Application of bacillus velezensis TCS001 and lipopeptide crude extract thereof in prevention and treatment of magnaporthe oryzae and activation of resistance of rice to magnaporthe oryzae

Abstract

The invention provides application of bacillus velezensis TCS001 and / or lipopeptide crude extract of the bacillus velezensis TCS001 in biological prevention and treatment of magnaporthe grisea and application of inducing rice to resist the magnaporthe grisea, the preservation number of the bacillus velezensis TCS001 is CGMCC No.8921, and a new biological agent is provided for prevention and treatment of the magnaporthe grisea. It is found for the first time that after the bacillus velezensis TCS001 and / or the lipopeptide crude extract of the bacillus velezensis TCS001 are sprayed to the rice, the expression level of rice resistance immunity related genes is remarkably increased, and it is indicated that the bacillus velezensis TCS001 and / or the lipopeptide crude extract of the bacillus velezensis TCS001 activate the resistance reaction of the rice to magnaporthe oryzae.

Description

Technical Field

[0001] The present invention relates to the field of plant protection biological pesticides, relates to the application of Bacillus velezensis TCS001 as an active ingredient in controlling Magnaporthe oryzae of rice, relates to the antibacterial effect of the crude lipopeptide extract of Bacillus velezensis TCS001 on Magnaporthe oryzae of rice, and also relates to the effect of activating rice to resist Magnaporthe oryzae by Bacillus velezensis TCS001 and / or the crude lipopeptide extract of Bacillus velezensis TCS001. Background Art

[0002] Approximately half of the world's population takes rice as the staple food, and rice is often damaged by insects and pathogenic bacteria during the planting process. Rice blast caused by the fungus Magnaporthe oryzae is one of the most common rice diseases, which can cause serious yield losses. Currently, the main control methods include screening for resistant varieties and applying chemical fungicides. However, the field environment is complex, and it is difficult for a single resistant variety to face direct or indirect infections by multiple pathogenic bacteria; in addition, due to the long-term and large-scale use of chemical fungicides, not only will it exacerbate the generation of drug resistance of pathogenic bacteria, but the reduction in the number of soil microorganisms and the decline in soil fertility caused by drug residues pose a threat to the ecological environment. Therefore, it is particularly urgent to develop efficient, safe and green alternatives.

[0003] Bacillus velezensis is a new type of biocontrol bacterium that can synthesize a large number of lipopeptide substances with strong antibacterial activity. Bacillus velezensis is widely distributed and can survive under adverse conditions such as drought and salinity. There are few related products, and it has broad development prospects.

[0004] Bacillus velezensis TCS001 applied in this application was isolated from Bohai Sea sludge. The original strain was Bacillus sp. CT2628, and after mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit number is CGMCC No. 8921, which was first published in Patent ZL201410168402.2. The inventors of this application found in previous studies that although Bacillus velezensis TCS001 has a certain inhibitory effect on Botrytis cinerea of cucumber, Didymella bryoniae of cucumber, Sclerotinia sclerotiorum of rape, Cercospora cucumerina of cucumber, Fusarium oxysporum f. sp. vasinfectum of cotton and Cercospora musae of banana. However, there are significant differences in the inhibitory effects of Bacillus velezensis TCS001 on different plant pathogenic bacteria. This application is the first to study the antibacterial effects of Bacillus velezensis TCS001 and the fermentation filtrate of Bacillus velezensis TCS001 on Magnaporthe oryzae of rice.

[0005] In previous research by the inventors of this application, it was found that the fermentation filtrate of Bacillus velezensis TCS001 (excluding the bacteria themselves and only containing its secondary metabolites) has high antibacterial activity against various plant pathogenic fungi. It is speculated that the fermentation of Bacillus velezensis TCS001 may produce a variety of secondary metabolites with antibacterial activity (refer to the invention patent that has been submitted: A mixture containing lipopeptide compounds isolated from the fermentation broth of Bacillus velezensis TCS001 and its application, application number: CN202311517534.7). This application is the first to study the antibacterial effect of the crude lipopeptide extract of Bacillus velezensis TCS001 on Magnaporthe oryzae, the causal agent of rice blast disease.

[0006] It is known in the art that there are many species of Bacillus velezensis, and there is no specific statistical quantity. However, there are few reports on Bacillus velezensis that can induce plant resistance and have stress resistance and growth promotion effects. This application is the first to study the effect of Bacillus velezensis TCS001 and the crude lipopeptide extract of Bacillus velezensis TCS001 on activating the resistance of rice to Magnaporthe oryzae. Summary of the Invention

[0007] The purpose of the present invention is to provide the use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 for biological control of Magnaporthe oryzae. The preservation number of Bacillus velezensis TCS001 is CGMCC No.8921. To provide a new biological agent for the control of rice blast disease.

[0008] Another purpose of the present invention is to provide the use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 in antagonizing Magnaporthe oryzae. The preservation number of Bacillus velezensis TCS001 is CGMCC No.8921. It is characterized in that the antagonism against Magnaporthe oryzae is the formation of appressoria of antagonistic Magnaporthe oryzae, and / or inhibition of the growth and development of the mycelium of Magnaporthe oryzae, and / or reduction of the pathogenicity of Magnaporthe oryzae.

[0009] The purpose of the present invention is to provide the use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 in the preparation of pesticides for controlling rice blast disease. The preservation number of Bacillus velezensis TCS001 is CGMCC No.8921.

[0010] Preferably, the rice blast is one or more of seedling rice blast, leaf blast, collar blast, node blast, panicle neck blast, rachis blast or grain blast, etc.

[0011] The object of the present invention is to provide the use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 in inducing rice to resist Magnaporthe oryzae, and the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

[0012] The object of the present invention is to provide the use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 in the preparation of a drug for inducing rice to resist rice blast, and the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

[0013] Preferably, the use is to activate the rice resistance response protein.

[0014] Preferably, the use is to increase the expression level of genes related to rice resistance immunity.

[0015] Preferably, the genes related to rice resistance immunity are one or more of OsKS4, OsNAC4, OsPR1a, OsChitinase1, etc.

[0016] Preferably, the use is to activate the accumulation of reactive oxygen species in rice.

[0017] Preferably, the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 10 mg / L - 500 mg / L.

[0018] Preferably, the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 10 mg / L - 200 mg / L.

[0019] The object of the present invention is to provide a method for controlling Magnaporthe oryzae, including the step of treating with Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001, and the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

[0020] Preferably, the step of treatment is contact treatment.

[0021] The object of the present invention is to provide a method for preventing and controlling rice blast, which includes applying Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 to rice crops, and the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

[0022] Another object of the present invention is to provide the use of the crude lipopeptide extract of Bacillus velezensis TCS001, which has a significant antibacterial effect on Magnaporthe oryzae of rice.

[0023] Preferably, the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 10 mg / L - 100 mg / L.

[0024] Another object of the present invention is to provide the use of the crude lipopeptide extract of Bacillus velezensis TCS001, which can activate rice to resist Magnaporthe oryzae of rice.

[0025] Preferably, the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 100 mg / L - 200 mg / L.

[0026] Preferably, the Bacillus velezensis TCS001 strain of the present invention is as described in Example 1. By using the streak plate method, the Bacillus velezensis strain TCS001 is streaked and inoculated on an LB solid medium, and cultured in a bacterial incubator at 28°C for 48 h to obtain a single colony of Bacillus velezensis TCS001.

[0027] Preferably, the fermentation broth of Bacillus velezensis TCS001 of the present invention is as described in Example 1. The seed liquid of Bacillus velezensis TCS001 is inoculated into the fermentation medium of Bacillus velezensis TCS001, and cultured on a shaker at 28°C and 173 rpm for 72 h to prepare the fermentation broth of Bacillus velezensis TCS001.

[0028] Preferably, the fermentation filtrate of Bacillus velezensis TCS001 of the present invention is as described in Example 1. The prepared fermentation broth of Bacillus velezensis TCS001 is centrifuged at 8000 rpm for 10 min at 4°C. After completion, the fermentation supernatant is taken and filtered 3 times with a 0.22 μm filter membrane to prepare the fermentation filtrate of Bacillus velezensis TCS001.

[0029] Preferably, the lipopeptide crude extract of Bacillus velezensis TCS001 according to the present invention is as described in Example 2. The fermentation broth of Bacillus velezensis TCS001 is placed at 4°C and centrifuged at 8000 rpm for 10 min. The supernatant is collected and filtered through a 0.22 μm filter membrane three times to obtain a sterile fermentation filtrate. Then, the pH value of the supernatant is adjusted to 2.0 with 6 mol / L hydrochloric acid and left standing overnight. After that, the acid-precipitated solution is centrifuged again at 8000 rpm for 10 min at 4°C, the supernatant is discarded, and the precipitate is retained. Finally, the precipitate is dried with a drying lamp, and the obtained product is the lipopeptide crude extract of Bacillus velezensis TCS001.

[0030] Compared with the prior art, the present invention has the following beneficial technical effects:

[0031] Through confrontation experiments, the present invention first discovers that Bacillus velezensis TCS001 has a significant antibacterial effect on Magnaporthe oryzae, and the antibacterial effect is 96.7%.

[0032] The present invention first discovers that the fermentation filtrate of Bacillus velezensis TCS001 has a significant antibacterial effect on Magnaporthe oryzae.

[0033] The present invention first discovers that the lipopeptide crude extract of Bacillus velezensis TCS001 has a significant antibacterial effect on rice blast.

[0034] The present invention first discovers that Bacillus velezensis TCS001 can activate the immune response of rice and resist the infection of Magnaporthe oryzae.

[0035] The present invention first discovers that the fermentation filtrate of Bacillus velezensis TCS001 can activate the immune response of rice and resist the infection of Magnaporthe oryzae.

[0036] The present invention first discovers that the lipopeptide crude extract of Bacillus velezensis TCS001 can activate the immune response of rice and resist the infection of Magnaporthe oryzae.

[0037] The present invention first discovers that after the fermentation broth prepared from Bacillus velezensis TCS001 strain is induced by spraying, the expression level of rice resistance and immune-related genes is increased, and the rice resistance response protein is activated, indicating that Bacillus velezensis TCS001, and / or the lipopeptide crude extract of Bacillus velezensis TCS001 activates the resistance response of rice to Magnaporthe oryzae and activates the accumulation of reactive oxygen species in rice. Description of the Drawings

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0039] Figure 1 Antibacterial effect of Bacillus velezensis TCS001 against Magnaporthe oryzae in confrontation;

[0040] Figure 2 Antibacterial activity of the fermentation filtrate of Bacillus velezensis TCS001 against Magnaporthe oryzae;

[0041] Figure 3 Inhibitory rate of the fermentation filtrate of Bacillus velezensis TCS001 in in vitro antibacterial test;

[0042] Figure 4 Antibacterial effect of the crude lipopeptide extract of Bacillus velezensis TCS001 in in vitro antibacterial test;

[0043] Figure 5 Inhibitory rate of the crude lipopeptide extract of Bacillus velezensis TCS001 in in vitro antibacterial test;

[0044] Figure 6 Observation by scanning electron microscope on the effect of the crude lipopeptide extract of Bacillus velezensis TCS001 on the mycelial morphological structure of Magnaporthe oryzae;

[0045] Figure 7 Observation by optical microscope on the morphological changes of appressoria of Magnaporthe oryzae treated with the crude lipopeptide extract of Bacillus velezensis TCS001;

[0046] Figure 8 Inhibitory rate of the crude lipopeptide extract of Bacillus velezensis TCS001 on the formation of appressoria of Magnaporthe oryzae;

[0047] Figure 9 Disease incidence after inoculating rice leaves with Magnaporthe oryzae treated with crude lipopeptide extracts of Bacillus velezensis TCS001 at different concentrations;

[0048] Figure 10 Disease incidence in the test of inoculating rice seedlings treated with fermentation broth of Bacillus velezensis TCS001 at different concentrations with a spore suspension of Magnaporthe oryzae;

[0049] Figure 11 Relative lesion area of rice seedlings inoculated with a spore suspension of Magnaporthe oryzae after being treated with fermentation broth of Bacillus velezensis TCS001 at different concentrations;

[0050] Figure 12Treatment with fermentation broths of Bacillus velezensis TCS001 at different concentrations activates the immune response of rice resistance-related proteins;

[0051] Figure 13 Treatment with fermentation broths of Bacillus velezensis TCS001 at different concentrations activates the reactive oxygen species burst in rice. Specific implementation mode

[0052] Example 1

[0053] Determination of the in vitro antibacterial activity of Bacillus velezensis TCS001 and its fermentation filtrate.

[0054] Culture medium

[0055] LB solid medium: agar powder 18.0 g / L, tryptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, pH 7.0, sterilized at 121 °C for 15 min.

[0056] MLB liquid medium: tryptone 7.0 g / L, yeast powder 2.0 g / L, NaCl 6.0 g / L, MgCl·6H2O 0.5 g / L, KCl 0.06 g / L, glucose 2 g / L, pH 7.0, sterilized at 121 °C for 15 min.

[0057] Sporulation fermentation medium for Bacillus velezensis TCS001: soluble peanut cake powder 28.9 g / L, glucose 30 g / L, soluble starch 30 g / L, FePO4 0.202 g / L, KCl 0.06 g / L, NaCl 6 g / L, MgCl2·6H2O 0.5 g / L, K2HPO4 3 g / L, KH2PO4 1.5 g / L, CaCO3 0.5 g / L, MnSO4 0.05 g / L, pH 7.0, 31% liquid loading (refer to the invention patent submitted: a culture medium and its culture process for industrial production of Bacillus velezensis TCS00 fermentation broth, application number: CN202311517533.2).

[0058] CM-C medium (1000 mL): 20-fold nitrate solution 50 mL, 1000-fold trace element solution 1 mL, glucose 10 g / L, peptone 2 g, yeast extract 1 g, casein amino acid 1 g, 1000-fold vitamin solution 1 mL.

[0059] 20-fold nitrate solution (1000 mL): NaNO3 120 g, KCl 10.4 g, MgSO4·7H2O 10.4 g, KH2PO4 30.4 g, add ddH2O to 1000 mL.

[0060] 1000x Vitamin Solution (100 mL): Biotin 0.01 g, Pyridoxin 0.01 g, Thiamine 0.01 g, Riboflavin 0.01 g, PABA (p-aminobenzoic acid) 0.01 g, Nicotinic acid 0.01 g, add ddH2O to 100 mL.

[0061] 1000x Trace Element Solution (100 mL): ZnSO4·7H2O 2.2 g, H3BO3 1.1 g, MnCl2·4H2O 1.1 g, FeSO4·7H2O 0.5 g, CoCl2·6H2O 0.17 g, CuSO4·5H2O 0.16 g, Na2MoO4·5H2O 0.15 g.

[0062] Activation of Bacillus velezensis TCS001: The Bacillus velezensis strain TCS001 was streaked on an LB solid medium by the streak plate method and cultured in a bacterial incubator at 28 °C for 48 h to obtain single colonies of Bacillus velezensis TCS001, which were used for the preparation of the Bacillus velezensis TCS001 seed solution and the plate confrontation test with Magnaporthe oryzae.

[0063] Preparation of Bacillus velezensis TCS001 seed solution: Prepare 150 mL of MLB liquid medium and sterilize it at 121 °C for 20 min; inoculate the single colonies of Bacillus velezensis TCS001 into the MLB liquid medium and culture it on a shaker at 145 rpm and 27 °C for 16 h.

[0064] Preparation of Bacillus velezensis TCS001 fermentation broth: Inoculate the Bacillus velezensis TCS001 seed solution into the Bacillus velezensis TCS001 spore-producing fermentation medium at an inoculation amount of 1%, and culture it on a shaker at 173 rpm and 28 °C for 72 h to obtain the Bacillus velezensis TCS001 fermentation broth.

[0065] Preparation of Bacillus velezensis TCS001 fermentation filtrate: Centrifuge the above-prepared Bacillus velezensis TCS001 fermentation broth at 4 °C and 8000 rpm for 10 min, and after completion, take the fermentation supernatant and filter it 3 times with a 0.22 μm filter membrane to prepare the Bacillus velezensis TCS001 fermentation filtrate for standby.

[0066] Confrontation test: Using a sterile inoculation loop and inoculation needle, a 8-mm sterile punch was used to punch holes in the mycelium, and a Magnaporthe oryzae fungal cake was picked and inoculated in the center of a CM-C Petri dish. Subsequently, a single colony of Bacillus velezensis TCS001 was streaked with an inoculation loop and placed 2 cm away from the center of the fungal cake, repeated 3 times, and rotated 45 °C each time. Another CM-C medium containing Magnaporthe oryzae was used as a blank control plate, and each treatment was repeated 3 times.

[0067] Antibacterial activity test of the fermentation filtrate of Bacillus velezensis TCS001: Using the poisoned medium method, the fermentation filtrate of Bacillus velezensis TCS001 was prepared into medicated media with ratios of 1 / 5, 1 / 10, 1 / 20, 1 / 40, 1 / 60, 1 / 80, 1 / 100, and 1 / 120 (fermentation filtrate of Bacillus velezensis TCS001 / water volume ratio) in CM-C medium. After thoroughly shaking the above 8 concentrations of medicated media, they were poured into Petri dishes with a diameter of 60 mm to make medicated plates with corresponding concentrations. Another CM-C medium without the medicament was used as a blank control plate, and each treatment was repeated 3 times.

[0068] Under sterile conditions, a sterile punch with a diameter of 8 mm was used to cut a fungal cake from the vigorously growing mycelium at the edge of the colony of the cultivated Magnaporthe oryzae pathogen. The fungal cake was inoculated to the center of each medicated plate with a sterile inoculation needle. It was incubated in the dark in an incubator at 25 °C in an inverted position. When the colony grew to 3 / 4 of the plate area, the diameter was measured using the cross method. The average value obtained from the two measurements was the colony diameter, and the inhibition rate (I) was calculated.

[0069] I = [(D1 - D2) / (D1)] × 100%

[0070] D1: Colony diameter (mm) formed in the blank control plate;

[0071] D2: Colony diameter (mm) formed in the medicated plate of the test agent;

[0072] Experimental results

[0073] The antibacterial effect of the plate confrontation of Bacillus velezensis TCS001 and the antibacterial rate of the fermentation filtrate of Bacillus velezensis TCS001 are as Figures 1 to 3 shown.

[0074] From Figure 1 , Figure 2 and Figure 3 (Table 1), it can be seen that for Magnaporthe oryzae, the antibacterial effect of the 4-point plate confrontation of Bacillus velezensis TCS001 strain was 96.7%; the antibacterial effect of the fermentation filtrate of Bacillus velezensis TCS001 was the best when diluted 5 times (see Figure 2 ), and the antibacterial rate against Magnaporthe oryzae reached 83.1% (see Figure 3 , Table 1).

[0075] Table 1 Antibacterial activity of the fermentation filtrate of Bacillus velezensis TCS001 against Magnaporthe oryzae

[0076]

[0077] Example 2

[0078] Determination of the in vitro antibacterial activity of the crude lipopeptide extract of Bacillus velezensis TCS001 and its effects on the mycelial morphological structure and appressorium formation of Magnaporthe oryzae

[0079] Culture medium

[0080] Culture medium for producing crude lipopeptide extract of Bacillus velezensis TCS001: soluble starch 10.5 g / L, peanut cake powder 18.5 g / L, NaCl 3.0 g / L, liquid loading 32%, pH 6.0, sterilized at 121 °C for 15 min

[0081] Extraction of crude lipopeptide extract of Bacillus velezensis TCS001: Use an inoculation loop to pick a single colony from the Bacillus velezensis TCS001 strain cultured for 48 h, inoculate it into a 250 mL conical flask containing 150 mL of MLB culture solution, and culture it in a constant temperature shaker at 145 rpm and 27 °C for 16 h to prepare a seed solution. Subsequently, inoculate the seed solution into a 250 mL conical flask containing 80 mL of the culture medium for producing crude lipopeptide extract of Bacillus velezensis TCS001 at an inoculation amount of 3%, and culture it in a constant temperature shaker at 25 °C and 164 rpm for 48 h. After the culture is completed, a fermentation broth with a high content of crude lipopeptide extract of Bacillus velezensis TCS001 is obtained, and the crude lipopeptide extract of Bacillus velezensis TCS001 is extracted by the acid precipitation method

[0082] The specific steps are as follows: First, place the fermentation broth of Bacillus velezensis TCS001 at 4 °C, centrifuge it at 8000 rpm for 10 min, collect the supernatant and filter it 3 times with a 0.22 μm filter membrane to obtain a sterile fermentation filtrate; then adjust the pH value of the supernatant to 2.0 with 6 mol / L hydrochloric acid and let it stand overnight; then centrifuge the acid-precipitated solution at 4 °C and 8000 rpm for 10 min again, discard the supernatant, retain the precipitate, and finally dry the precipitate with a drying lamp. The obtained product is the crude lipopeptide extract of Bacillus velezensis TCS001

[0083] Antibacterial Activity Test of Lipopeptide Crude Extract from Bacillus velezensis TCS001 against Magnaporthe oryzae: Using the mycelial growth rate method, first, the lipopeptide crude extract from Bacillus velezensis TCS001 was prepared into a mother liquor with a mass fraction of 5% using N,N-dimethylformamide (DMF), and then gradient diluted. The medicated CM-C medium with concentrations of 100 mg / L, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, and 10 mg / L was prepared by sequential dilution with sterile water. The CM-C without the agent was used as the blank control, and 3 replicates were set for each treatment. An 8-mm-diameter mycelial disc of Magnaporthe oryzae was inoculated in the center of the petri dish, with the mycelial surface facing down, and incubated in an inverted position at (25 ± 0.1) °C. When the diameter of the Magnaporthe oryzae colony in the control group reached 3 / 4 of the petri dish diameter, the cross-cross method was used to measure the diameter of the Magnaporthe oryzae colony, and the inhibition rate I was calculated.

[0084] I = [(D1 - D2) / (D1 - 8)] × 100%

[0085] D1: The diameter of the colony formed in the blank control petri dish (mm);

[0086] D2: The diameter of the colony formed in the petri dish with the test agent (mm);

[0087] According to D1 as the diameter of the blank control colony, mm; D2 as the diameter of the colony in the medicated treatment group, mm.

[0088] Scanning of the Mycelial Morphology of Magnaporthe oryzae

[0089] Preparation of Scanning Electron Microscope Samples:

[0090] The Magnaporthe oryzae at different treatment concentrations was divided into small pieces of 0.5 × 0.3 cm in size. The small pieces were placed in a 2-mL centrifuge tube, and 2.5% glutaraldehyde was added and incubated overnight at 4 °C. The glutaraldehyde was sucked off with a pipette, and the samples were washed with 0.1 M PBS buffer at pH 7.0 for 15 min and washed 3 times. The samples were successively soaked in ethanol solutions with concentration gradients of 30%, 50%, 70%, 80%, 90%, and 95% for 15 min each. The ethanol solution was sucked off with a pipette, and the samples were eluted with 100% ethanol for 20 min and repeated 2 times. The ethanol was poured out, and the samples were soaked in a mixed solution of ethanol and isoamyl acetate (V / V = 1 / 1) for 30 min. The samples were soaked in 100% isoamyl acetate for 2 h. The conductive adhesive was pasted on the sample stage, and the samples were gently clamped at the edges with tweezers and pasted onto the conductive adhesive. They were dried in a laminar flow cabinet, gold-plated at the critical point, and finally observed with a scanning electron microscope.

[0091] The appressorium is an important organ during the process of pathogenic fungi infecting the surface of rice leaves. After the conidia of the pathogen fall onto the surface of rice leaves, the germ tubes of the conidia germinate, and then appressoria are produced at the tips of the germ tubes. The appressoria generate turgor pressure, pierce the surface of rice leaves, and then produce hyphae to infect the epidermal cells of the leaves. Finally, the infecting hyphae spread to adjacent cells, and lesions form on the leaf surface in about a week.

[0092] Collection of Magnaporthe oryzae conidia: Add sterile water to the CM-C medium, scrape the conidia off the medium with an inoculation loop, pipette and aspirate repeatedly, and filter the spore suspension through two layers of Micocloth. Observe the concentration of the spore suspension, count using a hemocytometer, and adjust the spore suspension concentration to 1 - 2×10 4 conidia / mL.

[0093] Germination of Magnaporthe oryzae spores: Drop the spore suspension on a hydrophobic glass slide, place the glass slide in a petri dish, cover the lid of the petri dish with 3 - 5 layers of moist filter paper, and incubate at 25°C with humidity for 16 h.

[0094] Experimental results

[0095] The antibacterial effect of the crude lipopeptide extract of Bacillus velezensis TCS001 is as Figure 4 shown in the antibacterial rate as Figure 5 (Table 2). The results show that when the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 100 mg / L, the antibacterial effect on Magnaporthe oryzae is the strongest, and the inhibition rate reaches 81.2%.

[0096] Effect on mycelial growth is as Figure 6 , the mycelia of Magnaporthe oryzae without treatment with the crude lipopeptide extract of Bacillus velezensis TCS001 are relatively smooth on the surface. When Magnaporthe oryzae is co-cultured with the crude lipopeptide extract of Bacillus velezensis TCS001, shrinkage and fracture phenomena appear on the surface of the mycelia of Magnaporthe oryzae. It shows that after treatment with the crude lipopeptide extract of Bacillus velezensis TCS001, Magnaporthe oryzae affects the normal growth of the mycelia, thus achieving the antibacterial effect.

[0097] Effect on the formation of appressoria of Magnaporthe oryzae is shown by Figure 7 and Figure 8 . It can be seen that the higher the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001, the greater the effect on the formation of appressoria. When the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 100 mg / L, the inhibition rate of appressorium formation reaches 90.5%.

[0098] Table 2 Determination results of the antibacterial activity of the crude lipopeptide extract of Bacillus velezensis TCS001 against Magnaporthe oryzae

[0099] Example 3

[0100] Experiment on inoculating Magnaporthe oryzae on rice leaves treated with crude lipopeptide extracts of Bacillus velezensis TCS001 at different concentrations.

[0101] Collect rice leaves at the 3 - 4 leaf stage for standby. Use a sterile scissors to cut the rice leaves into leaf segments of 5 cm and place them in a petri dish. Before inoculating the crude lipopeptide extracts of Bacillus velezensis TCS001, use a 10 μL sterile pipette tip to puncture wounds between the veins of each leaf. It is appropriate that the wounds do not penetrate the leaf. At each wound, inoculate a mycelial cake of Magnaporthe oryzae treated with 50 mg / L or 100 mg / L crude lipopeptide extracts of Bacillus velezensis TCS001, and spray water to keep it moist. After inoculation, perform dark moisture - keeping treatment for 24 h. After 24 h, culture it under light at 28 °C for 3 - 5 d, and always maintain a relative humidity of more than 90% during this period. Count the disease incidence.

[0102] Experimental results

[0103] It can be seen from Figure 9 that after treatment with the crude lipopeptide extracts of Bacillus velezensis TCS001, the lesion area decreases, and the pathogenicity of Magnaporthe oryzae is significantly weakened, indicating that the crude lipopeptide extracts of Bacillus velezensis TCS001 have an obvious effect of reducing the pathogenicity of Magnaporthe oryzae.

[0104] Example 4

[0105] Use the fermentation broth of Bacillus velezensis TCS001 with a high spore - producing content prepared in Example 1 to carry out an experiment on inoculating a spore suspension of Magnaporthe oryzae on rice seedlings induced by treatment with different concentrations of the fermentation broth of Bacillus velezensis TCS001.

[0106] Cultivation of rice seedlings:

[0107] Soak TP309 seeds at 30 °C for 2 d and germinate them at 37 °C for 1 - 2 d until the bud length reaches 0.5 cm. Select evenly germinated seeds and sow 6 - 7 seeds in each pot (17 cm pot). Keep 3 strong seedlings at the 2 - leaf stage, change the nutrient solution every 3 - 5 d, prevent pests regularly, and avoid stress. Use the seedlings at the 4 - leaf stage to the tillering stage for the experiment.

[0108] Cultivation of Magnaporthe oryzae:

[0109] Culture it in the dark at 28 °C for 2 d (inverted) on CM - C medium, and then culture it under light at 26 °C for 5 d (upright).

[0110] Collection of conidia of Magnaporthe oryzae:

[0111] Add sterilized water to the CM-C medium. Use an inoculation loop to scrape the conidia of Magnaporthe oryzae off the medium, repeatedly pipette and aspirate with a pipettor, transfer them into a 50 mL centrifuge tube, shake, and finally filter the conidial suspension of Magnaporthe oryzae through two layers of Miracloth.

[0112] Statistics of the concentration of the conidial suspension of Magnaporthe oryzae:

[0113] Count using a hemocytometer and adjust the concentration of the spore suspension to 1×10 6 spores / mL.

[0114] Select rice TP309 at the four-leaf stage to the tillering stage and seal it with a transparent PVC film roll. Nine days before inoculating Magnaporthe oryzae, spray the leaves with the fermentation broth of Bacillus velezensis TCS001 diluted 40-fold or 80-fold (use sterile water for the control group) three times at intervals of 3 days. When inoculating, spray the conidial suspension of Magnaporthe oryzae and cover it with a wet plastic wrap to keep it moist. After dark treatment at 22°C for 1 day, culture it under a light-dark cycle (16 h / 8 h). Pinpoint lesions appeared on the wild-type control after 3 days. Take pictures and record when the lesions are obvious. Stick the back of the leaf on a double-sided tape A4 paper, scan it after covering with a PVC film, and measure the lesion area with a leaf area meter.

[0115] Experimental results

[0116] From Figure 10 and Figure 11 it can be seen that after the in vivo rice leaves were induced and treated with the fermentation broth of Bacillus velezensis TCS001 (sprayed on the leaves three times at intervals of 3 days), the relative lesion area was less, and the relative lesion area treated with the 40-fold dilution was the least. It shows that Bacillus velezensis TCS001 and / or the crude extract of lipopeptides of Bacillus velezensis TCS001 in the fermentation broth of Bacillus velezensis TCS001 have the ability to activate the immune response of rice, stimulate the defense response of rice, and enhance the ability of rice to resist Magnaporthe oryzae.

[0117] Example 5

[0118] Use the fermentation broth of Bacillus velezensis TCS001 with a high spore content prepared in Example 1 to carry out the immune response of the relevant proteins of the defense response of rice activated by the induction treatment of the fermentation broth of Bacillus velezensis TCS001 (sprayed on the leaves three times at intervals of 3 days).

[0119] Samples of rice sprayed with the fermentation broth of Bacillus velezensis TCS001 diluted 40-fold or 80-fold respectively for 2 days were placed in a -80°C refrigerator for later use. Total RNA was extracted from the collected samples using Trizol reagent. Then, 1 μg was taken and reverse transcribed into cDNA using M-MLV reverse transcriptase and Olig(dT)18. Primers for quantitative qRT-PCR were designed using the online software GenScript qRT-PCR (www.genscript.com / tools / real-time-pcr-tag man-primer-design-tool), and the primers are shown in Table 3. The internal reference was rice Ubiquitin (LOC_Os03g13170). Real-time fluorescence quantitative PCR was performed on rice blast defense-related genes.

[0120] Reaction system:

[0121] TB Green Master 5 μL, Primer-F (10 μmol / L) 0.2 μL, Primer-R (10 μmol / L) 0.2 μL, cDNA 2 μL, distilled water 2.6 μL.

[0122] Reaction conditions:

[0123] Pre-denaturation at 95°C for 30 s; denaturation at 95°C for 20 s, extension at 60°C for 30 s, 40 cycles. The relative expression levels of genes were calculated using the 2-ΔΔCT method. -ΔΔCT method to calculate the relative expression levels of genes.

[0124] Table 3

[0125]

[0126] The results are as Figure 12 shown. After continuous spraying and induction treatment with the fermentation broth of Bacillus velezensis TCS001 three times, the expression levels of rice defense-related genes (OsKS4, OsNAC4, OsPR1a, and OsChitinase1) were significantly increased, indicating that Bacillus velezensis TCS001 in the fermentation broth of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 can increase the expression levels of rice resistance and immunity-related genes and activate the resistance and immune response of rice against Magnaporthe oryzae.

[0127] Example 6

[0128] The fermentation broth of Bacillus velezensis TCS001 treatment activates the burst of reactive oxygen species in rice.

[0129] The rice plants treated by the fermentation broth of Bacillus velezensis TCS001 (sprayed on the leaves three times at an interval of 3 days) were punched on both sides of the main vein with a puncher of 0.5 cm, and the obtained leaf discs were placed in hydrogen peroxide for dark overnight treatment. Then, three leaf discs of each sample were randomly selected and placed in a 1.5 mL test tube containing 100 μL of luminol, 1 μL of horseradish peroxidase and 1 μL of chitin. Finally, they were quickly placed into a Glomax 20 / 20 Luminometer instrument, and the fluorescence was detected every 10 s for a total of 25 min. Each sample was repeated 3 times.

[0130] The detection results by luminol chemiluminescence method showed that ( Figure 13 ), compared with the wild type, the rice plants treated with the fermentation broth of Bacillus velezensis TCS001 by continuous spraying three times had an accelerated growth rate of reactive oxygen species induced by chitin, and its accumulation rate was significantly higher than that before treatment. The results showed that Bacillus velezensis TCS001 in the fermentation broth of Bacillus velezensis TCS001, and / or the crude extract of lipopeptides of Bacillus velezensis TCS001 enhanced the basic defense ability of rice against Magnaporthe oryzae.

[0131] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. The present invention has been described in detail only with reference to the preferred embodiments. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered by the scope of the claims of the present invention.

Claims

1. Use of Bacillus velezensis TCS001, and / or fermentation broth of Bacillus velezensis TCS001, and / or fermentation filtrate of Bacillus velezensis TCS001, and / or crude lipopeptide extract of Bacillus velezensis TCS001 for biological control of Magnaporthe oryzae, wherein the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

2. Use of Bacillus velezensis TCS001, and / or the fermentation broth of Bacillus velezensis TCS001, and / or the fermentation filtrate of Bacillus velezensis TCS001, and / or the crude lipopeptide extract of Bacillus velezensis TCS001 in antagonizing Magnaporthe oryzae, wherein the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921, characterized in that, The antagonistic Magnaporthe oryzae is to antagonize the formation of appressoria of Magnaporthe oryzae, and / or inhibit the growth and development of hyphae of Magnaporthe oryzae, and / or reduce the pathogenicity of Magnaporthe oryzae.

3. Use of Bacillus velezensis TCS001, and / or fermentation broth of Bacillus velezensis TCS001, and / or fermentation filtrate of Bacillus velezensis TCS001, and / or crude lipopeptide extract of Bacillus velezensis TCS001 in the preparation of pesticides for controlling rice blast, wherein the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

4. The use according to claim 3, characterized in that, The rice blast includes one or more of seedling blast, leaf blast, collar blast, node blast, panicle neck blast, branch blast or grain blast, etc.

5. Use of Bacillus velezensis TCS001, and / or fermentation broth of Bacillus velezensis TCS001, and / or fermentation filtrate of Bacillus velezensis TCS001, and / or crude lipopeptide extract of Bacillus velezensis TCS001 to induce rice to resist Magnaporthe oryzae, wherein the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

6. Use of Bacillus velezensis TCS001, and / or fermentation broth of Bacillus velezensis TCS001, and / or fermentation filtrate of Bacillus velezensis TCS001, and / or crude lipopeptide extract of Bacillus velezensis TCS001 in the preparation of drugs for inducing rice to resist rice blast, wherein the preservation number of Bacillus velezensis TCS001 is CGMCC No. 8921.

7. The use according to claim 5 or 6, characterized in that, The use is to increase the expression level of genes related to rice resistance and immunity; further preferably, the use is to activate rice resistance response proteins.

8. The use according to claim 7, characterized in that, The genes related to rice resistance and immunity are one or more of OsKS4, OsNAC4, OsPR1a, OsChitinase1, etc.

9. Use according to claim 5 or 6, characterized in that, The use is to activate the accumulation of reactive oxygen species in rice.

10. The use according to any one of claims 1-9, characterized in that, The concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 10 mg / L - 500 mg / L, preferably, the concentration of the crude lipopeptide extract of Bacillus velezensis TCS001 is 10 mg / L - 200 mg / L.

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