Aspergillus tumefaciens strain and application thereof in prevention and treatment of tobacco mosaic virus
By screening and applying the strain MFS2328, which has high temperature tolerance and its secondary metabolites, the problem of the reduction of the activity of existing fungal agents under high temperature conditions is solved, and effective prevention and treatment of tobacco mosaic virus and the extension of the shelf life of the fungal agents is achieved.
Patent Information
- Application Number
- CN202510319193.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-13
AI Technical Summary
The existing fungal agents used to prevent and control tobacco mosaic viruses have reduced activity under high temperature conditions, resulting in short shelf life and difficult to meet the actual application needs.
A strain of Aspergillus wool from seawater, MFS2328, a strain of marine clumps, was screened and applied, which had better high temperature tolerance and obtained its secondary metabolites by fermentation and extraction, for the preparation of bacterial agents for the prevention and control of tobacco mosaic viruses.
The survival rate of MFS2328 strain under high temperature conditions was significantly higher than that of other strains, and its fermentation supernatant and secondary metabolic crude extract had a significant inhibitory effect on tobacco mosaic virus, extending the shelf life of the bacterial agent.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of screening and application of functional microorganisms, and particularly relates to a strain of Aspergillus penicillioides and its application in preventing and controlling tobacco mosaic virus. Background Art
[0002] Tobacco mosaic virus disease is a plant virus disease caused by Tobacco mosaic virus (TMV). It occurs widely in tobacco-growing areas around the world, causing serious losses and being one of the most serious diseases harming tobacco. TMV is mainly transmitted through sap friction and can infect more than 350 host plants including solanaceous crops such as tobacco, tomato, potato, eggplant, pepper, and Solanum nigrum. It can occur throughout the whole growth period from the seedbed to the field. The cured leaves have uneven colors, resulting in a decline in quality and a reduction in yield. At present, there have been many studies on the biological control of tobacco mosaic virus in China, but most of them are limited to the screening of biocontrol strains, mainly focusing on bacteria screening, and there is little in-depth research on the isolation and extraction of antagonistic substances produced by fungi.
[0003] Fungi are important resources for developing microbial pesticides, and a large amount of scientific research efforts have been invested by countries around the world. Microbial pesticides are isolated and purified from natural products, which can directly act on pest and disease control or be used as lead compounds to synthesize new pesticides. These pesticides can not only retain the advantages of low toxicity and low residue of the original bioactive substances, but also overcome the deficiencies of poor stability and low activity of some natural products, thus fully exploiting the potential of bioactive substances.
[0004] Currently, there are more than 70 registered biopesticides with terrestrial fungi or their metabolites as active ingredients. These microbial pesticides are widely used in the control of plant diseases, insect pests, and field weeds. China has also achieved remarkable results in application and basic research. Although the current output of biopesticides in China only accounts for 9% of the total pesticide output, considering the long-term interests of humanity and the development trend of pesticides, the development potential of biopesticides is very large. The living environment of marine fungi is relatively harsh (high salt, high pressure, anoxic, low nutrition, no light, etc.). In order to survive, they have produced some secondary metabolites with unique structures during the long-term evolution process. Especially for marine animal-plant symbiotic fungi, to strive for the maximum survival, they actively participate in the metabolic activities of animals and plants by secreting various bioactive metabolites, playing an important role in the anti-infection and anti-phagocytosis ecological defenses of animals and plants.
[0005] Although there have been reports of strains with antagonistic activity against tobacco mosaic virus, it has been found during use that the high-temperature resistance effect of the reported strains is not good, resulting in a reduction in the activity of the prepared microbial agents during storage. Summary of the Invention
[0006] The object of the present invention is to provide a strain of Aspergillus floccosus and its application in controlling tobacco mosaic virus, that is, a strain of Aspergillus floccosus from seawater and its application in controlling tobacco common mosaic virus. The bioactive secondary metabolites of this strain can also be applied to the biological control of tobacco common mosaic virus, thus making up for the deficiencies of the existing technology.
[0007] The Aspergillus floccosus MFS2328 strain provided by the present invention, which is from seawater, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 18, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 40803.
[0008] The MFS2328 strain screened by the present invention is used to control tobacco common mosaic virus and can also be used to prepare products with the function of controlling tobacco common mosaic virus;
[0009] As a specific record of an embodiment, the said product is a microbial inoculant.
[0010] The present invention also provides the metabolites of the MFS2328 strain. The MFS2328 strain is fermented, ethyl acetate is added to the supernatant after fermentation for extraction, the obtained organic phase is concentrated under reduced pressure on a rotary evaporator, and after elution with methanol, it is evaporated to dryness at room temperature to obtain the crude extract of the metabolites of the MFS2328 strain.
[0011] The said fermentation is to inoculate the MFS2328 strain into a PDB liquid medium and ferment at 28°C.
[0012] The above-mentioned metabolites are used to control tobacco common mosaic virus disease.
[0013] The present invention obtains the MFS2328 strain with biological activity against tobacco common mosaic virus, which can be applied to the control of tobacco common mosaic virus disease. Its fermentation supernatant and crude extract have obvious inhibition rates against TMV. At the same time, the MFS2328 strain has better high-temperature tolerance. Description of the Drawings
[0014] Figure 1 : Colony culture morphology diagram of the strain MFS2328, where the left figure is the front photo of the strain culture dish, and the right figure is the front photo of the strain culture dish;
[0015] Figure 2 : Inhibition effect diagram of the fermentation supernatant of the strain MFS2328 on TMV,
[0016] Figure 3 : Inhibitory effect diagram of the crude extract of the secondary metabolite of strain MFS2328 on TMV,
[0017] Figure 4 : Photo of strain MFS2328 cultured in PDB liquid for 14 days,
[0018] Figure 5 : Photo of strain MFS2328 cultured on rice medium solid for 7 days,
[0019] Figure 6 : HPLC chromatogram of the secondary metabolite crude extract of strain MFS2328 (the upper figure is for liquid fermentation culture, and the lower figure is for solid fermentation culture). Detailed implementation mode
[0020] In the research on biocontrol agents against tobacco mosaic virus, it was found that the currently used biocontrol agents against tobacco mosaic virus have a short shelf life for the preparations prepared as active strains due to poor heat resistance.
[0021] From the perspective of biocontrol of tobacco mosaic virus, the present invention screens antagonistic marine fungal strains with excellent control effects and strong heat resistance, isolates and extracts the antagonistic active substances produced by them, and applies them to the control of tobacco mosaic virus disease.
[0022] The present invention will be further described below in conjunction with examples and drawings, but it is not limited in any way. Any modification or improvement based on the teachings of the present invention falls within the protection scope of the present invention.
[0023] Example 1: Isolation, purification and identification of Aspergillus floccosus MFS2328 strain. The seawater sample collected from the red beach of the Liaohe River estuary in Panjin, Liaoning in October 2020 was placed on PDA medium (culture medium preparation method: Peel the potatoes, cut 200 g into small pieces, add 1 L of seawater, boil for 30 min, filter with 4-6 layers of gauze, add 20 g of glucose, dissolve, and supplement the lost water with distilled water. For solid medium, add 1.5-2% agar, autoclave at 121 °C for 20 min.). It was cultured at 28 °C, and the MFS2328 strain was obtained by single-spot isolation and purification. It was identified as Aspergillus floccosus by 18s rDNA ITS gene sequence analysis.
[0024] Example 2: Culture morphology, physical and chemical parameters, and temperature tolerance of Aspergillus floccosus MFS2328 strain
[0025] 1. Microscopic morphological characteristics of strain MFS2328
[0026] Observed under the microscope, the conidiophores of strain MFS2328 grow on stoloniferous hyphae, with a diameter of 5 - 8 μm; the sporangium is spherical, with a diameter of 22 - 30 μm; the sporogenous cells are single-layered on the sporangium, without septa or with 1 septum, 2.3 - 4.1 × 8 - 12 μm; the conidia are arranged in chains, spherical, with spines, and a diameter of 3 - 7 μm.
[0027] 2. Culture conditions of strain MFS2328
[0028] The colony was cultured on PDA medium for 7 days, with a diameter of 9 cm, flat, and the aerial hyphae were dense and dark yellow; the conidiophores were light brown and moderately produced ( Figure 1 ). After culturing in PDA liquid medium, the cells were in a cake shape. The optimal growth temperature of strain MFS2328 is 28 °C, the optimal pH value is 10, the optimal culture time is 8 days, and the optimal medium is seawater PDA medium.
[0029] 3. Temperature tolerance of the strain
[0030] The temperature tolerance experiment was carried out on the MFS2328 strain screened in the present invention and the strain MFS2119 with the preservation number of CGMCC NO.40163, which has been reported to have biological activity against tobacco mosaic virus. The two activated strains were respectively inoculated into PDA seawater medium and cultured at 28 °C for 5 - 7 days until the colonies grew significantly. An appropriate amount of sterile water was aspirated and added to the activated colonies, and the surface of the colonies was scraped to prepare a bacterial suspension, and its concentration was adjusted to 10×10 6 CFU / mL. The bacterial suspension was placed in a constant temperature water bath at 45 °C, and samples were taken at 0.5 h, 1 h, and 2 h respectively. The heat-treated bacterial suspension was spread on the medium and cultured at 28 °C for 5 - 7 days, and the colony-forming units (CFU) were calculated. Three replicates were set, and the survival rate was calculated.
[0031] The results showed that the Aspergillus flocculosus MFS2328 strain screened in the present invention had better tolerance to the temperature of 45 °C. After treatment at this temperature for 0.5 h and 1 h, the survival rate of the MFS2328 strain was significantly higher than that of the strain MFS2119. And the inhibitory effect on tobacco mosaic virus TMV at 45 °C also showed that the MFS2328 strain had a better effect.
[0032] Table 1: Survival rate table of the strain treated at 45 °C for different times (%)
[0033]
[0034] Note: * indicates significant difference (P < 0.05)
[0035] Example 3: Inhibitory effect of MFS2328 strain on tobacco mosaic virus TMV
[0036] The inhibitory effect of the obtained strain on TMV was determined by the local lesion method. Weigh 1 g of fresh TMV-infected leaves, add 10 mL of sterilized phosphate buffer, grind them into a homogenate, filter with sterilized gauze, and take the supernatant as the inoculum. Inoculate the strain into PDB liquid medium for large-scale fermentation culture, take the fermentation supernatant, which is used as the anti-TMV bacterial liquid for standby. Mix the bacterial liquid and the TMV inoculum in equal amounts, and mix the PDB liquid medium and the TMV inoculum in equal amounts. After standing for 10 min at room temperature, rub-inoculate Samsun-NN tobacco, and spray an appropriate amount of carborundum on the leaf surface before inoculation. Inoculate the left half leaf with the mixture of the bacterial liquid and the TMV inoculum, and inoculate the right half leaf with the mixture of the PDB medium and the TMV inoculum as a control, and inoculate 200 μL on each half leaf. After inoculation, rinse the leaf surface with water, and repeat 3 times. Observe the results 3 days after inoculation, and count the number of lesions to calculate the inhibition rate. The results showed that the fermentation broth of the strain had a good inhibitory effect on TMV, and the lesion inhibition rate reached 88% (Table 2, Figure 2 ), and the crude extract of the secondary metabolites of the strain had a lesion inhibition rate of 68.17% on TMV (Table 3, Figure 3 ), indicating that the biocontrol strain produced resistance substances that inhibited the activity of TMV during its growth and metabolism.
[0037] Inhibition rate (%) = [1 - (average number of lesions in treatment / average number of lesions in control)] × 100.
[0038] Table 2: Inhibitory effect of the fermentation supernatant of MFS2328 strain on TMV
[0039]
[0040] Table 3: Inhibitory effect of the crude extract of the secondary metabolites of MFS2328 strain on TMV
[0041]
[0042]
[0043] Example 4: Detection of the anti-TMV effect of the fermentation product of MFS2328 strain
[0044] The tested marine fungus MFS2328 strain was inoculated into liquid PDB medium ( Figure 4 ) and solid rice medium ( Figure 5) Incubate statically at 28°C for about 40 days. After fermentation, take the supernatant of the liquid PDB medium and extract it three times with an equal volume of ethyl acetate. Directly add an equal volume of ethyl acetate to the solid-fermented rice culture of the bacteria and extract it three times. The obtained organic phases are respectively concentrated under reduced pressure on a rotary evaporator, eluted with methanol, and evaporated to dryness at room temperature to obtain the crude secondary metabolites of the MFS2328 strain from liquid fermentation and solid fermentation.
[0045] Dissolve the crude fermentation extracts of the MFS2328 strain obtained by the two culture methods in an appropriate amount of methanol, and perform high-performance liquid chromatography (HPLC) analysis. Compare and analyze according to the number of chromatographic peaks and the ultraviolet absorption curve to analyze the richness and content of the secondary metabolites of the MFS2328 strain under the two different culture methods of liquid and solid Figure 6 ). Judging from the results, there are slight differences in the richness of the secondary metabolites of the MFS2328 strain under the two culture methods, and there are four identical main chromatographic peaks at 235 nm.
[0046] Table 4: HPLC standard program table
[0047] Time (min) Flow rate (mL / min) Methanol Water 0-5 0.8 10％ 90％ 5-35 0.8 10％-100％ 90％-0 35-45 0.8 100％ 0 45-50 0.8 100％-10％ 0-90％ 50-60 0.8 10％ 90％
[0048] Example 5: Bioassay of the secondary metabolite active substance of the marine fungus MFS2328 strain against Myzus persicae
[0049] Inoculate the marine fungus MFS2328 strain into the liquid PDB medium and incubate statically at 28°C for about 40 days. After fermentation, take the supernatant of the liquid PDB medium and extract it three times with an equal volume of ethyl acetate. The obtained organic phases are respectively concentrated under reduced pressure on a rotary evaporator, eluted with methanol, and evaporated to dryness at room temperature to obtain the crude secondary metabolites of the MFS2328 strain from liquid fermentation.
[0050] Use the method of dipping insects and leaves to test the virulence of the fungal crude extract against Myzus persicae. Line a petri dish (9 cm in diameter) with filter paper, add 1 mL of distilled water to maintain appropriate humidity. Immerse the tobacco leaves of appropriate size in the solution for 10 s and then dry them, and then put them into the petri dish. Keep the petiole of the tobacco leaf moist with absorbent cotton and water. Immerse the tobacco leaf with aphids in the diluted crude extract solution for 10 S, and pick 30 aphids (wingless aphids of the same size and healthy) into the petri dish.
[0051] Prepare a stock solution of 10000 mg / L of the crude extract with acetone, dilute it in a gradient with clear water, and prepare solutions of 5000 mg / L, 2500 mg / L, 1250 mg / L, 625 mg / L, and 312.5 mg / L respectively, CK (acetone). Set 3 replicates for each, check and count the mortality of Myzus persicae under the crude extract of each concentration at 24 h, and calculate the LC50, regression equation and correlation coefficient.
[0052] Table 5: Analysis table of LC50 of crude extract of secondary metabolites of MFS2328 strain against Myzus persicae
[0053] Regression equation LC50 (mg / L) Confidence interval Correlation coefficient y = -7.113 + 2.920x 272.95 152.23-373.95 0.90
[0054] The results showed that the active components of the crude extract of secondary metabolites of the marine fungus MFS2328 strain had high toxicity to Myzus persicae, and the insecticidal rate could reach 100% at the treatment concentration of 5000 mg / L. Through statistical analysis, the LC50 of the fermentation broth extract of strain MFS2328 against Myzus persicae was 472.15 mg / L, and the virulence regression equation was: y = -7.113 + 2.920x, R = 0.90, and the 95% confidence limit was 152.23 - 373.95.
Claims
1. A fungus, characterized in that: The deposit number of the Aspergillus fusca is CGMCC NO.40803.
2. Use of the Aspergillus fusus described in claim 1 in preventing and controlling tobacco common mosaic virus.
3. Use of the Aspergillus fusus described in claim 1 in the preparation of products for preventing and controlling tobacco common mosaic virus.
4. A microbial agent for preventing and controlling tobacco common mosaic virus, characterized in that: The microbial agent contains the live bacteria of Aspergillus niger according to claim 1.
5. The microbial agent according to claim 4, characterized in that The microbial agent also contains the fermentation metabolites of Aspergillus fusus according to claim 1.
6. The microbial agent according to claim 5, characterized in that The fermentation metabolite is obtained by fermenting the strain, adding ethyl acetate to the fermentation supernatant for extraction, concentrating the obtained organic phase under reduced pressure on a rotary evaporator, eluting with methanol and evaporating to dryness at room temperature to obtain the metabolite.
7. The microbial agent according to claim 6, characterized in that The fermentation is carried out by inoculating the strain into PDB liquid culture medium at 28°C.
8. A method for preventing and treating tobacco common mosaic virus disease, characterized in that: The method described is to use the microbial agent described in any one of claims 4-7 for prevention and control.