Bacillus velezensis capable of preventing and treating peanut southern blight and application of bacillus velezensis
By screening out the fermentation broth of Bacillus Bacillus strain ZS5-1 from the highly pathogenic white silkworms and applying it to the crop planting soil, the problem of chemical pesticide pollution in the prevention and control of peanut white silkworms was solved, and effective prevention and control of the highly pathogenic white silkworms was achieved, and the health and yield of peanut crops were significantly improved.
Patent Information
- Application Number
- CN202411817867.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-14
AI Technical Summary
The prevention and control measures for peanut white silk disease mainly rely on chemical pesticides, which lead to environmental pollution and pesticide residue problems. No peanut materials that are effective against high pathogenic white silk disease bacteria were found.
Many biocontrol strains were screened from the highly pathogenic white silkworm strain ZY2, especially the Bacillus Bacillus strain ZS5-1, and the fermentation broth was applied to the crop planting soil to achieve the prevention and treatment of peanut white silkworm disease.
In potted and field experiments, Bacillus vellus strain ZS5-1 significantly inhibited the white schizophrenia bacteria, with the prevention and treatment effects reaching 60.72% and 33.73%. Microscopic observations showed that this strain could cause the white schizophrenia bacteria to shorten the mycelium, twist and tangle, constriction and fracture, and protoplasm digestion.
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Figure CN119614434A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of crop disease prevention and control, and more particularly to a Velez subtilis bacillus capable of preventing and controlling peanut white rot and application thereof. Background Art
[0002] Peanut is widely planted in my country and is one of the important oil crops in my country. In recent years, peanut white rot has become the main soil-borne disease of peanuts, occurring in most peanut production areas, with a trend of increasing severity year by year, causing significant economic losses.
[0003] The pathogen of peanut white rot is a necrotrophic pathogenic fungus. The asexual generation is Sclerotium rolfsii Sacc., which belongs to the asexual spore-forming filamentous fungi; the sexual generation is Agroathelia rolfsii Cruz i.
[0004] Studies have found that there is pathogenicity differentiation in peanut Sclerotium rolfsii. According to the disease index of the affected peanuts, the strains can be divided into high, medium and weak pathogenicity types. Highly pathogenic strains are distributed in many production areas in my country. Recent studies have found that highly pathogenic Sclerotium rolfsii can cause materials that are resistant to medium pathogenic strains to become susceptible to the disease. At present, no peanut materials resistant to highly pathogenic Sclerotium rolfsii have been found.
[0005] At present, the prevention and control measures for peanut white rot are still mainly chemical control. The pesticides registered on the China Pesticide Information Network (http: / / www.chinatesticide.org.cn / ) for the prevention and control of peanut white rot mainly include thiophanate-methyl, azoxystrobin, tebuconazole and thiram, etc. Long-term and large-scale use of these pesticides will cause environmental pollution and pesticide residue problems, threatening the health of humans and animals.
[0006] In order to ensure the sustainable and stable development of the peanut industry, it is necessary to explore biological control strains against white rot. However, there is no research on biological control strains for highly pathogenic strains. Summary of the invention
[0007] Starting with the highly pathogenic Sclerotium rolfsii strain ZY2, our team conducted screening and efficacy research on biocontrol bacteria, and screened out several effective biocontrol bacteria.
[0008] Based on the above research, the present invention provides a Bacillus velezensis that can prevent and control peanut white rot, characterized in that it was deposited in the China Center for Type Culture Collection (CCTCC) on October 16, 2024, with the accession number: CCTCC M 20242222.
[0009] The present invention also provides application of the above-mentioned Bacillus Velezii in preventing and controlling peanut white rot.
[0010] The present invention also provides a method for preventing and controlling white rot of peanut, comprising the step of applying the fermentation liquid of the Velez bacillus to the soil where crops are planted.
[0011] In a specific embodiment, the method comprises the following steps:
[0012] S1: culturing the Bacillus Velez subtilis to prepare a fermentation broth or a sterile fermentation broth of the Bacillus Velez subtilis;
[0013] S2: applying the fermentation liquid of Bacillus Velez subtilis to the soil where the crops are planted.
[0014] In a specific embodiment, in S1, the Bacillus velez is cultured in LB medium.
[0015] In a specific embodiment, in S2, the crops are irrigated with the fermentation liquid of Bacillus Velez subtilis.
[0016] In a specific embodiment, the crop is peanut.
[0017] The strain ZS5-1 of the present invention has a control effect of 60.72% on ZY2 in potted experiments, and the field test results show that the control effect reaches 33.73%. Microscopic observation found that ZS5-1 can cause ZY2 hyphae to shorten, twist and entangle, constrict and break, and protoplasm to dissolve. Culture medium tests found that ZS5-1 can produce protease, amylase, pectinase, cellulase and xylanase, but no chitinase. The strain has good biocontrol potential for highly pathogenic white rot fungi, and provides a good biocontrol resource for preventing and controlling peanut white rot.
[0018] Biomaterial Deposit
[0019] The Bacillus velezensis of the present invention was deposited in the China Type Culture Collection (CCTCC) of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on October 16, 2024, with the deposit number: CCTCC M 20242222. The taxonomic name is Bacillus velezensis. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 (A) and Gram staining photos of strain ZS5-1.
[0021] Figure 2 This is the cluster analysis diagram of 16S rDNA of strain ZS5-1.
[0022] Figure 3This is the plate inhibitory effect of strain ZS5-1 on Saccharomyces rolfsii ZY2.
[0023] Figure 4 The morphological effects of strain ZS5-1 on S. rolfsii. A and C are the optical microscopic photos and cell electron microscopic photos of the hyphae of the control S. rolfsii ZY2, respectively; B and D are the optical microscopic photos and cell electron microscopic photos of the hyphae of S. rolfsii ZY2 confronting ZS5-1, respectively.
[0024] Figure 5 This is a study on the enzyme production of strain ZS5-1.
[0025] Figure 6 This is a pot experiment for controlling Sclerotium rolfsii ZY2. A is a photo of a representative plant of the control group; B is a photo of a representative plant treated with the fermentation liquid of strain ZS5-1; and C is the disease statistics of each group.
[0026] Figure 7 This is a field test of strain ZS5-1 against Sclerotium rolfsii ZY2. A is a representative photo of the control group; B is a representative field photo of root irrigation with the fermentation liquid of strain ZS5-1 at OD600=1; B is a representative field photo of root irrigation with the fermentation liquid of strain ZS5-1 at OD600=2. DETAILED DESCRIPTION
[0027] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0028] 1. Identification and preservation of Bacillus Velez
[0029] The strain ZS5-1 was cultured on LB solid medium for 48 h. The colony morphology was as follows Figure 1 As shown in A, it presents milky white round colonies with slightly wrinkled surface and irregular growth around them. Gram staining is positive ( Figure 1 B).
[0030] The results of physiological and biochemical tests showed that the strain ZS5-1 was positive in Gram staining, VP test, and catalase reaction (Table 1), which was consistent with the biochemical metabolic characteristics of Bacillus.
[0031] Table 1 Some physiological and biological characteristics of strain ZS5-1
[0032]
[0033] Note: + indicates positive, - indicates negative
[0034] The genomic DNA of the strain was extracted, 16S rDNA was amplified, and sequenced. The sequence was uploaded to NCBI's GENBANK with the access number SRR31534375. The sequence was clustered with the existing sequences, and the results were as follows Figure 2 As shown, the ZS5-1 strain and Bacillus velezensis CR-502(T) clustered on the same branch.
[0035] Combined with morphological characteristics, the strain was identified as Bacillus velezensis.
[0036] The above strain was deposited in the China Type Culture Collection (CCTCC) of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on October 16, 2024, with the accession number: CCTCC M20242222. The taxonomic name is Bacillus velezensis.
[0037] 2. Sclerotium rolfsii in confrontation with ZS5-1
[0038] The bacterial strain that has an inhibitory effect on S. rolfsii ZY2 was inoculated into 10 mL of LB liquid culture medium placed in a 50 mL sterile culture tube and incubated in a shaker at 30°C and 200 r·min. -1 After shaking culture for 24 h, the OD of the bacterial solution was measured using the NanodropOne ultra-microbiometer (ThermoFisher company, USA). 600 Value, diluted to OD 600 The value is 1. 20 μL of the diluted bacterial culture solution was inoculated on the PDA culture medium and inoculated at the four corners of the ZY2 mycelium block 3 cm away from the center of the culture medium. The culture medium inoculated only with ZY2 mycelium block but not with bacteria was used as the control and cultured in the dark at 30°C.
[0039] The diameter of ZY2 colonies on the confrontation medium was measured, as Figure 3 As shown, ZS5-1 can significantly inhibit the colony size of S. rolfsii ZY2.
[0040] After ZS5-1 and S. rolfsii ZY2 were cultured in confrontation for 5 days, the difference was compared with that of ZY2 without confrontation culture ( Figure 4 A), observation of the ZY2 hyphae in confrontation culture under an inverted fluorescence microscope showed that the mycelium had increased septa, shortened compartments, and deformed branches; some were deformed, twisted and tangled, and the top of the mycelium was swollen. At the same time, the protoplasm was dissolved and overflowed, resulting in the loss of vitality, resulting in the appearance of empty shell mycelium ( Figure 4 B).
[0041] Transmission electron microscopy observations revealed that the biocontrol bacteria had a significant effect on the mycelial morphology of S. rolfsii. Figure 4 As shown in C and D, the cell wall of Sclerotium is thickened, the protoplasm is shrunk, and the organelles are unevenly distributed.
[0042] 3. Determination of enzyme production capacity of ZS5-1
[0043] ZS5-1 was inoculated on different enzyme assay media and cultured. In the assay media for protease, amylase, pectinase and xylanase, the strain was able to decompose these substances and produce a large transparent circle at the inoculation site ( Figure 5 -ABCF); while the transparent zone produced on the cellulase assay medium is smaller ( Figure 5 -E); only a faint transparent circle on the chitinase assay medium ( Figure 5 -D); ZS5-1 can produce protease, amylase, pectinase, xylanase and cellulase, but cannot produce chitinase.
[0044] 4. Pot Plant Test
[0045] Mix nutrient soil and vermiculite in a ratio of 2:1, put them into non-woven bags, seal them and sterilize them at 121℃ for 2h, cool them and place them in a cool and ventilated place for later use. Soak the Zhonghua 21 peanut seeds in 2.5% sodium hypochlorite solution for 2min, wash them with distilled water 4-5 times, soak them in 28℃ sterile water for 4h, germinate them in a 28℃ constant temperature incubator for two days, transplant them into sterile soil, and culture them in a growth room at 28℃ with 16h light and 8h dark.
[0046] The selected bacterial strains were inoculated into LB culture medium and cultured at 30°C and 200 r·min -1 Incubate with shaking for 24 h and adjust the bacterial concentration to OD 600 The value is 1, and the bacterial solution is poured around the roots of peanuts after 4 weeks of growth at 50mL / plant, and the peanut plants poured with LB culture solution are used as controls. After 7 days of root irrigation, fresh oat grains carrying white rot fungus are inoculated at the base of the plant stems, 2 grains / plant. After inoculation, moisturize and culture, and conduct a disease severity survey one week later to calculate the disease index. The severity levels of peanut plants inoculated with white rot fungus are as follows: Level 0: Plants are asymptomatic; Level 1: Only lesions are produced at the base of the stem; Level 2: There are lesions at the base of the stem, and 1 / 3 of the plants show systemic symptoms (wilt, death, wilting, etc.); Level 3: 2 / 3 of the plants show systemic symptoms (wilt, death, wilting, etc.); Level 4: The whole plant dies or wilts.
[0047] Disease index = {[∑(disease-prone plants at each level × corresponding level value)] / (total number of plants surveyed × highest disease level)}×100
[0048] Control effect (%) = (treatment disease index - control disease index) / control disease index × 100% The results are as follows Figure 6As shown, the plants inoculated with only the S. rolfsii ZY2 strain had serious disease, with a disease index of 77.78; the peanut plants treated with the fermentation liquid of the ZS5-1 strain had a very light disease, with most plants having disease levels 1 and 2, with a disease index of 30.56, and a high control effect of 60.72%.
[0049] 5. Field control test
[0050] Peanut variety Zhonghua 21 was sown on May 7, 2024 at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Xinzhou District, Wuhan City, Hubei Province. 75 days after sowing, the ZS5-1 bacterial solution was sprayed on the base of the plant stems. 5 days later, oat grains carrying ZY2 were inoculated at the base of the stems, 10 grains / plant. After inoculation, the field was sprayed with water for 3 days to keep moist. The disease severity was investigated 6 days after inoculation. The experiment set up 2 treatments and 1 control, including treatment A: ZS5-1 bacterial solution sprayed on the base of the stems (OD 600 =1), B: Spray the stem base with ZS5-1 solution (OD 600 =2), C: sterile LB medium (control), each group set up 3 replicates, each cell 7.5m 2 About 100 peanut plants.
[0051] The results are as follows Figure 7 As shown, the bacterial strain ZS5-1 was sprayed on the base of the stems of peanut plants in the field and then inoculated with the peanut white rot fungus ZY2. The disease indexes of the peanut plants in treatment A and treatment B were 35.62 and 33.89, respectively, and the disease severity of the control treatment sprayed with LB culture medium was 51.14; the control effects were 30.35% and 33.73%, respectively, indicating that the strain ZS5-1 has a certain control effect on the highly pathogenic strain ZY2 of peanut white rot in the field.
[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A Bacillus velezensis ZS5-1 for preventing and treating peanut white rot, characterized in that: It was deposited in the China Center for Type Culture Collection (CCTCC) on October 16, 2024, with the accession number: CCTCC M20242222.
2. Use of the Bacillus Velezii described in claim 1 in preventing and treating peanut white rot.
3. A method for preventing and treating peanut white rot, characterized in that: The method comprises the step of applying the fermentation liquid of Bacillus Velez subtilis according to claim 1 to soil for planting crops.
4. The method according to claim 3, characterized in that The following steps are involved: S1: culturing the Bacillus Velez subtilis to prepare a fermentation broth or a sterile fermentation broth of the Bacillus Velez subtilis; S2: applying the Bacillus Velez fermentation liquid to the crop planting soil.
5. The method according to claim 4, characterized in that In S1, the Bacillus velez was cultured in LB medium.
6. The method according to claim 4, characterized in that In S2, the crops are irrigated with the fermented liquid of Bacillus Velez subtilis.
7. The method according to any one of claims 3 to 6, characterized in that: The crop is peanut.