Endophyte G-5 for improving tobacco quality

Abstract

The invention discloses an endophyte G-5 for improving tobacco quality. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) The strain is preserved in China General Microbiological Culture Collection Center on July 05, 2024, and the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the strain separated by the method can improve the tobacco quality and has the advantages of being free of time and season limitation, rapid, oriented, easy to separate and the like, the selected fungus is relatively simple in structure, metabolic components are simple, stable and oriented, and the endophytic fungus strain can be used for industrial production of tobacco, improves the content of nicotine in the tobacco and meets market requirements.

Description

Technical Field

[0001] The invention relates to the field of tobacco endophytes, and in particular to an endophyte G-5 for improving tobacco quality. Background Art

[0002] Tobacco is an important leaf-producing economic crop in my country. In recent years, there have been some problems in tobacco planting and production, such as continuous cropping obstacles, pests and diseases, and poor stress resistance. Endophytes in tobacco have many positive effects on the cultivation, growth and production of tobacco, and have great application value. Studies have shown that endophytic fungi in tobacco have positive effects such as promoting tobacco plant growth, enhancing tobacco disease resistance, improving plant resistance to metal stress, degrading nicotine, and increasing aroma. On the other hand, endophytic fungi in tobacco have the advantage of producing the same secondary metabolites as the host in the long-term symbiotic process with tobacco. And by controlling secondary metabolites, the quality of tobacco can be further improved.

[0003] As one of the main varieties in Yunnan Province, Yunyan 87 has great economic value in Yunnan Province and nationwide. Nicotine is an important secondary metabolite of tobacco, which affects the quality of tobacco and the taste of cigarettes. However, due to uncontrollable factors such as climate, tobacco plants often suffer from low temperature, little sunlight and heavy rain in the early growth period, which leads to a decrease in the nicotine content of tobacco, thus affecting the quality of tobacco. In order to effectively improve the quality of tobacco, measures such as increasing nitrogen application, selecting varieties, controlling the number of leaves, and controlling planting density have been widely adopted in China. However, these measures have also aggravated problems such as soil compaction and continuous cropping obstacles. Therefore, regulating nicotine metabolism through tobacco endophytes has become an important way to improve the quality of Yunyan 87. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the object of the present invention is to provide an endophyte G-5 and for improving the quality of tobacco smoke.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] An endophyte G-5 for improving tobacco quality, the deposit number of the strain is: CGMCC N0.41400; it was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 5, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the method for obtaining it is as follows: Step 1, take healthy Yunyan 87 plants grown under three growth conditions: field, laboratory hydroponics, and laboratory sterile soil culture, and separate the endophytic fungi from the roots, stems, and leaves. The specific separation process is: sterile water washing, tissue surface disinfection; tissue inoculation;

[0007] Step 2: solid culture: culture temperature 28°C, culture time 5-7 days; liquid culture: inoculate the above endophytic fungal strain into liquid culture medium, culture on a shaker, culture temperature 30°C, rotation speed 220rpm, culture time 2-3d, separate and culture the obtained strains and purify them using the dilution plate method to obtain the endophytic bacteria G-5 described in the present application;

[0008] Furthermore, in step 2: it includes solid culture medium and liquid culture medium, the solid culture medium uses potato dextrose agar medium (PDA) (46 g / L), sterilized by high pressure steam at 121°C for 20 min, and adding streptomycin (10 mg / L); liquid culture medium: potato dextrose broth (25 g / L), sterilized by high pressure steam at 121°C for 20 min, and adding streptomycin (10 mg / L).

[0009] Furthermore, the identification method is as follows: the rDNA ITS of the strain is amplified by PCR using the universal fungal primers ITS1 / ITS4, and the ITS PCR amplification product is sequenced. The obtained sequence is searched by BLAST in the GenBank nuclear sequence database, and it is found that the ITS region of the test strain has the highest homology with the reported Fusarium oxysporum, Aspergillus lentulus, and Aspergillus terreus, respectively. Combined with morphological identification, the species of the endophytic fungus G-5 is determined.

[0010] Furthermore, the physical and chemical properties of the endophyte G-5 are as follows: Aspergillus lentulus 001g-5, the colony is grayish white and fluffy, the back of the cultured colony is white or grayish white, the hyphae are separated and bifurcated, and the spore production is small. The solid culture colony has a low aggregation degree and is flat on the surface of the culture medium. The liquid culture ball is large in size and difficult to separate.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] The invention discloses an endophyte G-5 for improving tobacco quality. The isolated strain can produce the same chemical components as host cells, and has the advantages of being fast and easy to separate without being restricted by time and season. The metabolic components of the fungus are simple, stable and directional. The technology can provide ideas for the development of endophytic fungi in tobacco and the improvement of tobacco quality, and also provide a scientific basis for the correlation between the synthesis of effective components of endophytes and their host cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Tobacco seedling trays and sowing methods;

[0014] Figure 2 Morphology of endophytic fungi of 1 strain Yunyan 87;

[0015] Figure 3 1 Endophytic fungal fermentation diagram of Yunyan 87;

[0016] Figure 4 It is the HPLC-nicotine graph. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0018] like Figure 1-4 As shown: An endophyte G-5 for improving tobacco quality, the deposit number of the strain is: CGMCCN0.41400; it was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 5, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the method for obtaining it is as follows: Step 1, take healthy Yunyan 87 plants grown under three growth conditions: field, laboratory hydroponics, and laboratory sterile soil culture, and separate the endophytic fungi from the roots, stems, and leaves. The specific separation process is: sterile water washing, tissue surface disinfection; tissue inoculation;

[0019] Step 2: solid culture: culture temperature 28°C, culture time 5-7 days; liquid culture: inoculate the above endophytic fungal strain into liquid culture medium, culture on a shaker, culture temperature 30°C, rotation speed 220rpm, culture time 2-3d, separate and culture the obtained strains and purify them using the dilution plate method to obtain the endophytic bacteria G-5 described in the present application;

[0020] Furthermore, in step 2: it includes solid culture medium and liquid culture medium, the solid culture medium uses potato dextrose agar medium (PDA) (46 g / L), sterilized by high pressure steam at 121°C for 20 min, and adding streptomycin (10 mg / L); liquid culture medium: potato dextrose broth (25 g / L), sterilized by high pressure steam at 121°C for 20 min, and adding streptomycin (10 mg / L).

[0021] Furthermore, the identification method is as follows: the rDNA ITS of the strain is amplified by PCR using the universal fungal primers ITS1 / ITS4, and the ITS PCR amplification product is sequenced. The obtained sequence is searched by BLAST in the GenBank nuclear sequence database, and it is found that the ITS region of the test strain has the highest homology with the reported Fusarium oxysporum, Aspergillus lentulus, and Aspergillus terreus, respectively. Combined with morphological identification, the species of the endophytic fungus G-5 is determined.

[0022] Furthermore, the physical and chemical properties of the endophyte G-5 are as follows: Aspergillus lentulus 001g-5, the colony is grayish white and fluffy, the back of the cultured colony is white or grayish white, the hyphae are separated and bifurcated, and the spore production is small. The solid culture colony has a low aggregation degree and is flat on the surface of the culture medium. The liquid culture ball is large in size and difficult to separate.

[0023] Example: Isolation and purification of endophytic fungi from Yunyan 87

[0024] Isolation of endophytic fungi includes:

[0025] A. Material processing: The leaves, stems and roots of Yunyan 87 plants harvested under different conditions were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots and fibrous roots;

[0026] B. Material disinfection: Tobacco leaves: soak in 75% alcohol for 30 seconds, soak in 0.1% mercuric chloride for 15 seconds, and rinse with sterile water for 3 times; Tobacco stems: wipe the epidermis with 75% alcohol, and keep the cortex and pith; Roots: soak in 75% alcohol for 60 seconds, soak in 0.1% mercuric chloride for 20 seconds, and rinse with sterile water for 3 times. The sterile water after the last rinse is collected as a control and applied to the blank culture medium to check the disinfection of the samples;

[0027] C. Inoculation, separation and purification: Add sterile water to the tobacco leaf part for grinding, and evenly spread the grinding liquid and residue on the surface of the culture medium; cut the tobacco stem part into 5-8mm small pieces with an inoculation knife and spread them on the surface of the culture medium; cut the thick root part into 3-5mm small sections with an inoculation knife, and spread the cut surface down on the surface of the culture medium; add sterile water to the fibrous root part for grinding, and evenly spread the grinding liquid and residue on the surface of the culture medium. All materials are placed in a 28℃ constant temperature incubator for 5-7 days to observe the growth of the colony. The purification method used is the dilution plate method. Due to the different reproduction rates of different fungi, the faster growing strains can be separated and purified first, and the slower growing strains can be separated and purified after they are stable.

[0028] Fermentation culture of endophytic fungi of Yunyan 87

[0029] Pick a small amount of mycelium from the culture medium of Yunyan 87 endophytic fungi and add it to 100ml liquid culture medium, put it into a 250ml conical bottle, seal the bottle mouth with a film, and culture it on a shaker at a temperature of 30℃, a speed of 220rpm, and a culture time of 2 to 3 days;

[0030] The liquid culture medium has a formula of: potato dextrose broth (25 g / L), sterilized by high pressure steam at 121° C. for 20 min, and then adding streptomycin after the temperature drops to room temperature, wherein the concentration of streptomycin is 10 mg / L.

[0031] Determination of nicotine as a secondary metabolite of endophytic fungi by HPLC

[0032] Preparation of standard solution: Take 1 mg of nicotine standard, weigh accurately, add 1 ml of methanol to dissolve.

[0033] Preparation of sample solution: Filter the mycelium from the fermentation liquid of endophytic fungi, take 10 ml of the bacterial liquid, freeze-dry it, dissolve the dry matter in 1.5 ml of methanol, and filter it through a 0.45 μm filter membrane to obtain the sample solution.

[0034] Chromatographic detection conditions of nicotine standard and endophytic fungal secondary metabolites: The detection instrument is Agilent HPLC-1260 Infinity high performance liquid chromatograph; chromatographic column C18 (4.6mm×250mm, 5μm); the mobile phase is acetonitrile / ammonium formate solution (10% acetonitrile, the solvent is 0.02mol / L ammonium formate solution) as A, acetonitrile as B, and gradient elution is performed according to the table; the flow rate is 1ml / min; the column temperature is 35℃; the detection wavelength is 260nm. Gradient elution table:

[0035]

[0036] Determination: HPLC was used to detect the components of the tobacco endophytic fungus fermentation broth extract. The results are as follows: Figure 4 The results showed that the peak time of nicotine standard was 15.539, the fermentation broth extract of Aspergillus lentulus 001g-5 had a peak at 15.746, and the retention time of the fermentation broth extract of an endophytic fungus was basically consistent with that of the nicotine standard, indicating that the metabolites of this endophytic fungus contained nicotine.

[0037] By comparing with known standards through the high performance liquid chromatography method, it was shown that the secondary metabolites of this endophytic fungus contained a large amount of nicotine, which provided a possibility for increasing the nicotine content in flue-cured tobacco, without destroying the planting environment and saving time, materials and other costs.

[0038] Compared with the prior art, the strains isolated by the method of the present invention can produce the same chemical components as the host cells, and have the advantages of being fast, directional, and easy to separate without time and season restrictions. The structure of the fungi involved is relatively simple, and the metabolic components are simple, stable, and directional. This technology can provide ideas for improving the quality of tobacco leaves, and also provide a scientific basis for the correlation between endophytes and the synthesis of effective components of their host cells.

[0039] Those skilled in the art may make various modifications to the above content without departing from the spirit and scope of the present invention as defined in the claims. Therefore, the scope of the present invention is not limited to the above description, but is determined by the scope of the claims.

[0040] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. An endophyte G-5 for improving tobacco quality, characterized in that: The deposit number of the strain is: CGMCCN0.41400; it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 5, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the method for obtaining it is as follows: Step 1, take healthy Yunyan 87 plants growing naturally in the field, and isolate endophytic fungi from six parts, namely, thick roots, fibrous roots, stems, upper leaves, middle leaves, and lower leaves; Step 2, solid culture: culture temperature 28 ° C, culture time 5 to 7 days; liquid culture: the above endophytic fungal strain is inoculated into liquid culture medium, cultured on a shaker, culture temperature 30 ° C, rotation speed 220 rpm, culture time 48 to 72 hours, and the obtained strain is separated to obtain the endophyte G-5 described in the present application.

2. The endophyte G-5 according to claim 1, characterized in that: In step 2: It includes solid culture medium and liquid culture medium. The solid culture medium uses potato dextrose agar medium (PDA) (46g / L), which is sterilized by high-pressure steam at 121°C for 20 minutes and streptomycin (10mg / L) is added; the liquid culture medium: potato dextrose broth (25g / L), which is sterilized by high-pressure steam at 121°C for 20 minutes and streptomycin (10mg / L) is added.

3. The endophyte G-5 according to claim 1, characterized in that: The identification method is as follows: using fungal universal primers ITS1 / ITS4 to perform PCR amplification of rDNAITS of the strain, and sequencing the ITS PCR amplification product, and performing BLAST search on the obtained sequence in the GenBank nuclear sequence database, it was found that the ITS region of the test strain had the highest homology with the reported Fusarium oxysporum, Aspergillus lentulus, and Aspergillus terreus, respectively. Combined with morphological identification, the species of endophytic fungus G-5 was determined.

4. The endophyte G-5 according to claim 3, characterized in that: The physicochemical properties of the endophyte G-5 are as follows: Aspergillus lentulus 001g-5, the colony is grayish white and fluffy, the back is white or grayish white, the hyphae are separated and bifurcated, and the spore production is small.

Images