Brevibacillus forts LJQJ21 and application thereof in prevention and treatment of plant diseases
By developing Brevibacillus fortis LJQJ21, this strain can effectively inhibit plant pathogenic oomycetes and promote plant growth, solve the environmental and health problems brought by chemical pesticides, and provide a non-toxic and environmentally friendly biopesticide resource.
Patent Information
- Application Number
- CN202411918910.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-06-13
AI Technical Summary
When preventing and controlling plant diseases, existing chemical pesticides have problems such as pesticide residues, enhanced resistance to pathogenic bacteria and beneficial biological killing. Moreover, the proportion of biological pesticides in my country is relatively low, and non-toxic and environmentally friendly biological pesticide resources are needed.
A Brevibacillus fortis LJQJ21 strain was developed. This strain has a strong ability to inhibit plant pathogenic oomycetes, can secrete antibacterial substance Edeine, and promote the growth of pepper seedlings at low concentrations.
This strain has a significant inhibitory effect on various plant pathogenic oomycetes, can effectively prevent and control fruit rot caused by Phytophthora capsia, and significantly promote the growth of pepper seedlings, and has potential bio-defense microbial resource value.
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Abstract
Description
Technical Field:
[0001] The present invention relates to a strain of Brevibacillus fortis LJQJ21 and its application in preventing and controlling plant oomycete diseases, belonging to the field of microbial pesticide research and development. Background Art:
[0002] There are diverse crop species in China, and they often suffer from diseases during production. Chemical pesticides are widely used for controlling various diseases due to their wide application range, rapid action effect, and convenient use. However, the long-term unreasonable and high-dose use of chemical pesticides has led to serious pesticide residues, causing poisoning of humans and livestock, enhancing the drug resistance of pathogenic bacteria, and killing beneficial organisms. Compared with chemical pesticides, using antagonistic microorganism (bacteria and fungi) preparations or biological pesticides containing antibacterial active ingredients to control plant diseases and promote the healthy growth of plants has become an important strategy for the green and sustainable development of agriculture. So far, the biological pesticides registered in China only account for about 5% of the total pesticides. Considering the sustainable development of agriculture and food safety, it is extremely urgent and necessary to develop non-toxic, environmentally friendly, and sustainable biological pesticides and their resource reserves.
[0003] The active ingredients in the microbial fungicides registered in China mainly include Bacillus spp., Pseudomonas, and Trichoderma. Among them, Bacillus spp. include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, and Paenibacillus polymyxa, etc. These products can be used to control diseases such as crop Fusarium diseases, crop gray mold, crop root rot, rice sheath blight, rice blast, rice bacterial leaf streak, tomato root-knot nematode, and cruciferous soft rot.
[0004] Brevibacillus is a genus of aerobic Gram-positive bacteria that are characterized by rapid growth, simple nutrition requirements, strong spore-forming ability, high resistance, and easy colonization in soil. They are widely distributed in nature and can also be found in soil, fresh water, seawater, and other habitats. In 1996, Shida et al. reclassified the strains originally belonging to the Bacillus brevis group based on 16S rRNA gene sequence homology and established the genus Brevibacillus. Common species mainly include B. brevis, B. laterosporus, B. formosus, etc. In 2019, Eric and Christopher further analyzed whole-genome homology and other factors, and divided the B. brevis population into three new species: B. fortis, B. porteri, and B. schisleri. Studies on Brevibacillus strains at home and abroad have shown that Brevibacillus strains have antibacterial, insecticidal (nematocidal), enzyme-producing, antitumor, plant growth-promoting, stress-resistant, and plant immunity-stimulating effects. Currently, research on Brevibacillus in agriculture mainly includes three aspects: 1) Research on the production of antibacterial and insecticidal substances by Brevibacillus. According to the biosynthetic pathway, the antibacterial substances produced by Brevibacillus are divided into ribosomally synthesized peptides and non-ribosomally synthesized peptides. Since most of the currently known antibacterial substances are non-ribosomally synthesized, this subgroup is further divided into short peptides and lipopeptides based on the presence of lipids. The main antibacterial substances produced by Brevibacillus are: Tostadin, Gramicidin AC, Edeine, Spergualin, Tyrocidines, BT peptide, BL-A60, Tauramamide, Brevibacillin B, Brevistin, etc. These substances have specific antibacterial activities against various bacteria, fungi, etc. For example, the BL-A60 substance produced by Brevibacillus laterosporus has a good inhibitory effect on Phytophthora infestans of crops; the Edeines produced by Brevibacillus fortis NRS-1210 have a good inhibitory effect on Fusarium oxysporum f. sp. cepae; the Edeine A and B produced by Brevibacillus brevis HNCS-1 have a good inhibitory effect on the tea tree pathogens Gloeosporium theaesinensis, Elsinoeleucospira, Phyllosticta theaefolia, Fusarium sp., and Cercospora theae.In addition, Brevibacillus laterosporus is a pathogen of various invertebrates and has insecticidal activity against various pests such as Lepidoptera, Diptera, and Coleoptera. Its insecticidal mechanism may be similar to that of the parasporal crystal protein of Bacillus thuringiensis. In addition, it may also involve chitinase, protease, lipopeptide, and spore surface protein, etc. 2) Related research on the production of elicitors by Brevibacillus to induce disease resistance in plants. Qiu Dewen et al. isolated a 116-amino acid protein elicitor PeBL1 from Brevibacillus laterosporus A60. PeBL1 can induce the up-regulation of defense-related genes PR-Ia, PR-5, PDF1.2, NPR1, and PAL in tobacco to varying degrees, indicating that PeBL1 induces systemic resistance in tobacco through the combined action of salicylic acid and jasmonic acid / ethylene signal transduction pathways. 3) Growth-promoting effects of Brevibacillus on plants. Li Yanfang et al. found that the 20-fold diluted sterile fermentation broth of Brevibacillus laterosporus strain B8 can promote the early germination, rooting, and growth of tomato seedlings; strain B8 can secrete indole-3-acetic acid (IAA) during cultivation, which is the main reason for its growth promotion. After inoculation using a partitioned petri dish, Brevibacillus laterosporus strain MG64 can significantly increase the shoot and root biomass of perennial ryegrass, increasing by about 2 times. Brevibacillus laterosporus AMCC100018 from the ocean can promote the growth of cucumbers, increasing cucumber yield by 9.15%, and Brevibacillus laterosporus AMCC100017 can significantly promote the growth of the apple rootstock Malus robusta Rehd.
[0005] The rice disease prevention and control research team of the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences has been committed to the exploration, reserve, research, development, demonstration, and popularization of biocontrol resources for plant diseases since the 1990s. They have successively developed biological pesticides "Wenquning" and "Yebaning" for controlling rice diseases, and the Bacillus amyloliquefaciens B1619 water dispersible granule for controlling Fusarium wilt and nematode diseases of solanaceous vegetables. The research team isolated the strain Brevibacillus fortis LJQJ21 from the rhizosphere soil of protected peppers in Jiangsu Province and registered it in the China General Microbiological Culture Collection Center on July 24, 2024, with the preservation number CGMCC No. 31435. Strain LJQJ21 has a strong inhibitory effect on the mycelial growth of various plant pathogenic oomycetes, mainly including Phytophthora capsici, Phytophthora infestans, Peronophythora litchi, and Pythium aphanidermatum, and also has a strong inhibitory effect on the germination of zoospores. High-performance liquid chromatography and mass spectrometry detection found that the fermentation supernatant of strain LJQJ21 in the culture medium BEPSY contains the pentapeptide antibiotic Edeine, and the microbial agent with the fermented bacterial liquid as the active ingredient has a good effect on preventing and controlling the infection of Phytophthora capsici on fruits. In addition, when using OD 600Soaking seeds with the cell suspension of B. fortis LJQJ21 at 0.1 has a significant promoting effect on pepper seedlings. In summary, Brevibacillus fortis LJQJ21, as a microbial pesticide resource strain with both antibacterial and growth-promoting functions, has great application value and development prospects in agricultural production. SUMMARY OF THE INVENTION:
[0006] The object of the present invention is to provide a strain of Brevibacillus fortis LJQJ21, which has a strong ability to inhibit plant pathogenic oomycetes and is a potential resource for the research and development of microbial pesticides. The genomic sequence of strain LJQJ21 has been uploaded to the NCBI nucleic acid database, and the GenBank accession number is CP176370. This strain has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is: CGMCC No. 31435.
[0007] The Brevibacillus fortis LJQJ21 described in the present invention has a strong effect of inhibiting plant pathogenic oomycetes and has a strong inhibitory effect on the mycelial growth of Phytophthora capsici, Phytophthora infestans, Peronophythora litchi, and Pythium aphanidermatum.
[0008] The Brevibacillus fortis LJQJ21 described in the present invention has the ability to secrete the antibacterial substance Edeine. Mass spectrometry detection shows that it can secrete Edeine A and B, and Edeine has the effect of inhibiting plant pathogenic oomycetes and the germination of their zoospores.
[0009] The Brevibacillus fortis LJQJ21 described in the present invention has a significant effect of promoting the growth of pepper seedlings when the cell concentration is OD 600 = 0.1.
[0010] The present invention provides a microbial agent, and its effective active ingredient is composed of the spore fermentation broth of Brevibacillus fortis LJQJ21 in the BEPSY medium. The main proportioning components are: the spore liquid of strain LJQJ21 (spore content 1×10 8 CFU / mL), preservative (sodium benzoate) 0.05 - 0.1%, pH 6.0 - 7.0.
[0011] The use of the microbial agent described in the present invention can be used to prevent and control the pepper fruit rot caused by Phytophthora capsici.
[0012] Brevibacillus fortis LJQJ21 of the present invention has a good inhibitory effect on plant pathogenic oomycetes, can secrete the antibacterial substance Edeine, has the effect of promoting the growth of pepper seedlings, can be used to control plant diseases caused by oomycetes, is a potential biocontrol microbial resource, and has the potential application value of being developed into a microbial agent.
[0013] The advantages of the present invention are as follows: Brevibacillus fortis LJQJ21 involved in the present invention has an inhibitory effect on 4 kinds of plant pathogenic oomycetes; the strain LJQJ21 can secrete antibacterial substances Edeine A and B, and Edeine can significantly inhibit the germination of oomycete zoospores; when the OD of the strain LJQJ21 600 = 0.1, it can promote the growth of pepper seedlings, and the biocontrol agent prepared from the fermented spore solution of its BEPSY medium can effectively control the occurrence of pepper fruit blight; Brevibacillus fortis LJQJ21 is a potential biocontrol microbial resource and has the value and potential of being developed into a microbial agent and applied in the field.
[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments. Description of the drawings:
[0015] Figure 1 Inhibitory effect of Brevibacillus fortis LJQJ21 on 4 kinds of plant pathogenic oomycetes
[0016] Figure 2 Mass spectrometry identification of pentapeptide antibacterial substance Edeine produced by Brevibacillus fortis LJQJ21
[0017] Figure 3 Inhibitory effect of antibacterial substance Edeine on the germination of Phytophthora capsici zoospores
[0018] Figure 4 Growth promotion effect of Brevibacillus fortis LJQJ2l bacterial suspension on pepper seedlings
[0019] Figure 5 Control effect of Brevibacillus fortis LJQJ21 biological agent on Phytophthora capsici of pepper fruits Specific implementation manners:
[0020] Example 1 Isolation of Brevibacillus fortis LJQJ21
[0021] In July 2019, Brevibacillus fortis LJQJ21 was isolated from the rhizosphere soil of greenhouse peppers in Sihong County, Jiangsu Province. The specific process is as follows: Weigh 10 g of pepper rhizosphere soil, add 90 mL of sterilized water, and dilute it by 10-fold gradient to obtain 10 -5 and 10 -6 dilution solutions; Take 330 μl and spread it on LB plates respectively. The plates are cultured in an incubator at 28 °C for 2 - 3 days. Pick single colonies and transfer them to LB plates to purify single cells, and store them in glycerol at -70 °C. The composition of LB medium is: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder, 15 - 20 g of agar, and 1000 mL of distilled water, pH 7.0.
[0022] This strain was named LJQJ21. Its colonies on LB plates are sticky and yellow. The vegetative cells of the strain are rod-shaped and produce spores. The optimal growth temperature is 28 - 35 °C. The whole genome of this strain has been sequenced, and the sequence has been deposited on the NCBI website. The GenBank accession number is: CP176370. Based on the whole genome sequence, this strain belongs to Brevibacillus fortis. The strain Brevibacillus fortis LJQJ21 was registered in the China General Microbiological Culture Collection Center on July 24, 2024, and the preservation number is CGMCC No. 31435.
[0023] Example 2 Inhibitory effect of Brevibacillus fortis LJQJ21 on phytopathogenic oomycetes
[0024] Tested pathogen strains: Phytophthora capsici, Phytophthora infestans, Peronophythora litchi, and Pythium aphanidermatum, preserved by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. The oomycetes are cultured on PDA medium (200 g of potato, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water, pH 6.5 - 7.0) at 28 °C for 5 days for standby.
[0025] Activate Brevibacillus fortis LJQJ21 on LB medium, then transfer it into 50 mL of LB culture solution and culture it with shaking (150 rpm / min) at 28 °C for 2 days for standby. Place a mycelial disc (5 mm in diameter) of phytopathogenic oomycetes in the middle of a blank PDA plate. Centered on the pathogen, add 5 μL of LJQJ21 bacterial solution at a distance of 2.5 cm from the pathogen. Each treatment is repeated in 3 plates. Set LB culture solution as the control. Incubate at a constant temperature of 26 °C. Evaluate the antibacterial effect of the antagonist on the pathogen when the colonies on the control plate are fully grown.
[0026] The test results are as Figure 1As shown in the figure, Brevibacillus fortis LJQJ21 has inhibitory effects on four plant pathogenic oomycetes, and the inhibitory effects of strain LJQJ21 on Phytophthora capsici, Peronophythora litchi, and Phytophthora infestans are relatively significant.
[0027] Example 3 Identification of the pentapeptide antibacterial substance Edeine produced by Brevibacillus fortis LJQJ21
[0028] Since Edeine is structurally similar to Tyramine (p-hydroxyphenethylamine), referring to the literature records of Johnson et al., using Tyramine as the standard substance, an Edeine quantitative standard curve was made by high performance liquid chromatography: accurately weigh and prepare a 1000 mg / L Tyramine standard working solution, and then dilute it successively to standard working solutions with concentrations of 200 mg / L, 100 mg / L, 50 mg / L, 20 mg / L, 10 mg / L, and 1 mg / L. Measure their absorption peak areas, make a standard curve and obtain the regression equation Y = 12.436X - 10.447, and the correlation coefficient R 2 = 0.9999.
[0029] Activate Brevibacillus fortis LJQJ21 on an LB plate, pick a single colony and inoculate it into an LB liquid medium for shaking culture for 16 h. Pipette 500 μL and inoculate it into 50 mL of BEPSY liquid fermentation medium, and shake culture at 28 °C and 180 r / min for more than 48 h. Centrifuge at 8000 r / min to remove the bacteria, and filter the supernatant through a 0.22 μm pore size filter membrane. Separate and determine Edeine by high performance liquid chromatography. Agilent XDB-C18, 5 nm 25 CM chromatographic column, and the chromatographic conditions are as follows: the mobile phase is methanol: 0.1% TFA water = 10:90, the flow rate is 1 mL / min, isocratic for 10 min, the column temperature is 35 °C, and the detection light length is 272 nm. Recover the Edeine chromatographic peak in high performance liquid chromatography, concentrate it and perform mass spectrometry molecular weight analysis. The results show that what is produced by Brevibacillus fortis LJQJ21 in the BEPSY fermentation medium are Edeine A and B ( Figure 2 ), and their molecular weights are 754.4587 and 796.4836 respectively. According to the peak area of the high performance liquid chromatography chart and based on the standard curve regression equation Y = 12.436X - 10.447, calculate the Edeine content of strain LJQJ21 in the BEPSY fermentation medium to be 473.64 mg / L. Composition of BEPSY medium: 5 g of yeast powder, 10 g of sucrose, 5 g of beef extract, 3 g of peptone, add distilled water to a constant volume of 1 L, pH 7.0.
[0030] References:
[0031] [1] Johnson E T, Bowman M J, Dunlap C A. Brevibacillus fortis NRS-1210 produces Edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f.sp. cepae[J]. Antonie van Leeuwenhoek, 2020, 113(7): 973-987. DOI: 10.1007 / s10482-020-01404-7.
[0032] Inhibitory effect of the antibacterial substance Edeine on the germination of zoospores of Phytophthora capsici
[0033] Activate Phytophthora capsici in V8 vegetable juice medium for 3 days. Take the edge mycelial block and place it in fresh V8 liquid medium, and continue to culture for 3 days. Then take it out and replace it with sterile water, and stimulate it at 4°C for 30 min. Observe the formation of zoospores under a microscope, collect a large number of zoospores, dilute them to a concentration of 1×10 7 zoospores / mL, add different concentrations of Edeine, and measure its inhibitory effect on the germination of zoospores.
[0034] Refer to Example 3, ferment Brevibacillus fortis LJQJ21 using medium BEPSY, separate and purify the antibacterial substance Edeine by high performance liquid chromatography, and quantify it through a standard curve. Measure the inhibitory effect of different concentrations (160, 80, 40, 20, 10 μg / mL) of Edeine on the germination of zoospores of Phytophthora capsici. The experimental results are as Figure 3 shown: After culturing at 26°C for 22 hours, when the concentration of Edenie is higher than 40 μg / mL, it has a significant inhibitory effect on zoospores.
[0035] Example 5 Growth promotion effect of Brevibacillus fortis LJQJ21 on pepper seedlings
[0036] Preparation of Brevibacillus fortis LJQJ21 bacterial suspension. Activate Brevibacillus fortis LJQJ21 on an LB plate from an -80°C refrigerator at 28°C; culture for 2 - 3 days to form single colonies. Inoculate the above single cells into an LB liquid medium, culture at 28°C and 180 rpm / min for 24 hours, centrifuge at 5000 rpm for 10 min, collect the thalli, wash the thalli twice with 10 mM magnesium sulfate, resuspend with sterile water, and then dilute the thalli concentration to OD600 of 2.0, 1.0, 0.1, and 0.01 respectively, and prepare them freshly as needed.
[0037] Select plump pepper seeds (variety: Sujiao No. 5), wash the seeds with sterile water, disinfect them with 70% alcohol for 3 - 5 min, and then wash the seeds with sterile water again. Soak the seeds in different concentrations of LJQJ21 bacterial suspension overnight, take them out and dry them on sterile filter paper, sow them into 1 / 2MS square plates, 5 plants per plate, with 3 replicates per treatment (3 plates), place them in an incubator at 28°C, with 12 h of light and 12 h of darkness, culture for 18 d, remove the ungerminated seeds, measure the fresh weight of the remaining pepper seedlings, and conduct statistical analysis. The experimental results are as Figure 4 shown: When the OD of the bacterial suspension is 0.1, LJQJ21 has a very significant growth-promoting effect on peppers, and the average fresh weight per plant can reach 42.6 mg, which is significantly increased by 91.03% compared to the control fresh weight.
[0038] Example 6 Preparation of Brevibacillus fortis LJQJ21 biological bactericide
[0039] A. Strain preparation and activation
[0040] Activate Brevibacillus fortis LJQJ21 on an LB plate from an -80°C refrigerator at 28°C; culture for 2 - 3 days to form single colonies.
[0041] B. Preparation of fermentation broth
[0042] Inoculate the above single strain into an LB liquid medium, culture at 28°C and 180 rpm / min for about 24 hours, and then transfer it again into a 300 mL / 1L Erlenmeyer flask BEPSY liquid fermentation medium at an inoculation amount of 3%, culture at 28°C and 180 rpm / min, and release it when more than 90% of the thalli in the fermentation broth form spores for standby.
[0043] C. Preparation of biological bactericide: Put the fermented spore broth into a storage tank, add 0.05 - 0.1% of the preservative sodium benzoate, stir evenly, and adjust the pH to 6.0 to form a biological control bactericide containing Brevibacillus fortis LJQJ21 (the spore content is about 1×108 CFU / mL).
[0044] Control effect of Brevibacillus fortis LJQJ21 bacterial agent on Phytophthora capsici of pepper fruits
[0045] The Brevibacillus fortis LJQJ21 biological bacterial agent was provided by Example 6.
[0046] Control effect test for Phytophthora capsici of pepper fruits: Select long pepper fruits with consistent growth and good health, disinfect their surfaces with alcohol swabs, and place them in experimental boxes respectively. Subsequently, inoculate Phytophthora capsici discs on the fruit surfaces. At the same time, conduct spray treatments with different concentrations (2-fold dilution and 5-fold dilution) of Brevibacillus fortis LJQJ21 bacterial agent. Each treatment has 3 replicates, and each replicate has 6 pepper fruits. After 7 days, investigate the rotting and disease occurrence at the oomycete inoculation sites on the fruits, record the diseased area, and calculate the control effect. Control effect (%) = [(diseased area of the control - diseased area of the treatment) / diseased area of the control] × 100%.
[0047] The experimental results are as Figure 5 shown: The average diseased area of the rotted parts of pepper fruits in the control treatment was 4.49 cm 2 , while after treatment with different concentrations of Brevibacillus fortis LJQJ21 bacterial agent, the development of disease spots on the rotted parts of pepper fruits was significantly reduced. Among them, the inhibition rate of the 2-fold dilution of the biocontrol bacterial agent on the diseased area of pepper fruits was 48.85%, and the inhibition rate of the 5-fold dilution of the biocontrol bacterial agent on the diseased area of pepper fruits was 40.99%.
Claims
1. A strain of Bacillus brevis having growth-promoting and antibacterial activities, characterized in that The bacterium is Brevibacillus fortisLJQJ21, which was registered with the China General Microbiological Culture Collection on July 24, 2024, with the collection number CGMCC No.31435.
2. Brevibacillus fortis LJQJ21 according to claim 1, characterized in that The fungus has a strong inhibitory effect on a variety of plant pathogenic oomycetes, mainly including pepper phytophthora, litchi downy mildew, infested phytophthora, and aphaniderma; in addition, the strain can secrete the pentapeptide antibacterial substances Edeine A and B, and Edeine can significantly inhibit the germination of pepper phytophthora zoospores.
3. A method for preparing a Brevibacillus fortis LJQJ21 preparation having a biocontrol function, characterized in that: 3.1 The liquid fermentation medium is BEPSY medium, and its formula is as follows: 5g yeast powder, 10g sucrose, 5g beef extract, 3g peptone, distilled water to 1L, pH 7.0; 3.2 Preparation of biocontrol agent: When 90% of the bacteria in the fermented liquid form spores, the fermented spore liquid is placed in a storage tank, and 0.05-0.1% of sodium benzoate, a preservative, is added, stirred evenly, and the pH is adjusted to 6.0-7.0 to form a biocontrol agent containing Brevibacillus fortis LJQJ21 (spore content is about 1×10 8 CFU / mL).
4. The biocontrol fungus preparation prepared according to claim 3 can be used to prevent and control oomycete diseases caused by Phytophthora capsici.