CN116751687A - Endophyte G-5 for improving tobacco quality - Google Patents
Endophyte G-5 for improving tobacco quality Download PDFInfo
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- CN116751687A CN116751687A CN202310655286.6A CN202310655286A CN116751687A CN 116751687 A CN116751687 A CN 116751687A CN 202310655286 A CN202310655286 A CN 202310655286A CN 116751687 A CN116751687 A CN 116751687A
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- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 36
- 241000208125 Nicotiana Species 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 11
- 241000233866 Fungi Species 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 18
- 238000009630 liquid culture Methods 0.000 claims description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 8
- 239000001965 potato dextrose agar Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- 241000228212 Aspergillus Species 0.000 claims description 4
- 241001465318 Aspergillus terreus Species 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 241000223221 Fusarium oxysporum Species 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 108091023242 Internal transcribed spacer Proteins 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 230000000877 morphologic effect Effects 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 241000894007 species Species 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 229960002715 nicotine Drugs 0.000 description 13
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 12
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229930000044 secondary metabolite Natural products 0.000 description 6
- 239000008223 sterile water Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 238000000227 grinding Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- 235000019504 cigarettes Nutrition 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229960002523 mercuric chloride Drugs 0.000 description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000009335 monocropping Methods 0.000 description 2
- 230000008092 positive effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241001149711 Aspergillus lentulus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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Abstract
The application discloses endophyte G-5 for improving tobacco quality and application thereof. The strain separated by the method can improve the quality of tobacco, has the advantages of rapidness, orientation, easiness in separation and the like without time and season limitation, and has the advantages of relatively simple structure, simple metabolic components, stability and orientation.
Description
Technical Field
The application relates to the field of endophytes of tobacco, in particular to an endophyte G-5 for improving the quality of tobacco.
Background
Tobacco is an important leaf cash crop in China, and in recent years, tobacco planting production has some problems, such as: continuous cropping obstacle, disease and insect damage, poor stress resistance and the like. The endophyte of tobacco has a great number of positive effects on the cultivation, growth, production and other aspects of tobacco, and has great application value. Researches show that the endophytic fungi in tobacco have the positive effects of promoting the growth of tobacco plants, enhancing the disease resistance of the tobacco, improving the metal stress resistance of the plants, degrading nicotine, improving the aroma and the like. On the other hand, endophytic fungi of tobacco have the advantage of producing the same secondary metabolites as the host during long-term symbiotic processes with tobacco. And the quality of the tobacco is further improved through controlling the secondary metabolite.
The Yunyan 87 is one of main cultivated varieties in Yunnan province, and has great economic value in the Yunnan province and the whole country. Nicotine is an important secondary metabolite of tobacco, affects the quality of tobacco and the taste of cigarettes, however, due to uncontrollable factors such as climate, the condition that tobacco plants suffer from low-temperature low-irradiation raininess in the early stage of growth often occurs, so that the content of nicotine in tobacco is reduced, thereby affecting the quality of tobacco, and in order to effectively improve the quality of tobacco, the following processes are widely adopted in domestic fields: increasing nitrogen application amount, optimizing variety, controlling leaf number, controlling planting density and the like, but the measures also exacerbate the problems of soil hardening, continuous cropping obstacle and the like. Therefore, regulating nicotine metabolism by tobacco endophytes is an important way to improve the quality of cloud tobacco 87.
Disclosure of Invention
In order to solve the problems in the prior art, the application aims to provide endophyte G-5 for improving the quality of tobacco leaves.
In order to achieve the above purpose, the technical scheme of the application is as follows:
an endophyte G-5 for improving tobacco quality is obtained by the following steps: step one, taking healthy Yunyan 87 plants in three growth conditions of field, laboratory water culture and laboratory sterile soil culture, and separating endophytic fungi from rootstock and leaf respectively. The separation process comprises the following steps: washing with sterile water, and sterilizing the tissue surface; inoculating the tissue;
step two, solid culture: the culture temperature is 28 ℃, and the culture time is 5-7 days; liquid culture: inoculating the endophytic fungus strain into a liquid culture medium, shake culturing in a shaking table at 30 ℃ and 220rpm for 2-3 d, and purifying the obtained strain by a dilution coating flat plate method to obtain endophytic fungus G-5;
further, in the second step: comprises a solid culture medium and a liquid culture medium, wherein the solid culture medium is Potato Dextrose Agar (PDA) (46 g/L), the sterilization is carried out for 20min by high-pressure steam at 121 ℃, and streptomycin (10 mg/L) is added; liquid medium: potato dextrose broth (25 g/L), autoclaved at 121℃for 20min, and streptomycin (10 mg/L) was added.
Further, the identification method comprises the following steps: PCR amplification of rDNA ITS is carried out on the strain by using fungus universal primers ITS1/ITS4, sequencing is carried out on the ITS PCR amplification products, BLAST search is carried out on the obtained sequences in a GenBank accounting sequence database, the ITS region of the test strain is found to have highest homology with reported fusarium oxysporum (Fusarium oxysporum), aspergillus lentulus and aspergillus terreus (Aspergillus terreus), and morphological identification is combined to determine the strain G-5 of the endophytic fungus.
Further, the physicochemical properties of the endophyte G-5 are as follows: aspergillus lentulus001g-5, the bacterial colony is in an off-white villus shape, the back of the cultured bacterial colony presents white or off-white, the hypha is separated and branched, and the spore production is less. The aggregation degree of the solid culture colonies is low, and the solid culture colonies are integrally paved on the surface of a culture medium. The liquid culture bacteria have large sphere volume and are not easy to separate.
Compared with the prior art, the application has the beneficial effects that:
the application relates to endophyte G-5 for improving tobacco quality; the separated strain can produce the same chemical components as the host cells, has the advantages of rapidness, easiness in separation and the like without time and season limitation, and has simple, stable and directional fungus metabolism components.
Drawings
Fig. 1: tobacco seedling tray and seeding mode;
fig. 2: morphology of 1 strain of cloud 87 endophytic fungi;
fig. 3:1, fermenting a graph of the endophytic fungi of the Yunyan 87;
fig. 4: HPLC-nicotine profile.
Detailed Description
The technical scheme of the application is further described in detail below with reference to the attached drawings and the detailed description:
as shown in fig. 1-4:
EXAMPLE 1 isolation and purification of the endophytic fungi of Yunyan 87
The separation of endophytic fungi comprises:
A. and (3) material treatment: the leaves, stems and roots of the Yunyan 87 plants collected under different conditions are divided into six parts of upper leaves, middle leaves, lower leaves, stems, thick roots and fibrous roots;
B. material disinfection: tobacco leaf: soaking in 75% alcohol for 30 s-0.1% mercuric chloride for 15 s-washing with sterile water for 3 times; tobacco stems: wiping epidermis-remaining cortex and center column with 75% alcohol; root: soaking in 75% alcohol for 60 s-0.1% mercuric chloride for 20 s-washing with sterile water for 3 times, collecting sterile water after the last washing as a control, and coating into a blank culture medium to test the disinfection condition of a sample;
C. inoculating, separating and purifying: adding sterile water into the tobacco leaf part for grinding treatment, and uniformly laying grinding liquid and residues on the surface of a culture medium; cutting the stem part of the cigarette into small pieces with the diameter of 5-8 mm by an inoculating knife, and laying the small pieces on the surface of a culture medium; cutting the thick root part into small sections with the diameter of 3-5 mm by an inoculating knife, and laying the section on the surface of a culture medium downwards; adding sterile water into the fibrous root part for grinding treatment, uniformly laying grinding fluid and residues on the surface of a culture medium, placing all materials into a constant temperature incubator at 28 ℃ for culturing for 5-7 days, and observing the growth condition of bacterial colonies. The method adopted in the purification is a dilution coating flat plate method, and the strain with faster growth can be preferentially separated and purified due to different propagation speeds of different fungi, and the strain with slower growth is separated and purified after the strain is stabilized.
EXAMPLE 2 fermentation culture of Yunyan 87 endophytic fungi
Selecting a small amount of mycelia from the Yunyan 87 endophytic fungi culture medium cultured in the embodiment 1, adding the mycelia into 100ml of liquid culture medium, filling the liquid culture medium into a 250ml conical flask, sealing a bottle mouth, performing shake culture on a shaking table at the culture temperature of 30 ℃ and the rotation speed of 220rpm, and culturing for 2-3 d;
the formula of the liquid culture medium comprises the following components: potato dextrose broth (25 g/L), autoclaved at 121℃for 20min, and streptomycin at a concentration of 10mg/L was added after the temperature had been reduced to room temperature.
EXAMPLE 3 HPLC method for detecting the Secondary metabolite Nicotine of endophytic fungi
Preparation of standard solution: taking 1mg of nicotine standard substance, precisely weighing, and adding 1ml of methanol for dissolution.
Preparation of sample solution: filtering the endophytic fungus fermentation broth in example 2, removing mycelium, taking 10ml of fungus liquid, freeze-drying, dissolving the dried product with 1.5ml of methanol, and filtering with a 0.45 μm filter membrane.
Nicotine standard and endophytic fungus secondary metabolite chromatographic detection conditions: the detection instrument is an Agilent HPLC-1260 Infinicity high performance liquid chromatograph; column C18 (4.6 mm. Times.250 mm,5 μm); the mobile phase was eluted with a gradient of acetonitrile/ammonium formate solution (10% acetonitrile, 0.02mol/L ammonium formate solution as solvent) as A and acetonitrile as B as specified in the table; the flow rate is 1ml/min; the column temperature is 35 ℃; the detection wavelength was 260nm. Gradient elution table:
and (3) measuring: the components of the fermentation broth extract of the endophytic fungi in tobacco are detected by HPLC, and the result shows that the fermentation broth extract with the peak time of 15.539,Aspergillus lentulus001g-5 of the nicotine standard substance has a peak at 15.746, and the retention time of the fermentation broth extract of the endophytic fungi in 1 strain is basically consistent with that of the nicotine standard substance, which indicates that the metabolite of the endophytic fungi in 1 strain contains nicotine.
By comparing the high performance liquid phase method with the known standard substances, the secondary metabolite of the 1-strain endophytic fungi contains a large amount of nicotine, thereby providing possibility for improving the nicotine content in the flue-cured tobacco. The planting environment is not destroyed, and the cost of time, materials and the like is saved.
Compared with the prior art, the strain separated by the method can generate the same chemical components as host cells, has the advantages of rapidness, orientation, easiness in separation and the like without time and season limitation, and relates to fungi with a relatively simple structure, and the metabolic components are simple, stable and oriented. The technology can provide thought for improving tobacco quality and scientific basis for the relativity of the synthesis of the active ingredients of the endophyte and the host cell.
Various modifications may be made to the above disclosure by those skilled in the art without departing from the spirit and scope of the application as defined by the appended claims. The scope of the application is therefore not limited to the above description, but is instead determined by the scope of the claims.
The foregoing is merely illustrative of specific embodiments of the present application, and the scope of the application is not limited thereto, but any changes or substitutions that do not undergo the inventive effort should be construed as falling within the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope defined by the claims.
Claims (4)
1. An endophyte G-5 for improving tobacco quality is characterized by comprising the following steps: firstly, taking healthy cloud tobacco 87 plants naturally growing in the field, and separating endophytic fungi from six parts of thick roots, fibrous roots, stems, upper leaves, middle leaves and lower leaves;
step two, solid culture: the culture temperature is 28 ℃, and the culture time is 5-7 days; liquid culture: the endophyte strain is inoculated into a liquid culture medium, shake culture is carried out on a shaking table at the culture temperature of 30 ℃ and the rotation speed of 220rpm for 48-72 hours, and the obtained strain is separated, thus obtaining the endophyte G-5.
2. The endophyte G-5 of claim 1, wherein: in the second step: comprises a solid culture medium and a liquid culture medium, wherein the solid culture medium is Potato Dextrose Agar (PDA) (46 g/L), the sterilization is carried out for 20min by high-pressure steam at 121 ℃, and streptomycin (10 mg/L) is added; liquid medium: potato dextrose broth (25 g/L), autoclaved at 121℃for 20min, and streptomycin (10 mg/L) was added.
3. The endophyte G-5 of claim 1, wherein: the identification method comprises the following steps: PCR amplification of the strain with fungus universal primers ITS1/ITS4 and sequencing of the ITSPCR amplified product, BLAST search of the obtained sequence in GenBank accounting sequence database, found that ITS region of the test strain was the highest in homology with reported Fusarium oxysporum, aspergillus candidum, aspergillus terreus (Aspergillus terreus), and species G-5 of endophyte, respectively, in combination with morphological identification.
4. The endophyte G-5 of claim 3, wherein: the physicochemical properties of the endophyte G-5 are as follows: aspergillus lentus 001g-5, the colony is gray villus, the back surface is white or gray, the hypha is separated and branched, and the spore production is less.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310655286.6A CN116751687A (en) | 2023-06-05 | 2023-06-05 | Endophyte G-5 for improving tobacco quality |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310655286.6A CN116751687A (en) | 2023-06-05 | 2023-06-05 | Endophyte G-5 for improving tobacco quality |
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| Publication Number | Publication Date |
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| CN116751687A true CN116751687A (en) | 2023-09-15 |
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