Long bacillus lysinoform agent, preparation method and application thereof

Abstract

The application discloses a bacillus licheniformis longus agent, a preparation method and application, and the agent is prepared from a bacillus licheniformis longus strain C1 and comprises the following steps: culturing the bacillus licheniformis longus strain through a tryptone soy agar culture medium until yellow single colonies appear; inoculating the yellow single colonies into serum, and performing seed culture on the bacillus licheniformis longus liquid culture medium until a logarithmic growth phase is reached to obtain a bacterial liquid; inoculating the cultured bacterial liquid into a production device according to the bacillus licheniformis longus strain, and performing production culture on the bacillus licheniformis longus production culture medium until a logarithmic growth phase is reached to prepare a bacillus licheniformis longus biocontrol agent; performing centrifugal treatment on the obtained bacillus licheniformis longus biocontrol agent, taking supernatant, and performing filtration on the supernatant to prepare a bacillus licheniformis longus biocontrol fermentation liquid. The bacillus licheniformis longus agent is applied to antagonizing pyricularia oryzae and preventing and controlling rice blast.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological pesticides, and particularly relates to a bacillus lysiniformis bacterial agent, a preparation method and application thereof. BACKGROUND

[0002] Microorganisms are widely distributed in nature, and have become a research hotspot due to their advantages such as non-toxicity, harmlessness, non-pollution, high efficiency and non-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 usually exists in soil, water and plant surfaces. Bacillus lysiniformis has attracted widespread attention due to its unique metabolic characteristics and potential industrial, agricultural and environmental applications. Bacillus lysiniformis has a significant inhibitory effect on various plant pathogenic microorganisms, fungi and nematodes. In addition, these bacteria can 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 biological medicine have gradually increased, and they have become an important part of modern sustainable agriculture and ecological system management.

[0003] Magnaporthe oryzae mainly damages the leaves, stems and panicles of rice. At the early stage of the disease, the lesion appears as water-soaked dark green or brown. As the disease progresses, the lesion gradually expands, forming obvious brown or black spots. The leaves of the affected parts may appear dry and yellow, which hinders photosynthesis and affects plant growth. The pathogen can invade downward through the stem, causing stem rot, and in severe cases, the plant may be prostrate. Rice blast also affects the development of rice panicles, causing neck disease, which increases the empty shell rate of rice. Finally, the diseased plants may appear wilting and dead, which seriously affects the yield and quality of rice. Rice blast is the most serious disease in rice production, causing a 10-20% reduction in yield in epidemic years, and a 40-50% or even 100% reduction in yield in severe cases.

[0004] The current methods for preventing and treating rice blast are mainly: (1) selecting resistant varieties: according to the epidemic characteristics of rice blast and the local disease occurrence, selecting rice varieties resistant to rice blast or planting rice varieties with strong resistance to disease, which can effectively reduce the occurrence of rice blast. (2) Reasonable fertilization: avoid partial or excessive application of nitrogen fertilizer, and apply appropriate amount of potassium fertilizer and phosphorus fertilizer during the tillering stage of rice to enhance the disease resistance of plants and reduce the probability of occurrence of rice blast. (3) Timely irrigation: adopt reasonable water management measures to avoid over-dry or over-wet in the rice field. Keep shallow water layer during tillering stage to jointing stage, and appropriately dry the field during heading stage to control the spread of the pathogen and create unfavorable conditions for the prevention and treatment of rice blast. (4) Field management: remove field disease residues and weeds to reduce the source of the pathogen. Reasonable planting density can ensure field ventilation and light transmission, reduce humidity, and inhibit the reproduction and spread of rice blast pathogen. (5) Chemical control: at the early stage of rice blast, timely use of chemicals for prevention and treatment can select 75% triazole wettable powder 800-1000 times liquid, 40% isoprothiolane emulsion 500-800 times liquid or 20% rice blast amide suspension for foliar spraying, which has good control effect on rice blast. The above methods for preventing and treating 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 high maintenance cost; field management is labor-intensive and requires continuous attention, which may not be fully implemented by some farmers; although chemical control has quick effect, long-term use may cause environmental pollution and pesticide residues. The biological pesticide prepared from plant metabolites is not easy to make rice blast resistant, but the plant metabolites have poor stability, short half-life and high cost, for example: the half-life of exogenous jasmonic acid is less than 6h and the cost is as high as $120 / kg. In addition, the rapid evolution of rice blast itself and the complex composition of physiological race lead to rapid loss of variety resistance and narrow resistance spectrum (such as the detection rate of triazole-resistant strains reaching 38%). Therefore, the invention of a green and harmless, efficient and durable microbial agent meets the maximum demand of sustainable agricultural development and environmental protection in China. SUMMARY

[0005] To solve the above technical problems, the purpose of the present application is to provide a long lysinibacillus agent, a preparation method and its application. The long lysinibacillus agent is prepared by activating, seed culture and production culture of long lysinibacillus strain.

[0006] The purpose of the present application is realized by the following technical solutions:

[0007] A long lysinibacillus, the strain is Lysinibacillus macroides C1, which has been preserved in China General Microbiological Culture Collection Center on July 29, 2019, and the preservation number is CGMCC No. 18349.

[0008] A bacillus formosanus inoculant prepared from a bacillus formosanus strain.

[0009] A preparation method of a bacillus formosanus inoculant, comprising:

[0010] Step 1) activating the bacillus formosanus strain: the bacillus formosanus strain is cultured in a tryptone soy agar culture medium until yellow single colonies appear.

[0011] Step 2) culturing the yellow single colony seed: the yellow single colony is inoculated into serum, and the bacillus formosanus liquid culture medium is used for seed culture to the logarithmic growth phase to obtain a bacterial liquid.

[0012] Step 3) inoculating the cultured bacterial liquid into a production device at a seeding amount of 1% of the total volume of the bacillus formosanus strain, and using the bacillus formosanus production culture medium for production culture to the logarithmic growth phase to prepare the bacillus formosanus biocontrol inoculant.

[0013] Step 4) centrifuging the obtained bacillus formosanus biocontrol inoculant, and taking the supernatant after centrifugation, and filtering the supernatant to obtain the bacillus formosanus biocontrol fermentation liquid.

[0014] In the step 1), the bacillus formosanus strain is cultured in a tryptone soy agar culture medium by double-layer culture method, and the tryptone soy agar culture medium comprises 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar, and 1000mL of distilled water, and the pH is 7.5.

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

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

[0017] In the step 4), the bacillus formosanus biocontrol inoculant is centrifuged by a high-speed centrifuge, wherein the centrifugal speed is 7000-7500rpm, the centrifugal temperature is 4°C, and the centrifugal time is 12mins.

[0018] The bacillus formosanus inoculant is applied to antagonize Magnaporthe oryzae and prevent and control rice blast.

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

[0020] The bacillus licheniformis provided by the application has the advantages of simple production process, short culture time, low cost, and the strain can be prepared into a rice blast prevention and control biocontrol agent and a biocontrol fermentation liquor through a simple, fast and low-cost operation method.

[0021] The biocontrol agent and the biocontrol fermentation liquor prepared from the bacillus licheniformis have the characteristics of green, harmless, high efficiency, long duration, simple and convenient application, and are not easy to cause drug resistance of diseases and pests. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 a 、 1b is an antagonistic effect diagram of the bacillus licheniformis agent;

[0023] Figure 2 is a picture of the disease of the rice leaf treated by the co-inoculation treatment, the pre-induction treatment and the post-intervention treatment of the bacillus licheniformis agent;

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

[0025] Figure 4 is a result of detecting the relative expression amount of the gene related to the jasmonic acid pathway of the rice and the content of the jasmonic acid of the rice after spraying the bacillus licheniformis agent. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below with examples and drawings.

[0027] Example 1

[0028] A bacillus licheniformis, the strain is bacillus licheniformis C1.

[0029] A bacillus licheniformis agent prepared from the bacillus licheniformis strain through activation, seed culture and production culture. The preparation method of the bacillus licheniformis agent comprises:

[0030] Step 1) activation:

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

[0032] Step 2) seed culture:

[0033] The yellow single colony cultured in step 1) is inoculated into a serum bottle to seed culture in a long lysine Bacillus subtilis liquid medium to obtain a bacterial liquid in a logarithmic growth phase, the temperature of the seed culture is 32-34°C, and the pH is 7.0;

[0034] Step 3) production culture:

[0035] The bacterial liquid obtained after the seed culture in step 2) is inoculated into a production bottle at a inoculation amount of 5% of the total volume of the long lysine Bacillus subtilis strain, and is subjected to production culture in a long lysine Bacillus subtilis production medium to obtain a long lysine Bacillus subtilis biocontrol agent in a logarithmic growth phase, the temperature of the production culture is 32-34°C, the pH is 7.0, and the long lysine Bacillus subtilis biocontrol agent is obtained in a logarithmic growth phase;

[0036] Step 4) centrifugation:

[0037] The biocontrol agent of the long lysine Bacillus subtilis obtained in step 3) is put into a high-speed centrifuge, the centrifugation speed is 7500 rpm, the centrifugation temperature is 4°C, the centrifugation time is 10 mins, the supernatant is collected after centrifugation, the supernatant is filtered by a bacterial filter, and then a long lysine Bacillus subtilis biocontrol fermentation liquid is obtained, and the obtained biocontrol fermentation liquid is stored at 4°C for standby use.

[0038] The strain provided in the embodiment is identified as long lysine Bacillus subtilis through physiological and biochemical identification and molecular biology identification, and has the following main biological characteristics: the strain forms yellow circular colonies on a double-layer solid plate medium after 4 days of culture, the colony edge is neat and smooth, and the colony diameter is 0.3 mm; the strain is yellow in a liquid culture medium, the optimal growth temperature is 32°C, and the pH is 7.

[0039] The application of the long lysine Bacillus subtilis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast is as follows:

[0040] Plate assay test of Magnaporthe oryzae: a Magnaporthe oryzae cake is inoculated in the center of a PDA (potato 200 g is cut into pieces, boiled for 20 minutes, filtered with eight layers of gauze to obtain juice, glucose 20 g, agar 15 g, and water is added to 1000 mL) plate, 10 μL of long lysine Bacillus subtilis C1 bacterial suspension (concentration 1×108 CFU / mL, prepared by culturing long lysine Bacillus subtilis liquid medium for 24 hours) is inoculated at a distance of 2 cm from the cake, sterile water is inoculated as a control, and the above steps are repeated four times, and the inhibition rate of the long lysine Bacillus subtilis C1 on Magnaporthe oryzae is 71.3% after 5 days of culture at 28°C, and the antagonistic effect picture is shown in Figure 1 a 、 1b .

[0041] Inhibition rate = (control colony diameter - inoculation antagonistic bacteria colony diameter) / control colony diameter x 100%. After inoculation of Bacillus licheniformis agent, the mycelium growth rate of Magnaporthe grisea was slower than the control, and the mycelium was sparse, and the spore amount was significantly reduced.

[0042] Example Two

[0043] 1) Experimental materials

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

[0045] Pathogenic bacteria: Magnaporthe grisea physiological race 110-2, spores of Magnaporthe grisea were added to Tween water (0.05% Tween 20 solution) to prepare a spore suspension for use.

[0046] 2) Experimental method

[0047] 2.1) Effect of Bacillus licheniformis biocontrol agent 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] Four-leaf one-core period rice seedlings with uniform growth were randomly divided into five groups, and the corresponding treatments were carried out according to the experimental design in Table 1. The number of blast spots was calculated 7 days after inoculation of the spore suspension. The untreated group (CK) was used as the negative control, and the relative incidence rate (%) was calculated according to the formula: Incidence rate = number of spots in the experimental group / number of spots in the untreated group x 100.

[0050] Table 1 for co-inoculation treatment

[0051]

[0052] (2) Pre-induction treatment

[0053] Four-leaf one-core period rice seedlings with uniform growth were randomly divided into five groups, and the corresponding treatments were carried out according to the experimental design in Table 1. The number of blast spots was calculated 7 days after inoculation of the spore suspension. The untreated group (CK) was used as the negative control, and the relative incidence rate (%) was calculated according to the formula: Incidence rate = number of spots in the experimental group / number of spots in the untreated group x 100.

[0054] Table 2 for pre-induction treatment

[0055]

[0056]

[0057] (3) Post-intervention treatment

[0058] Take four-leaf one heart period of rice seedlings with consistent growth, randomly divided into 5 groups, according to the experimental design of table 1 to carry out corresponding treatment. 7 days after inoculation of spore suspension, the number of rice blast spots was calculated. The untreated group (CK) was used as the negative control, and the relative incidence rate (%) was calculated according to the formula: incidence rate = number of spots in the experimental group / number of spots in the untreated group x 100.

[0059] Table 3 post-intervention treatment

[0060]

[0061] 2.2) Combined analysis of transcriptome and metabolome

[0062] Select four-leaf one heart rice plants with consistent growth, randomly divided into long lysine bacillus biocontrol agent treatment group and control group (liquid medium) for foliar spraying, respectively, 2h after spraying, collect mixed samples of leaves (6 plants in each group, 3 biological replicates), freeze in liquid nitrogen, and simultaneously perform transcriptome sequencing (Illumina NovaSeq 6000 platform, PE150 mode) and non-targeted metabolome analysis (UPLC-QTOF-MS / MS system, positive and negative ion modes), screen key regulatory factors through differential gene expression analysis (DESeq2 software, |log2FC|≥1 and FDR<0.05) and metabolite multivariate statistical analysis (OPLS-DA model, VIP>1 and p<0.05), and analyze the resistance mechanism induced by long lysine bacillus biocontrol agent combined with KEGG pathway joint enrichment (MetaboAnalyst 5.0 platform).

[0063] 2.3) JA content determination and JA signaling pathway gene expression detection

[0064] Select four-leaf one heart rice plants with consistent growth, randomly divided into long lysine bacillus biocontrol agent treatment group and control group (liquid medium) for foliar spraying, respectively, 2h after spraying, collect mixed samples of leaves (6 plants in each group, 3 biological replicates). After freezing the leaves in liquid nitrogen, grind them thoroughly. Half of the samples are detected for jasmonic acid content by high performance liquid chromatography-mass spectrometry (LC-MS). Half of the samples are extracted for RNA, and then reverse transcription is performed to obtain synthetic cDNA template. The relative expression of JA signaling pathway genes is detected by real-time quantitative PCR system. The expression of rice reference gene UBQ is standardized by 2^-ΔΔCt algorithm, and the relative expression of each gene is calculated. Each reaction is set with 3 technical replicates, and the data is corrected for amplification efficiency by LinRegPCR software, and the significance of difference between groups is statistically analyzed (t-test, p<0.05).

[0065] 3) Experimental results

[0066] (1) Incidence rate statistical results

[0067] Table 4 shows the statistical results of the incidence rate of rice blast fungus in each group after inoculation with different treatments. Representative images of diseased rice leaves under the co-inoculation, pre-induction, and post-intervention treatments are also included. Figure 2 (As shown in the image). The results showed that foliar spraying of *Bacillus longiformis* biocontrol agent can improve rice resistance to rice blast.

[0068] Table 4

[0069]

[0070]

[0071] (2) Results of combined transcriptomic and metabolomic analysis

[0072] Two hours after foliar spraying, the transcriptome and metabolome results of rice leaves in the treatment and control groups are shown in the figure. Figure 3 .according to Figure 3 It can be seen that after spraying with the long-shaped lysine-containing Bacillus biocontrol agent, transcriptome data showed that the expression level of rice jasmonic acid pathway-related genes was significantly increased, and metabolome data showed that the jasmonic acid content was significantly increased. This indicates that spraying with the long-shaped lysine-containing Bacillus biocontrol agent enhances rice jasmonic acid synthesis to improve rice blast resistance and can be applied to the prevention and control of rice blast.

[0073] (3) Results of combined transcriptomic and metabolomic analysis

[0074] Two hours after foliar spraying, the relative expression levels of genes related to the jasmonic acid pathway and the jasmonic acid content in rice were measured in both the treatment and control groups. The results are as follows: Figure 4 As shown. According to Figure 4 It can be seen that after spraying the biocontrol agent of Bacillus longiformis, the expression level of genes related to the jasmonic acid pathway in rice increased significantly, indicating that spraying the biocontrol agent of Bacillus longiformis enhances the synthesis of jasmonic acid in rice to improve the resistance to rice blast, and can be applied to the prevention and control of rice blast.

[0075] The above experiments demonstrate that optimizing the culture medium composition and culture conditions (such as carbon source selection and pH adjustment) can significantly improve cell density and metabolic activity. This inoculant exhibits a significant inhibitory effect on *Magnaporthe oryzae*, the causal agent of rice blast, with control efficacy ranging from 45% to 72%. The biocontrol agent and fermentation broth prepared from *Bacillus longiformis* possess advantages such as good environmental friendliness, no phytotoxicity to humans, animals, and crops, and ease of application, and are unlikely to induce pesticide resistance in pests and diseases. This study provides an efficient solution for the biological control of rice blast, offering both ecological and economic benefits.

[0076] Although the embodiments of the present application have been disclosed with reference to the above embodiments, the above description is merely used to understand the present application and is not used to limit the present application. Any person skilled in the art, without departing from the spirit and scope of the present application, can make any modification and change in the form and details of the embodiments, but the patent protection scope of the present application should be subject to the scope defined by the appended claims.

Claims

1. The application of long-shaped Bacillus lysinoformis inoculant in antagonizing Magnaporthe oryzae and preventing and controlling rice blast, characterized in that, The strain is Lysinibacillus macrolides C1, which has been preserved in the China General Microbiological Culture Collection Center on July 29, 2019, and the preservation number is CGMCC No. 18349.

2. The application of the long-shaped Bacillus lysinoformis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast according to claim 1, characterized in that, The preparation method of the Lysinibacillus macrolides bacterial agent comprises the following steps: Step 1) activating the Lysinibacillus macrolides strain: the strain of the Lysinibacillus macrolides strain is cultured by a tryptone soy agar culture medium until yellow single colonies appear; Step 2) culturing the yellow single colony seed: the yellow single colony is inoculated into serum, and the Lysinibacillus macrolides liquid culture medium is used for seed culture to the logarithmic growth phase to obtain a bacterial liquid; Step 3) inoculating the cultured bacterial liquid into a production device at a inoculation amount of 1% of the total volume of the Lysinibacillus macrolides strain, and using the Lysinibacillus macrolides production culture medium for production culture to the logarithmic growth phase to prepare the Lysinibacillus macrolides biocontrol agent; Step 4) centrifuging the obtained Lysinibacillus macrolides biocontrol agent, and taking the supernatant after centrifugation, and filtering the supernatant to obtain the Lysinibacillus macrolides biocontrol fermentation liquid.

3. The application of the long-shaped Bacillus lysinoformis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast according to claim 2, characterized in that, In step 1), the strain of the Lysinibacillus macrolides strain is cultured by a tryptone soy agar culture medium by a double-layer culture method, wherein the tryptone soy agar culture medium used comprises tryptone 15 g, soybean peptone 5 g, sodium chloride 5 g, agar 15 g, distilled water 1000 mL, and pH = 7.

5.

4. The application of the long-shaped Bacillus lysinoformis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast according to claim 2, characterized in that, In step 2), the temperature for seed culture of the yellow single colony is 32-34°C, and the pH is 7.

0.

5. The application of the long-shaped Bacillus lysinoformis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast according to claim 2, characterized in that, In step 3), the culture temperature of the Lysinibacillus macrolides production culture medium is 32-34°C, and the pH is 7.

0.

6. The application of the long-shaped Bacillus lysinoformis agent in antagonizing Magnaporthe oryzae and preventing and controlling rice blast according to claim 2, characterized in that, In step 4), the Lysinibacillus macrolides biocontrol agent is treated by a high-speed centrifuge, wherein the centrifugal speed is 7000-7500 rpm, the centrifugal temperature is 4°C, and the centrifugal time is 12 mins.

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