinum , Fusarium wilt Fusarium oxysporum f. sp. niveum , Colletotrichum spp. Colletotrichum gloeospoioides , Alternaria apple Alternaria alternata , and Alternaria fragariae A. alternata One or any combination of two or more.

9. Use of the Bacillus megaterium or its lipopeptide extract according to claim 1 in preventing and treating Astragalus root rot.

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