Long lysinibacillus inoculant, preparation method and application of long lysinibacillus inoculant

Abstract

The invention discloses a long lysinibacillus inoculant, a preparation method and application, the inoculant is prepared from a long lysinibacillus strain C1, and the preparation method comprises the following steps: culturing the strain of the long lysinibacillus strain through a tryptone soybean agar culture medium until yellow single colonies appear; inoculating the yellow single colony into serum, and carrying out seed culture by using a long lysine bacillus liquid culture medium until a logarithmic phase to obtain a bacterial solution; and inoculating the cultured bacterial liquid into a production device according to a long lysine bacillus strain, and carrying out production culture by using a long lysine bacillus production culture medium to a logarithmic phase to prepare the long lysine bacillus biocontrol inoculant. And carrying out centrifugal treatment on the obtained biocontrol microbial inoculum of the long lysine bacillus, taking a supernatant, and filtering the supernatant to obtain the long lysine bacillus biocontrol fermentation broth. The invention further discloses application of the lysinibacillus elongate microbial inoculum in antagonism of rice blast bacteria and prevention and control of rice blast.

Description

Technical Field

[0001] The present invention relates to the technical field of biological pesticides, and particularly relates to a Bacillus lysiniformis bacterium agent, a preparation method thereof and an application thereof. Background Art

[0002] Microorganisms are widely present in nature and have become a research hotspot due to their advantages such as non-toxicity, harmlessness, pollution-free, high efficiency and low resistance in the process of preventing and controlling plant diseases and pests. Bacillus lysiniformis is a Gram-positive, rod-shaped, spore-forming bacterium belonging to the genus Bacillus. It is widely distributed in nature and is usually present in environments such as soil, water bodies and plant surfaces. Bacillus lysiniformis has received extensive attention due to its unique metabolic characteristics and potential industrial, agricultural and environmental protection applications. Bacillus lysiniformis has a significant inhibitory effect on a variety of plant pathogenic microorganisms, fungi and nematodes. In addition, these bacteria can also promote plant growth, improve the disease resistance of crops, and enhance the adaptability of plants to adversity. With the in-depth study of Bacillus lysiniformis, their applications in the fields of agriculture, environmental protection, feed additives, soil improvement and biomedicine have gradually increased, becoming an important part of modern sustainable agriculture and ecosystem management.

[0003] Rice blast mainly damages the leaves, stems and panicles of rice. In the initial stage of the disease, the lesions are water-soaked, dark green or brown. As the disease progresses, the lesions gradually expand, forming obvious brown or black spots. The leaves of the affected parts may become dry and yellow, resulting in the obstruction of photosynthesis and affecting the growth of the plants. The pathogen can invade downward through the stem, causing the stem to rot, and in severe cases, the plants may lodge. Rice blast also affects the development of the panicles, causing lesions at the neck of the panicles, resulting in an increase in the empty grain rate of the rice. Eventually, the diseased plants may wilt and die, seriously affecting the yield and quality of rice. It is the most serious disease in rice production, causing a 10-20% reduction in yield in epidemic years, and a 40-50% or more reduction in severe cases, and even a complete harvest failure.

[0004] Currently, the main methods for controlling rice blast are as follows: (1) Selecting disease-resistant varieties: According to the epidemic characteristics of rice blast and the local disease occurrence situation, selecting rice varieties resistant to rice blast or planting rice varieties with stronger disease resistance can effectively reduce the occurrence of rice blast. (2) Rational fertilization: Avoid partial or excessive application of nitrogen fertilizer, and appropriately apply potassium and phosphorus fertilizers during the tillering stage of rice to enhance the disease resistance of plants and reduce the occurrence probability of rice blast. (3) Timely irrigation: Adopt reasonable water management measures to avoid the paddy field being too dry or too wet. Keep a shallow water layer from the tillering stage to the jointing stage, and appropriately dry the field during the heading stage to control the spread of the pathogen and create unfavorable conditions for the control of rice blast. (4) Field management: Remove the diseased residues and weeds in the field to reduce the source of the pathogen. Reasonably plant densely to ensure good ventilation and light transmission in the field, reduce the humidity, and inhibit the reproduction and spread of the rice blast pathogen. (5) Chemical control: At the initial stage of the occurrence of rice blast, timely use chemicals for control. 800-1000-fold liquid of 75% tricyclazole wettable powder, 500-800-fold liquid of 40% isoprothiolane emulsifiable concentrate or 20% blastamide suspension can be selected for foliar spraying, which has a good control effect on rice blast. The above methods for controlling rice blast require professional knowledge and are prone to errors in actual operation; timely irrigation is difficult to control under extreme weather conditions and has a high maintenance cost; field management has a large workload and requires continuous attention, and some farmers may not be able to implement it comprehensively; although chemical control has a quick effect, long-term use may cause environmental pollution and pesticide residues. However, the biopesticide made from plant-derived metabolites is not easy to cause the rice blast fungus to develop resistance, but the plant-derived metabolites have poor stability, a short half-life and high costs. For example, the direct application of exogenous jasmonic acid has a half-life of <6h and a cost as high as $120 / kg. And due to problems such as the complex composition and rapid evolution of the physiological races of rice blast itself, the variety resistance is lost rapidly and the disease resistance spectrum is narrow (such as the detection rate of tricyclazole-resistant strains has reached 38%). Therefore, inventing a green, harmless, highly efficient and long-lasting microbial agent meets the greatest needs of the sustainable development of China's agriculture and environmental protection. Summary of the Invention

[0005] To solve the above technical problems, the object of the present invention is to provide a Lysinibacillus macroides microbial agent, a preparation method and its application. The Lysinibacillus macroides microbial agent is prepared by activating, seed culturing and production culturing a Lysinibacillus macroides strain.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A Lysinibacillus macroides, the strain is Lysinibacillus macroides C1, which was deposited in the China General Microbiological Culture Collection Center on July 29, 2019, and the deposit number is CGMCC No. 18349.

[0008] A Bacillus lysiniformis agent prepared from a Bacillus lysiniformis strain.

[0009] A method for preparing a Bacillus lysiniformis agent, comprising:

[0010] Step 1) Activate the Bacillus lysiniformis strain: Cultivate the strain of Bacillus lysiniformis through tryptic soy agar medium until yellow single colonies appear;

[0011] Step 2) Cultivate the yellow single colony seeds: Inoculate the yellow single colonies into serum and perform seed culture in a Bacillus lysiniformis liquid medium until the logarithmic growth phase to obtain a bacterial solution;

[0012] Step 3) Inoculate the cultured bacterial solution into a production device at an inoculation amount of 1% of the total volume of the Bacillus lysiniformis strain, and perform production culture in a Bacillus lysiniformis production medium until the logarithmic growth phase to obtain a biocontrol agent of Bacillus lysiniformis;

[0013] Step 4) Centrifuge the obtained biocontrol agent of Bacillus lysiniformis, take the supernatant after centrifugation, and filter the supernatant to obtain a biocontrol fermentation broth of Bacillus lysiniformis.

[0014] In the said step 1), the strain of Bacillus lysiniformis is cultivated through tryptic soy agar medium according to the double-layer culture method, and the used tryptic soy agar medium comprises 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 15 g of agar, 1000 mL of distilled water, and pH = 7.5.

[0015] In the said step 2), the temperature for cultivating the yellow single colony seeds is 32°C - 34°C, and the pH is 7.0.

[0016] Preferably, in the said step 3), the culture temperature of the Bacillus lysiniformis production medium is 32°C - 34°C, and the pH is 7.0.

[0017] In the said step 4), the biocontrol agent of Bacillus lysiniformis is centrifuged by a high-speed centrifuge, wherein the centrifugation speed is 7000 - 7500 rpm, the centrifugation temperature is 4°C, and the centrifugation time is 12 mins.

[0018] An application of a Bacillus lysiniformis agent in antagonizing Magnaporthe oryzae and controlling rice blast.

[0019] Compared with the prior art, one or more embodiments of the present invention may have the following advantages:

[0020] The production process of Lysinibacillus elongatus provided by the present invention is simple, with a short cultivation time and low cost. Moreover, this strain can be used to prepare a biocontrol agent and a biocontrol fermentation broth for preventing and controlling rice blast through simple, fast, and low-cost operation methods;

[0021] The biocontrol agent and the biocontrol fermentation broth prepared from Lysinibacillus elongatus are green, harmless, highly effective, long-lasting, simple and convenient to apply, and are not likely to cause pests and diseases to develop drug resistance. Brief Description of the Drawings

[0022] Figure 1a 、 1b is the antagonistic effect diagram of the Lysinibacillus elongatus agent;

[0023] Figure 2 are the disease pictures of rice leaves under the co-inoculation treatment, pre-induction treatment, and post-intervention treatment methods with the Lysinibacillus elongatus agent;

[0024] Figure 3 is the result diagram of the transcriptome and metabolome of rice leaves;

[0025] Figure 4 are the results of detecting the relative expression levels of genes related to the jasmonic acid pathway in rice and the jasmonic acid content in rice after spraying the Lysinibacillus elongatus agent. Detailed Embodiments

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings.

[0027] Example 1

[0028] A Lysinibacillus elongatus, and the strain is Lysinibacillus elongatus C1.

[0029] A Lysinibacillus elongatus agent prepared by activating, seed-culturing, and production-culturing a Lysinibacillus elongatus strain. The preparation method of the Lysinibacillus elongatus agent includes:

[0030] Step 1) Activation:

[0031] Cultivate the preserved strain of Lysinibacillus elongatus according to the double-layer plate culture method until yellow single colonies appear. The medium used is tryptone soy agar medium, and the formula is: 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 15 g of agar, 1000 mL of distilled water, pH = 7.5;

[0032] Step 2) Seed culture:

[0033] Inoculate the yellow single colony cultured in step 1) above into a serum bottle, and perform seed culture with a liquid medium for Lysinibacillus elongatus until the logarithmic growth phase to obtain a bacterial solution. The temperature for seed culture is 32°C to 34°C, and the pH is 7.0.

[0034] Step 3) Production culture:

[0035] Inoculate the bacterial solution obtained after seed culture in step 2) above into a production bottle at an inoculation amount of 5% of the total volume of the Lysinibacillus elongatus strain, and perform production culture with a production medium for Lysinibacillus elongatus until the logarithmic growth phase to obtain a biocontrol agent of Lysinibacillus elongatus. The temperature for production culture is 32°C to 34°C, and the pH is 7.0. The biocontrol agent of Lysinibacillus elongatus is obtained when cultured to the logarithmic growth phase.

[0036] Step 4) Centrifugation:

[0037] Put the biocontrol agent of Lysinibacillus elongatus obtained in step 3) into a high-speed centrifuge. The rotation speed for centrifugation is 7500 rpm, the centrifugation temperature is 4°C, and the centrifugation time is 10 minutes. After centrifugation, collect the supernatant, filter the supernatant with a bacterial filter, and then obtain a biocontrol fermentation broth of Lysinibacillus elongatus. The prepared biocontrol fermentation broth is stored at 4°C for standby.

[0038] The strain provided in this example is identified as Lysinibacillus elongatus through physiological and biochemical identification and molecular biological identification. The main biological characteristics are as follows: The strain is cultured on a double-layer solid plate medium for 4 days to form a yellow circular colony with a neat and smooth edge and a colony diameter of 0.3 mm; it is yellow when cultured in a liquid medium, with an optimal growth temperature of 32°C and a pH of 7.

[0039] Application of the Lysinibacillus elongatus bacterium agent in antagonizing Magnaporthe oryzae and controlling rice blast. The specific application method is as follows:

[0040] Magnaporthe oryzae plate assay: Inoculate a Magnaporthe oryzae bacterial cake in the center of a PDA (200 g of potato cut into pieces, boiled in water for 20 minutes, filtered with eight layers of gauze to obtain the juice, 20 g of glucose, 15 g of agar, added water to 1000 mL) plate. At the same time, spot inoculate 10 μL of the Lysinibacillus elongatus C1 bacterial suspension (concentration 1×108 CFU / mL, prepared by culturing in a liquid medium for Lysinibacillus elongatus for 24 hours) 2 cm away from the bacterial cake. Use sterile water spot inoculation as a control, repeat 4 times. After culturing at 28°C for 5 days, calculate the inhibition rate of Lysinibacillus elongatus C1 against Magnaporthe oryzae to be 71.3%. The picture of the antagonistic effect is as Figure 1a 、 1b shown.

[0041] Inhibition rate = (diameter of control colony - diameter of colony inoculated with antagonistic bacteria) / diameter of control colony × 100%. After inoculating with the Bacillus lysine formosus agent, the growth rate of the mycelia of Magnaporthe oryzae was slower than that of the control, and the mycelia were sparse, and the amount of spores was significantly reduced.

[0042] Example 2

[0043] 1) Experimental materials

[0044] Rice material: CO39, susceptible to most physiological races of Magnaporthe oryzae.

[0045] Pathogen: Physiological race 110-2 of Magnaporthe oryzae. Add the spores of Magnaporthe oryzae into warm distilled water (0.05% Tween 20 solution) to prepare a spore suspension for standby.

[0046] 2) Experimental methods

[0047] 2.1) Effects of the biocontrol agent of Bacillus lysine formosus on the incidence of rice blast under different treatment methods (co-inoculation treatment, pre-induction treatment, and post-intervention treatment):

[0048] (1) Co-inoculation treatment

[0049] Select rice seedlings at the four-leaf and one-heart stage with consistent growth vigor, randomly divide them into 5 groups, and perform corresponding treatments according to the experimental design in Table 1. Calculate the number of blast lesions 7 days after inoculating the spore suspension. Take the untreated group (CK) as the negative control, and calculate the relative incidence (%) according to the formula: Incidence = number of lesions in the experimental group / number of lesions in the untreated group × 100.

[0050] Table 1 is the co-inoculation treatment

[0051]

[0052] (2) Pre-induction treatment

[0053] Select rice seedlings at the four-leaf and one-heart stage with consistent growth vigor, randomly divide them into 5 groups, and perform corresponding treatments according to the experimental design in Table 1. Calculate the number of blast lesions 7 days after inoculating the spore suspension. Take the untreated group (CK) as the negative control, and calculate the relative incidence (%) according to the formula: Incidence = number of lesions in the experimental group / number of lesions in the untreated group × 100.

[0054] Table 2 is the pre-induction treatment

[0055]

[0056]

[0057] (3) Post-intervention treatment

[0058] Rice seedlings with consistent growth at the four-leaf and one-heart stage were randomly divided into 5 groups and subjected to corresponding treatments according to the experimental design in Table 1. The number of rice blast lesions was counted 7 days after inoculation with the spore suspension. The untreated group (CK) was used as the negative control, and the relative incidence (%) was calculated according to the formula: Incidence = number of lesions in the experimental group / number of lesions in the untreated group × 100.

[0059] Table 3 Post-intervention treatment

[0060]

[0061] 2.2) Integrated analysis of transcriptome and metabolome

[0062] Rice plants with consistent growth at the four-leaf and one-heart stage were selected and randomly divided into a treatment group with Lysinibacillus fusiformis biocontrol agent and a control group (liquid medium) for foliar spraying. Leaf mixed samples were collected 2 h after spraying (6 plants per group, 3 biological replicates). After quick freezing in liquid nitrogen, transcriptome sequencing (Illumina NovaSeq 6000 platform, PE150 mode) and non-targeted metabolome analysis (UPLC-QTOF-MS / MS system, positive and negative ion modes) were performed synchronously. Key regulatory factors were screened through differential gene expression analysis (DESeq2 software, |log2FC| ≥ 1 and FDR < 0.05) and multivariate statistical analysis of metabolites (OPLS-DA model, VIP > 1 and p < 0.05), and the resistance mechanism induced by Lysinibacillus fusiformis biocontrol agent was analyzed by combined enrichment of KEGG pathways (MetaboAnalyst 5.0 platform).

[0063] 2.3) Determination of JA content and detection of JA signaling pathway gene expression levels

[0064] Rice plants with consistent growth at the four-leaf and one-heart stage were selected and randomly divided into a treatment group with Lysinibacillus fusiformis biocontrol agent and a control group (liquid medium) for foliar spraying. Leaf mixed samples were collected 2 h after spraying (6 plants per group, 3 biological replicates). After quick freezing in liquid nitrogen, the leaves were ground thoroughly. Half of the samples were used to detect the jasmonic acid content by high performance liquid chromatography-mass spectrometry (LC-MS). After extracting RNA from the other half of the samples, cDNA templates were synthesized by reverse transcription, and the relative expression levels of JA signaling pathway genes were detected using a real-time quantitative PCR system. The relative expression levels of each gene were normalized using the rice internal reference gene UBQ by the 2^-ΔΔCt algorithm, and the relative expression levels of each gene were calculated. Three technical replicates were set for each reaction, and the statistical significance of differences between groups was analyzed after correcting the amplification efficiency using LinRegPCR software (t-test, p < 0.05).

[0065] 3) Experimental results

[0066] (1) Statistical results of incidence

[0067] Under different treatment methods, the statistical results of the incidence rate after inoculating each group with Magnaporthe oryzae are shown in Table 4. The disease pictures of representative rice leaves under the co-inoculation treatment, pre-induction treatment, and post-intervention treatment methods (as Figure 2 shown). The results show that foliar spraying of the biocontrol agent Lysinibacillus sphaericus can improve the resistance of rice to Magnaporthe oryzae.

[0068] Table 4

[0069]

[0070]

[0071] (2) Results of the combined transcriptome and metabolome analysis

[0072] Two hours after foliar spraying of the treatment group and the control group, the transcriptome and metabolome results of rice leaves are shown in Figure 3 . According to Figure 3 it can be seen that after spraying the biocontrol agent Lysinibacillus sphaericus, the transcriptome data shows that the expression levels of genes related to the jasmonic acid pathway in rice are significantly increased, and the metabolome data shows that the jasmonic acid content is significantly increased, indicating that spraying the biocontrol agent Lysinibacillus sphaericus enhances the synthesis of jasmonic acid in rice to improve the resistance to Magnaporthe oryzae, and can be applied to the prevention and control of rice blast.

[0073] (3) Results of the combined transcriptome and metabolome analysis

[0074] Two hours after foliar spraying of the treatment group and the control group, the results of detecting the relative expression levels of genes related to the jasmonic acid pathway in rice and the jasmonic acid content in rice are shown in Figure 4 . According to Figure 4 it can be seen that after spraying the biocontrol agent Lysinibacillus sphaericus, the expression levels of genes related to the jasmonic acid pathway in rice are significantly increased, indicating that spraying the biocontrol agent Lysinibacillus sphaericus enhances the synthesis of jasmonic acid in rice to improve the resistance to Magnaporthe oryzae, and can be applied to the prevention and control of rice blast.

[0075] The above experiments show that by optimizing the culture medium components and culture conditions (such as carbon source selection, pH regulation, etc.), the cell density and metabolic activity can be significantly improved. This bactericide shows a significant inhibitory effect on Magnaporthe oryzae in rice, and the test shows that the control effect can reach 45%-72%. The biocontrol agent and fermentation broth prepared from Lysinibacillus sphaericus have the advantages of good environmental friendliness, no phytotoxicity to humans, animals and crops, convenient application, etc., and are not likely to cause drug resistance in pests and diseases. This study provides an efficient solution for the biological prevention and control of rice blast, with both ecological and economic benefits.

[0076] Although the embodiments disclosed in the present invention are as described above, the content described is only an embodiment adopted for the convenience of understanding the present invention and is not intended to limit the present invention. Any person skilled in the art within the technical field to which the present invention pertains may make any modifications and changes in the form of implementation and details without departing from the spirit and scope disclosed in the present invention. However, the scope of patent protection of the present invention shall still be subject to the scope defined by the appended claims.

Claims

1. A Bacillus lysine in the form of a long shape, characterized in that, The strain is Lysinibacillus macroides C1, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 29, 2019, with the deposit number of CGMCC No. 18349.

2. A Lysinibacillus macroides bacterial agent prepared from the Lysinibacillus macroides strain described in claim 1.

3. The preparation method of the Bacillus lysine longus bacterium agent according to claim 2, characterized in that, The method includes: Step 1) Activate the Lysinibacillus macroides strain: Cultivate the strain of Lysinibacillus macroides through tryptic soy agar medium until yellow single colonies appear. Step 2) Cultivate the yellow single colony seeds: Inoculate the yellow single colonies into serum and conduct seed culture in Lysinibacillus macroides liquid medium until the logarithmic growth phase to obtain a bacterial solution. Step 3) Inoculate the cultured bacterial solution into the production device at an inoculation amount of 1% of the total volume of the Lysinibacillus macroides strain, and conduct production culture in Lysinibacillus macroides production medium until the logarithmic growth phase to prepare a biocontrol agent of Lysinibacillus macroides. Step 4) Centrifuge the obtained biocontrol agent of Lysinibacillus macroides, take the supernatant after centrifugation, and filter the supernatant to prepare a biocontrol fermentation broth of Lysinibacillus macroides.

4. The preparation method of the Bacillus lysiniformis agent according to claim 3, characterized in that, In step 1), the strain of Lysinibacillus macroides is cultivated through tryptic soy agar medium according to the double-layer culture method. The tryptic soy agar medium used includes 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 15 g of agar, 1000 mL of distilled water, and pH = 7.

5.

5. The preparation method of the Bacillus lysiniformis agent according to claim 3, wherein In step 2), the temperature for culturing the yellow single colony seeds is 32°C - 34°C, and the pH is 7.

0.

6. The preparation method of the Bacillus lysiniformis agent according to claim 3, characterized in that, In step 3), the culture temperature of the Lysinibacillus macroides production medium is 32°C - 34°C, and the pH is 7.

0.

7. The preparation method of the Bacillus lysiniformis agent according to claim 3, characterized in that, In step 4), the biocontrol agent of Lysinibacillus macroides is centrifuged by a high-speed centrifuge, where the centrifugation speed is 7000 - 7500 rpm, the centrifugation temperature is 4°C, and the centrifugation time is 12 mins.

8. Application of the Lysinibacillus macroides bacterial agent described in claim 2 in antagonizing Magnaporthe oryzae and controlling rice blast.

Images