Streptomyces griseogriseus YZ23120524 and application thereof in prevention and treatment of ginger blast
By using Streptocytica spores YZ23120524 isolated from ginkgo leaves as antibacterial agent for ginger plague, the toxic and side effects of unsatisfactory prevention and control of ginger plague in the prior art were solved, and efficient and safe biological control effects were achieved.
Patent Information
- Application Number
- CN202311816979.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-27
- Publication Date
- 2025-08-08
AI Technical Summary
The existing technology has not been effective in preventing and treating ginger plague. Chemical control has toxic side effects and residual problems, and biological control methods have not yet been effectively applied.
Streptomyces spores YZ23120524 was used, which was isolated from ginkgo leaves. It was used as an antibacterial agent for ginger plague. It was used by soaking seedlings and watering the roots during the seedling stage. The fermentation broth has a significant prevention and treatment effect on ginger plague, and has no toxic side effects and residues.
Streptomyces spores YZ23120524 has significant prevention and treatment effect on ginger plague, high safety, easy to be produced in industrialized, and has better prevention and treatment effect than chemical agents, reducing the risk of environmental pollution.
Smart Images
Figure CN120442433A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of ginger wilt prevention and treatment, and in particular to a Streptomyces sporangiophorus YZ23120524 and an application thereof in preventing and treating ginger wilt. Background Art
[0002] Ginger wilt (also known as ginger bacterial wilt), a major soilborne disease caused by Ralstonia solanacearum, has long been a major obstacle to ginger production. According to surveys, ginger-producing areas often suffer annual losses of 20% to 30% due to ginger bacterial wilt, with losses exceeding 80% in severe cases. Currently, the prevention and control of ginger bacterial wilt relies primarily on chemical control. Disease-resistant varieties and crop rotation and intercropping are also commonly used in production, but the results are unsatisfactory. Biological control, due to its safety and effectiveness, has become an important alternative to chemical control. In recent years, the use of endophytic bacteria, fungi, or actinomycetes has attracted widespread attention in the field of plant pathology and has become a hot topic in biological control research. Ginkgo endophytes, as an important biocontrol resource, have attracted widespread attention. Ginkgo biloba is a rare and widely cultivated tree species worldwide that rarely, if ever, becomes infected with pests and diseases during its growth. Research has shown that ginkgo extracts have a moderate inhibitory effect against numerous pests and diseases, including peach aphids, cabbage worms, thrips, pear scab, peach brown rot, tomato bacterial wilt, and eggplant Verticillium wilt. Based on the endosymbiotic theory proposed by Margulis et al., it is speculated that endophytes that evolved with ginkgo must also possess unique properties in controlling certain plant pests and diseases. Therefore, isolating strains from ginkgo that can control ginger wilt disease offers a new approach to combating the disease and improving ginger production efficiency. Summary of the Invention
[0003] The present invention provides a strain of Streptomyces sporeblight YZ23120524 and its use in preventing and controlling ginger wilt. The strain is isolated from ginkgo biloba leaves and exhibits a significant inhibitory effect on bacterial wilt pathogens, providing significant control effects on ginger wilt. Compared to chemical agents, the strain is non-toxic, non-residual, environmentally safe, and readily applicable.
[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0005] The present invention provides a Streptomyces pororaveus YZ23120524, which is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit date of December 8, 2023, and a deposit number of CCTCCNO: M20232508.
[0006] Preferably, the 16S rDNA sequence of the Streptomyces sporangiophora YZ23120524 is shown as SEQ ID NO.3.
[0007] The present invention also provides an application of Streptomyces ulmoides YZ23120524 in preventing and treating ginger blast.
[0008] The present invention also provides a ginger blast inhibitor, which is prepared by fermenting the above-mentioned Streptomyces sporangiophorus YZ23120524.
[0009] The present invention also provides a use of the ginger wilt inhibitor in preventing and treating ginger wilt.
[0010] Preferably, the ginger blast inhibitor is diluted 150 to 300 times when used.
[0011] Preferably, the ginger blast inhibitor is used by soaking seeds and watering them at the seedling stage.
[0012] Preferably, the seed soaking time is 0.5 to 2 hours.
[0013] Preferably, the irrigation period is 45 to 50 days after the ginger seedlings emerge, and the roots are watered.
[0014] Preferably, the amount of the watering is 400-600 mL / plant of the diluted ginger wilt inhibitor.
[0015] The invention provides a Streptomyces sporangiophorus YZ23120524 and application thereof in preventing and treating ginger blast.
[0016] The present invention's Streptomyces spore pale gray YZ23120524 is a new Streptomyces strain isolated and screened from ginkgo leaves, which has good inhibitory and preventive effects on bacterial wilt and ginger wilt. The present invention has the following beneficial effects: (1) The present invention's Streptomyces spore pale gray YZ23120524 has a strong inhibitory effect on ginger bacterial wilt on a plate, and its fermentation liquid has a good preventive and therapeutic effect on ginger wilt, and is more advantageous than the positive control chemical agent 72% agricultural streptomycin sulfate in preventing and treating ginger wilt. (2) The present invention's Streptomyces spore pale gray YZ23120524 is isolated from ginkgo leaves. Compared with chemical pesticides, it has no toxic side effects on ginger and soil ecology, no residue, good environmental safety, good development prospects and application potential, and is especially important for the prevention and treatment of ginger wilt. (3) In the separation method described in the present invention, the strain culture conditions are simple, easy to preserve, easy to industrialize and have good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The results of biocontrol bacteria screening in Example 1 are shown, where the arrows indicate the inhibition zones formed by strain YZ23120524.
[0018] Figure 2 This is a characteristic diagram of the colony morphology of strain YZ23120524 in Example 1 on a plate.
[0019] Figure 3 This is the agarose gel electrophoresis diagram of the PCR product of the strain YZ23120524 genome in Example 1, wherein lane M is a marker and lane 2 is the PCR reaction product of the strain YZ03102604 genome.
[0020] Preservation Instructions
[0021] Streptomyces pororaveus YZ23120524 is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The deposit date is December 8, 2023, and the deposit number is CCTCCNO: M 20232508. DETAILED DESCRIPTION
[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0023] The raw materials used in the present invention come from the following sources:
[0024] Gao's medium No. 1 (purchased from Qingdao Haibo Biotechnology Co., Ltd.); Ralstonia solanacearum was kindly provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences; ginger variety was Jiangyong Fragrant Ginger, a conventionally cultivated variety of the Shangjiangwei Fragrant Ginger Farmers' Professional Cooperative in Jiangyong County. Other biochemical reagents are listed in the table below:
[0025] Table 1 Sources of biochemical reagents
[0026]
[0027] Culture medium: Ralstonia solanacearum was activated and cultured using LB medium; the isolation medium for biocontrol bacteria was Gao's solid medium No. 1 (purchased from Qingdao Haibo Biotechnology Co., Ltd.); the medium for determining the antimicrobial active substances of biocontrol bacteria was Gao's liquid medium No. 1 (purchased from Qingdao Haibo Biotechnology Co., Ltd.).
[0028] Example 1
[0029] This example provides a strain of Streptomyces pororaveus YZ23120524, and the specific screening process is as follows:
[0030] 1. Ginkgo Leaf Sample Collection
[0031] Ginkgo leaf samples were collected from Hunan, Zhejiang, Jiangsu, and Chongqing, numbered, placed in ziplock bags, and stored in portable ice boxes, and brought back to the laboratory within 72 hours.
[0032] 2. Isolation of Ginkgo Endophytes
[0033] Select fresh ginkgo leaves, wash them with clean water, rinse them in running water for 0.5 hours, and place them in a beaker filled with distilled water.
[0034] Take 5g of each washed ginkgo leaf, first rinse it with 75% alcohol by volume for 1 minute, and then rinse it with sterile water three times; then rinse it with 0.1% sodium hypochlorite for 0.5min, and then rinse it with sterile water three times; the last rinse solution is placed in a sterilized small beaker.
[0035] The surface-sterilized material was rolled several times on a blank PDA culture medium, and the culture medium was placed in a 37°C incubator for 2 days to detect the disinfection effect.
[0036] After sterilization, blot the sample surface dry with sterile filter paper. In a clean bench, cut the ginkgo leaf samples into approximately 0.5 cm x 0.5 cm tissue blocks using sterile scissors. Place five tissue blocks per PDA and MMN plate, placing the cut surface onto the culture medium whenever possible. Finally, apply the final rinse solution from the sterilization process to the culture medium for incubation. These controls are numbered and recorded. After completion, incubate the samples in a 28°C incubator, observing and recording the growth of endophytic microorganisms daily.
[0037] Once colonies have grown on the surface or edge of the sample, use an inoculation needle to pick up the mycelium in a clean bench and transfer it to a new culture medium of the corresponding type. The culture medium is then numbered. The sample is then placed in a 28°C incubator in a darkened state and the colony growth is observed daily.
[0038] After the inoculated bacteria germinate, use spores to subculture multiple times until typical purified colonies are obtained. Transfer the purified strains to solid slant culture medium, preserving two slants per strain. Cultivate in a 28°C incubator. When the strains are growing vigorously, remove them and store them at 4°C. After two months, transfer them to new slant cultures.
[0039] 3. Screening of biocontrol strains
[0040] First, a colony standoff method was used to screen isolated biocontrol bacteria for those with inhibitory effects against bacterial wilt. The purified strains were inoculated using an inoculating loop onto LB plates pre-mixed with R. solanacearum. The plates were incubated at 28°C for 30 hours, and the appearance of inhibition zones was observed. The size of the inhibition zones was used to determine the antagonistic ability of the biocontrol bacteria. Strains with significant inhibitory effects were stored in a -80°C freezer with 30% glycerol.
[0041] In order to screen out biocontrol bacteria with strong extracellular antibacterial substances, the biocontrol actinomycetes with obvious antagonistic advantages that were initially screened were cultured in a shaking incubator at 28°C and 150r / min in Gao's No. 1 liquid medium. The obtained bacterial suspension was centrifuged at 7200rpm for 6min, and the supernatant was taken. The punching method was used to detect its inhibitory activity against Ralstonia solanacearum, that is, a NA culture plate mixed with Ralstonia solanacearum was punched with a puncher with a diameter of 11mm, and the supernatant culture solution after centrifugation and sterile NB liquid medium were added to the wells as controls. The experiment was repeated three times, and the diameter of the inhibition zone was measured after culturing at 37°C for 48h. One group of replicates was as follows: Figure 1 By comparing the size of the inhibition zone, Figure 1 The middle arrow points to the biocontrol bacteria with the best antagonistic effect against Ralstonia solanacearum.
[0042] After streak purification of the selected biocontrol bacteria, single colonies were selected and inoculated into the appropriate liquid culture medium. Cultured in a shaker (28°C, 180 rpm) for 24 hours, the culture was then aliquoted into 1.5 mL sterile centrifuge tubes at a 1:1 ratio with 60% sterile glycerol. After shaking thoroughly, the culture solution was stored at -80°C.
[0043] After the above screening, the biocontrol strain YZ23120524 was obtained. The diameter of the inhibition zone against bacterial wilt pathogen was 20 mm, which showed good inhibitory activity. Figure 2 .
[0044] 4. Molecular Biological Identification of Biocontrol Strain YZ23120524
[0045] Genomic DNA from biocontrol bacterium YZ23120524 was extracted according to the instructions provided with the bacterial and fungal genomic extraction kit. 16S rDNA sequences were amplified using a universal primer pair: forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1), and reverse primer 1492R: 5'-ACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2). The primer pair was synthesized by Shanghai Bioengineering Co., Ltd.
[0046] The PCR reaction system (50 μL system) is: 2.5 μL template DNA, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 0.75 μL dNTP (10 mM each), 5.0 μL 10× PCR buffer, 0.75 μL Taq enzyme (5 U / μL), and 40.0 μL ddH2O to 50 μL.
[0047] PCR reaction conditions: 94°C pre-denaturation for 5 min, 30 amplification cycles (94°C denaturation for 45 s, 56°C annealing for 45 s, 72°C extension for 105 s), and 10 min extension. After the reaction was completed, the PCR reaction products were detected by 1.0% agarose gel electrophoresis.
[0048] After electrophoresis detection, a specific fragment of about 1.4 kb was obtained, such as Figure 3 The detected PCR products were purified using a gel recovery kit and sent to Chengdu Luoning Biotechnology Co., Ltd. for sequencing. The sequencing sequences were subjected to BLAST comparison analysis in the GenBank database.
[0049] The 16S rDNA sequence of strain YZ23120524 was obtained by sequencing, as shown in SEQ ID NO.3:
[0050]
[0051] BLAST analysis of the GenBank database revealed that the strain was Streptomyces pororaveus, with a 99% homology. The strain was identified as Streptomyces pororaveus YZ23120524 and sent to the China Center for Type Culture Collection for preservation, receiving accession number CCTCCNO: M 20232508.
[0052] Table 2 BLAST alignment results
[0053]
[0054] Table 3 Classification results
[0055] kingdom Bacteria phylum Actinomycetota class Actinomycetes order Kitasatosporales family Streptomycetaceae genus Streptomyces species Streptomyces spororaveu It should be noted that there might be a misspelling in "Streptomycesspororaveu", and it is corrected to "Streptomyces spororaveu" in the translation.
[0056] Example 2
[0057] This embodiment provides a ginger blast inhibitor, and the specific preparation process is as follows:
[0058] Prepare Gao's No. 1 medium slant, inoculate the screened Streptomyces sporangiophorus YZ23120524, and culture in a 28°C incubator for 7 days to obtain slant bacteria for later use.
[0059] Prepare Gao's No. 1 liquid culture medium, divide it into Erlenmeyer flasks (250 mL, containing 80 mL of liquid) before sterilization, sterilize it at 121°C for 20 min, and after cooling, pick up the slant bacteria with an inoculation needle on a clean workbench, inoculate it into the Erlenmeyer flask containing the above-mentioned sterilized Gao's No. 1 liquid culture medium, and culture it in a shake flask at pH 7.2, 28°C, and 150 rpm for 7 days to obtain shake flask culture bacteria.
[0060] Prepare Gao's No. 1 liquid medium, sterilize at 121°C for 20 minutes, cool, inoculate with a shake flask culture, and then transfer to a fermenter for fermentation at a pH of 7.2, a temperature of 28°C, and a rotation speed of 150 rpm. Fermentation is terminated after 10 days. The resulting fermentation liquid is a ginger wilt inhibitor. The resulting ginger wilt inhibitor can be used to treat ginger wilt in the field. The inhibitor is diluted 200-fold and applied to ginger seeds after soaking for 1 hour and 45-50 days after ginger emergence. The application method is root irrigation, with an application rate of 500 mL / stalk.
[0061] Example 3
[0062] This example verifies the efficacy of the ginger blast inhibitor prepared in Example 2 by conducting a tobacco field experiment. The verification process is as follows:
[0063] Select a plot where ginger blast disease occurs year-round, dilute the ginger blast antibacterial agent prepared in Example 2 with water 200 times (v:v), soak the ginger seeds for 1 hour before sowing; 45 days after the ginger seedlings emerge, water the roots of the ginger seedlings with the 200-fold dilution of the antibacterial agent at a rate of 500 mL / stalk. At the same time, add 14 g / 667 m3 of 72% agricultural streptomycin sulfate. 2 As the control for the drug, the roots were irrigated once at the early stage of the disease and 10 days after the early stage of the disease, with the application rate of 50mL per root. The roots were irrigated with the same volume of water as the blank control. Each plot area was 17m 2 80 ginger plants were planted in plots with a row spacing of 35 cm × 60 cm. The plots were randomly arranged and replicated three times. Conventional ginger cultivation practices were followed. During the peak period of ginger wilt disease, all ginger plants were surveyed for disease status. The incidence rate and disease index were calculated to calculate the relative control efficacy of the antibacterial agent, Streptomyces sporeliensis YZ23120524, against ginger wilt in the field.
[0064] Incidence rate (%) = number of diseased plants / total number of plants surveyed × 100%
[0065] Disease index = [∑(number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100%
[0066] Control effect % = [1-(treatment disease index / control disease index)] × 100%
[0067] The results are shown in Table 4.
[0068] Table 4 Incidence and control effects of ginger wilt under different treatments
[0069]
[0070] As can be seen from Table 4, the relative control effects of the ginger blast inhibitor of the present invention and 72% agricultural streptomycin sulfate are 74.70% and 70.26% respectively. The ginger blast inhibitor has a higher control effect than 72% agricultural streptomycin sulfate by 4.44%.
[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A Streptomyces pororaveus strain YZ23120524, deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit date of December 8, 2023, and a deposit number of CCTCCNO: M20232508.
2. Streptomyces spore pale gray YZ23120524 according to claim 1, is characterized in that, The 16SrDNA sequence of the sporangiophore Streptomyces YZ23120524 is shown in SEQ ID NO.
3.
3. Use of the Streptomyces sporangiophora YZ23120524 according to claim 1 or 2 in preventing and treating ginger blast.
4. A ginger blast inhibitor, characterized in that: The antibacterial agent is prepared by fermenting the Streptomyces sporangiophorus YZ23120524 according to claim 1 or 2.
5. Use of the ginger blast inhibitor according to claim 4 in preventing and treating ginger blast.
6. The use according to claim 5, characterized in that The ginger blast inhibitor is diluted 150 to 300 times when used.
7. The use according to claim 6, characterized in that The ginger blast inhibitor is used by soaking seeds and watering them at the seedling stage.
8. The use according to claim 7, characterized in that The seed soaking time is 0.5 to 2 hours.
9. The use according to claim 8, characterized in that The irrigation period is 45 to 50 days after the ginger seedlings emerge, and the roots are watered.
10. The use according to claim 9, characterized in that The dosage for irrigation is 400-600 mL / plant of the diluted ginger wilt antibacterial agent.