CN117645932A - Marine aspergillus strain with tobacco black shank prevention and treatment effect - Google Patents
Marine aspergillus strain with tobacco black shank prevention and treatment effect Download PDFInfo
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Abstract
The invention provides an aspergillus strain from marine ciliate desert-grass, which has the preservation number of CGMCC NO.40801. The strain screened by the invention has biological control activity on tobacco black shank and can be applied to biological control of tobacco black shank.
Description
Technical Field
The invention belongs to the technical field of microorganism screening application, and particularly relates to a marine aspergillus strain with a tobacco black shank prevention and treatment effect.
Background
With the increasing attention of people to self health and living environment, the defects caused by the traditional chemical pest control measures have attracted great attention. The problems of increased pesticide residue in the environment, increased drug resistance of the pests, rampant re-occurrence of the pests and the like caused by the excessive use of chemical pesticides are most remarkable. With the further understanding of various social problems, environmental problems, and human health problems caused by chemical pesticides in various countries of the world, more and more chemical pesticides are restricted or prohibited. Biological pesticides, which are novel pesticides with low toxicity and low residue and are not easy to generate drug resistance by diseases and insect pests, naturally become hot spots for modern pesticide industry to pay attention to and try to develop, and research and industrialization of the biological pesticides are also strategic demands of the country.
Microbial pesticides are an important class of biopesticides, including agricultural antibiotics and live microbial pesticides. The microbial pesticide is separated and purified from natural products, can directly act on pest control, and can also be used as a lead compound to synthesize a novel pesticide. The pesticides not only can keep the advantages of low toxicity, low residue and the like of the original bioactive substances, but also overcomes the defects of poor stability, low activity and the like of certain natural products, so that the potential of the bioactive substances is fully exploited.
Fungi are important resources for developing microbial pesticides, and a great amount of scientific research force is put into research all over the world. The registered biopesticides containing land fungi or metabolites thereof as active ingredients are up to 70 or more, and these biopesticides are widely used for controlling plant diseases, insect pests and weeds in fields. The Chinese also has very outstanding results in application and basic research. Although the current yield of biopesticides in China only accounts for 9% of the total yield of pesticides, the development potential of biopesticides is huge in terms of long-term benefit of human beings and development trend of pesticides. The marine fungi are derived from marine environments and are not parasitic bacteria of land organisms, and in front of a biological pesticide development mode directly taking a living body of a microorganism as an effective pesticide or a sterilization component, the marine fungi have no advantages compared with the land fungi, but have great advantages in a pesticide development mode taking metabolism of the microorganism as an effective component.
The living environment of the marine fungi is harsh (high salt, high pressure, hypoxia, low nutrition, no light and the like), in order to obtain living space, secondary metabolites with unique structures are generated in the long-term evolution process, particularly the marine animal and plant co-epiphyte is used for striving for the maximum living of the marine fungi, the metabolism activities of the animal and plant are actively participated by secreting the metabolites with various biological activities, and important roles are played in the anti-infection and anti-phagocytic ecological defense of the animal and plant, and the marine fungus is considered as the best object for screening antibiotics. 70% -80% of the novel compounds isolated from marine fungi have a wide variety of biological activities and have attracted considerable attention from chemists and biologists, and the species include terpenes, peptides, alkaloids, ketones and esters, many of which are the active ingredients or precursors of certain pesticides and fungicides. Regarding the functions and the applications of the secondary metabolites of the marine fungi, researchers are focused on the development of medical drugs for treating human diseases, and the research on agricultural insecticidal and bactericidal functions of the metabolites of the marine fungi is less.
Tobacco black shank (Tobacco black shank), also known as "black stem crazy, black root", is an earth-borne oomycete disease widely distributed in the tobacco producing areas of the world, found for the first time in the java island of indonesia in 1896, and later several decades, was found in succession in most tobacco producing areas in india and the united states, and spread rapidly and caused serious losses. At present, the disease is commonly occurring in many countries and regions worldwide, particularly in temperate, subtropical and tropical regions. In 1950, the tobacco spread rapidly after the tobacco appears in Huang-Huai tobacco areas in China, especially in Anhui, henan and Shandong, and in recent years, the tobacco in south such as Yunnan, guizhou, sichuan and the like also occurs to different extents, so that the tobacco becomes one of the most serious rootstock diseases in tobacco production, and causes larger yield and output value loss. Tobacco black shank is difficult to prevent due to long-term surviving dormant structures (oospores and chlamydospores), plus a cryptic initial site of infection.
The breeding of disease-resistant varieties is dominant in the prevention and treatment of crop diseases, but the current lack of efficient disease-resistant varieties suitable for popularization and planting. The chemical control has quick effect and low cost, and in recent years, the dosage and the times are increasingly increased for controlling diseases, so that the diseases are controlled to a certain extent, but the chemical control also has a plurality of negative effects, such as environmental pollution, rising resistance level, ecological balance destruction and the like. In order to ensure the production safety of green and environment-friendly tobacco, the development of the high-efficiency biocontrol microbial inoculum is urgent as an important means for biological control. Therefore, the test is explored from the aspect of biological control, and marine fungus strains with activity on tobacco black shank are screened, so that a research basis and experimental materials are provided for developing biological agents for controlling the tobacco black shank by utilizing the strains and secondary metabolites thereof in the future.
Disclosure of Invention
The invention aims to provide a strain for preventing and treating tobacco black shank, namely an Aspergillus sp strain from marine ciliate desert-grass, which is used for preventing and treating tobacco black shank, and meanwhile, the screened strain can generate secondary metabolites with preventing and treating activity on tobacco black shank bacteria.
The invention firstly provides an Aspergillus sp strain MFZ2319 from marine centipede algae, which is preserved in China general microbiological culture Collection center for 8 months and 18 days in 2023, and the preservation address is: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is CGMCC No.40801.
The MFZ2319 strain screened by the invention is used for preventing and treating tobacco black shank and can also be used for preparing products with the function of preventing and treating tobacco black shank.
The product is a probiotic bacteria liquid product.
The invention also provides a metabolite of the MFZ2319 strain, and a specific preparation method thereof is as follows:
the fermentation supernatant of strain MFZ2319 was extracted with ethyl acetate and the resulting organic phase was concentrated under reduced pressure on a rotary evaporator, eluted with methanol and evaporated to dryness at room temperature to give a crude metabolic extract of strain MFZ 2319.
The fermentation is carried out by inoculating MFZ2319 strain into PDB seawater culture medium and fermenting at 28deg.C.
The metabolite is used for preventing and treating tobacco black shank.
The MFZ2319 strain obtained by the invention has biological control activity on tobacco black shank and can be applied to biological control of tobacco black shank.
Drawings
Fig. 1: colony morphology of strain MFZ2319 on PDA medium, wherein the left panel is front view of the medium and the right panel is rear view of the medium;
fig. 2: the antagonistic activity effect of the strain MFZ2319 on tobacco black shank bacteria is shown in the graph; the left side of the picture is MFZ2319 strain, and the right side of the picture is blank control; the middle of each graph is inoculated pathogenic bacteria, and two sides of each graph are strain secondary metabolites;
fig. 3: TLC profile of strain MFZ2319 secondary metabolite;
fig. 4: strain MFZ2319 secondary metabolite HPLC profile.
Detailed Description
The invention screens and obtains Aspergillus strain from marine centipede algae, and prepares the metabolite of secondary micromolecular compound from the strain, and the metabolite can also be applied to tobacco black shank prevention and control.
The strain of the present invention will be described in detail with reference to specific examples and drawings.
Example 1: isolation, identification and culture of strain
The method comprises the steps of culturing the obtained ciliate desert-grass on a PDA culture medium at 28 ℃ after collection in a first seawater bath of Qingdao city in Shandong province, separating and purifying the single bacterial plaque to obtain a MFZ2319 strain, and determining the MFZ2319 strain to be Aspergillus sp through 18s rDNA ITS gene sequence analysis and identification.
The preparation method of the PDA culture medium comprises the following steps: peeling potato, cutting 200g into small pieces, adding seawater 1L, boiling for 30min, filtering with 4-6 layers of gauze, adding 20g glucose, dissolving, and supplementing water with distilled water. The solid culture medium is added with 1.5-2% agar, and sterilized at 121deg.C under high pressure for 20min. )
The 18s rDNA ITS gene sequence is as follows:
GGCTCGCTATTCTTTTGCAGCGCTTACTGCGCGGCGAAAAACCTTACACACAGTGTCTTTTTGATACAGAACTCTTGCTTTGGTTTGGCCTAGAGATAGGTTGGGCCAGAGGTTTAACAAAACACAATTTAATTATTTTTACAGTTAGTCAAATTTTGAATTAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATATGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCAGAGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCCCCGGGTTTGGTATTGAGTGATACTCTTAGTCGGACTAGGCGTTTGCTTGAAAAGTATTGGCATGGGTAGTACTAGATAGTGCTGTCGACCTCTCAATGTATTAGGTTTATCCAACTCGTTGAATGGTGTGGCGGGATATTTCTGGTATTGTTGGCCCGGCCTTACAACAACCAAACAAGTTTGACCTCAAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGGCGGAGGAA(SEQ ID NO:1)。
after the selected MFZ2319 bacterial colonies are cultured on a PDA culture medium for 7 days, the bacterial colony has the diameter of 9cm, is flat, has thick hyphae, is white at the early stage of the front surface, is yellow at the later stage, and has dark pink ground color; the back is dark pink with the middle being darker (fig. 1). MFZ2319 strain has an optimal growth temperature of 28 ℃, an optimal pH value of 10.0 and an optimal culture time of 8d, and the optimal culture medium is a seawater PDB culture medium.
Microscopic morphological detection results show that the conidiophore of the MFZ2319 strain grows on thick-walled and expanded podocytes with hyphae, does not branch, expands at the top to form a circular top sac, and generates radial small peduncles on the surface of the top sac, which are columnar cells.
Example 2: prevention and treatment effect of secondary metabolite of MFZ2319 bacteria on tobacco black shank
The marine fungus MFZ2319 strain is inoculated in PDB liquid culture medium, and is subjected to standing culture for about 40d at the temperature of 28 ℃. After fermentation, the supernatant was extracted 3 times with an equal volume of ethyl acetate, and the obtained organic phase was concentrated under reduced pressure on a rotary evaporator, eluted with methanol and evaporated to dryness at room temperature to obtain a crude extract of the MFZ2319 strain metabolite.
The antibacterial activity of the crude extract is tested by adopting an agar diffusion method, 3 holes (with the aperture of 3 mm) are uniformly drilled along the central line of the culture dish, and the black shank germ cake is connected into the middle hole. Then, 50. Mu.L of the crude extract solution was added to the two side holes and placed in an incubator at 28℃for cultivation. Prochloraz was set as positive control, DMSO as negative control, 3 replicates were set. After 2-10 days, checking and measuring the antibacterial diameter, and statistically analyzing the antibacterial activity according to the measurement result.
The result shows that when the concentration of the secondary metabolism crude extract of the marine fungus MFZ2319 strain is 10mg/mL, the antibacterial diameter reaches 31.67mm, and the inhibition rate is 70.37%. The MFZ2319 strain screened by the invention has good control effect on tobacco black shank and has good popularization and application values.
Example 3: activity of marine fungus MFZ2319 Strain secondary metabolite on Aphis tabaci
The marine fungus MFC2319 strain is inoculated in PDB liquid culture medium, and is subjected to stationary culture for about 40d at the temperature of 28 ℃. After fermentation, the supernatant was extracted 3 times with an equal volume of ethyl acetate, and the obtained organic phase was concentrated under reduced pressure on a rotary evaporator, eluted with methanol and evaporated to dryness at room temperature to obtain a crude metabolic extract of MFC2319 strain.
The toxicity of the secondary metabolic crude extract on the myzus persicae was tested by the insect and leaf dipping method. Spreading filter paper in a culture dish (diameter of 9 cm), adding 1mL distilled water to maintain proper humidity, immersing tobacco leaf with proper size in the solution for 10s, air drying, placing in the culture dish, and moistening tobacco leaf handle with absorbent cotton. Tobacco leaves with aphids are immersed in the diluted crude extract solution for 10s, and 30 aphids (healthy wingless aphids of uniform size) are picked up in a petri dish.
The crude extract was made up with acetone as 10000mg/L mother liquor, diluted with clean water in a gradient, and 5000mg/L, 2500mg/L, 1250mg/L, 625mg/L and 312.5mg/L solutions, CK (acetone) were prepared, respectively. Each of the three replicates was counted and the data were processed with SPSS software to calculate LC50, regression equation and correlation coefficients at 24h for checking the death of the myzus persicae at each concentration of the crude extract.
The result shows that the active ingredient of the marine fungus MFC2319 strain secondary metabolism crude extract has certain toxicity to the myzus persicae, and the corrected mortality rate of the myzus persicae for 48h is 95.31% under the concentration of 10 mg/mL. Statistical analysis is carried out through SPSS software to obtain the LC50 of the strain MFC2319 fermentation liquor extract to the myzus persicae of 3.34mg/mL, and the virulence regression equation is: y= -3.8740+2.1517x, r=0.92.
The result shows that the MFC2319 strain screened by the invention has good control effect on myzus persicae and has good popularization and application values.
Example 4: enrichment analysis of secondary metabolites of marine fungus MFZ2319 Strain
The marine fungus MFZ2319 strain is inoculated in liquid PDB culture medium respectively, and is subjected to standing fermentation culture for about 40d at the temperature of 28 ℃. After fermentation, adding an equal volume of ethyl acetate into the supernatant of the liquid PDB culture medium to extract for 3 times, concentrating the obtained organic phase under reduced pressure on a rotary evaporator, eluting with methanol, and evaporating to dryness at room temperature to obtain a crude extract of MFZ2319 strain for liquid fermentation and solid fermentation secondary metabolism.
The obtained crude extract of strain MFZ2319 was dissolved in a suitable amount of methanol, analyzed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC), and analyzed by comparison according to the number of sample spots, the abundance and the number of chromatographic peaks on the thin layer plate and the uv absorption curve, and the abundance and the content of secondary metabolites of strain MFZ2319 were analyzed (fig. 3, fig. 4). Wherein the TLC development system was dichloromethane: methanol=10: 1 or 20:1, the color reagent is anisaldehyde-sulfuric acid.
Table 2: HPLC standard program table
| Time (min) | Flow rate (mL/min) | Methanol | Water and its preparation method |
| 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％ |
From the results, the strain MFZ2319 has higher abundance of secondary metabolite species, with 5 distinct main chromatographic peaks.
Claims (8)
1. The aspergillus strain is characterized by having a preservation number of CGMCC NO.40801.
2. Use of an aspergillus strain according to claim 1 for controlling tobacco black shank.
3. Use of an aspergillus strain according to claim 1 for the preparation of a product having a function of controlling tobacco black shank.
4. The use according to claim 3, wherein the product is a probiotic bacterial liquid product.
5. A product for controlling tobacco black shank, comprising the aspergillus strain of claim 1.
6. The article of manufacture of claim 5, further comprising a fermentation metabolite of the aspergillus strain of claim 1.
7. The preparation of claim 6, wherein the metabolic product is obtained by inoculating the aspergillus strain of claim 1 into a culture medium for fermentation, adding ethyl acetate into 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.
8. The article of manufacture of claim 7, wherein the culture medium is PDB seawater culture medium.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117511753A (en) * | 2023-11-23 | 2024-02-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Marine trichoderma strain and application thereof in prevention and treatment of tobacco mosaic virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117511753A (en) * | 2023-11-23 | 2024-02-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Marine trichoderma strain and application thereof in prevention and treatment of tobacco mosaic virus |
| CN117511753B (en) * | 2023-11-23 | 2024-04-26 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Marine trichoderma strain and application thereof in prevention and treatment of tobacco mosaic virus |
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