Endophytic fungus I-26 for promoting growth of tobacco seedlings and application of endophytic fungus I-26
By isolating and identifying Talaromyces miniuluteus 001I-26 and pouring its bacterial solution into tobacco seedlings, the problem of insufficient research on tobacco endophytic fungi in the prior art has been solved, significantly promoted the growth of tobacco seedlings, and provided a new method to improve the production quality of tobacco leaf.
Patent Information
- Application Number
- CN202510040006.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art has little research on tobacco in the study of tobacco endophytic fungi, and it is difficult to effectively promote the growth of tobacco seedlings.
An endophytic fungus I-26 was isolated and identified, belonging to Talaromyces miniuluteus, and the bacterial solution was prepared by liquid culture and poured into the upper ground and root matrix soil of the tobacco seedlings.
It has significantly promoted the growth of different varieties of tobacco seedlings, including plant height, maximum leaf length, maximum leaf width, root length, fresh weight and above ground fresh weight, providing a new way to promote the quality of tobacco leaf production.
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Figure CN119931842A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the research field of tobacco endophytic fungi, and in particular to an endophytic fungus I-26 for promoting tobacco seedling growth and application thereof. Background Art
[0002] Plant endophytes refer to microorganisms that live inside various tissues and organs or in the interstitial spaces of healthy plants at certain or all stages of their life history, generally including endophytic bacteria, endophytic fungi and endophytic actinomycetes.
[0003] At present, there are more than 120 species of endophytic bacteria isolated from plants, belonging to 54 genera, of which the most common are Pseudomonas, Bacillus, Enterobacter and Agrobacterium. In the study of endophytic actinomycetes, the most research is on Frankia, while the research on endophytic fungi is mostly on grasses, medicinal plants, marine plants, etc., but there are few studies on tobacco.
[0004] Endophytes live in tobacco for a long time, and the two have co-evolved. Studies have shown that tobacco endophytes can produce growth-promoting substances, help tobacco absorb mineral nutrients, and promote tobacco growth. Saito et al. found that Clostridium sp. contains a large amount of nitrogenase, which can play a nitrogen-fixing role in tobacco, provide inorganic nitrogen sources for tobacco, and thus improve the yield and quality of tobacco leaves. Yang Zhenfu et al. isolated 3 strains of Bacillus amyloliquefaciens from tobacco, which had a certain growth-promoting effect on tobacco seedlings. Jin Huiqing et al. showed that Fusarium strain YCEF005 and Acanthopanax strains YCEF053, YCEF191, YCEF193, and YCEF199 can improve the biomass and physiological indicators of tobacco plants. Therefore, tobacco endophytic fungi are a new way to improve tobacco leaf disease resistance, stress resistance, and improve tobacco leaf production quality.
[0005] In our study on the endophytic fungi flora of the dominant tobacco variety Yunyan 87 in Yunnan, a total of 103 endophytic fungi, 52 species, belonging to 29 genera, were isolated from Yunyan 87 tobacco plants picked in the field. Among them, there were 11 endophytic fungi of 10 genera in the upper leaves, 8 endophytic fungi of 7 genera in the middle leaves, 8 endophytic fungi of 6 genera in the lower leaves, 11 endophytic fungi of 9 genera in the stems, 21 endophytic fungi of 12 genera in the thick roots, and 7 endophytic fungi of 5 genera in the fibrous roots. After screening, a total of 7 endophytic fungi significantly promoted the biomass and trait indicators of tobacco seedlings of different tobacco varieties. Summary of the invention
[0006] In order to solve the problems existing in the above-mentioned prior art, the object of the present invention is to provide an endophytic fungus I-26 for promoting the growth of tobacco seedlings and its application.
[0007] To achieve the above object, the technical solution of the present invention is:
[0008] An endophytic fungus I-26 that promotes the growth of tobacco seedlings, the endophytic fungus being: Talaromyces minioluteus 001I-26; the deposit number of the strain is: CGMCC N0.41397; it was deposited on July 5, 2024 in the General Microbiology Center of the China Culture Collection Administration, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.
[0009] The strain was obtained by the following method:
[0010] A. Materials: The Yunyan 87 plants collected from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;
[0011] B. Disinfection: Upper leaves, middle leaves, and lower 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; Thick roots and fibrous 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 streaked on the plate as a control to check whether the sample is thoroughly disinfected;
[0012] C. Inoculation and separation: 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, and culture all the materials in a 28℃ constant temperature incubator for 5-7 days to observe the growth of the colony. 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.
[0013] Furthermore, the culture is specifically as follows: using potato dextrose agar medium (PDA) (46 g / L), adding streptomycin (10 mg / L) to prevent bacterial growth, the culture temperature is 28° C., and the culture time is 5 to 7 days;
[0014] The isolated endophytic fungi are inoculated into PDA culture medium for purification, and the purification is repeated 2 to 3 times until the colonies are single, and the functional endophytic fungi that promote the growth of tobacco seedlings are obtained after screening.
[0015] Furthermore, the endophytic fungus identification step is: using fungal universal primers ITS1 / ITS4 to perform PCR amplification of rDNA ITS of the strain, and sequencing the ITS PCR amplification product, performing BLAST search on the obtained sequence in the GenBank nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported Talaromyces minioluteus, and combined with morphological identification, the species of the endophytic fungus of the strain is determined.
[0016] Furthermore, the physical and chemical properties of the Talaromyces minioluteus 001I-26 are that the colonies are similar to those of Penicillium, the surface is discolored, brown and slightly concave, the hyphae are white to yellow, some of the hyphae are sparse to dense, and there are dark green spores on the surface.
[0017] Furthermore, the preparation method of the endophytic fungus I-26 bacterial liquid that promotes the growth of tobacco seedlings is to inoculate the endophytic fungus I-26 strain into 100 ml of liquid culture medium, put it into a 250 ml conical flask, seal the bottle mouth with a film, and culture it on a shaker with a culture temperature of 30° C., a rotation speed of 220 rpm, and a culture time of 48 to 72 hours; the liquid culture medium: potato glucose broth (25 g / L), added with streptomycin (10 mg / L) for culture.
[0018] The endophytic fungus I-26 and bacterial solution for promoting the growth of tobacco seedlings are used for pouring into the above-ground part and root substrate soil of different varieties of tobacco seedlings to promote the growth of tobacco seedlings.
[0019] Furthermore, the bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings were tested two weeks later.
[0020] Furthermore, the biomass and agronomic traits to be detected include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.
[0021] Compared with the prior art, the beneficial effects of the present invention are:
[0022] The endophytic fungus I-26 that promotes the growth of tobacco seedlings and its applications were inoculated into four different varieties of tobacco seedlings, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97. Based on the evaluation of seven indicators such as tobacco seedling biomass and agronomic traits, the seven strains of endophytic fungi were further confirmed to promote the growth of tobacco seedlings, providing basic support for their further application in production. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Tobacco seedling trays and sowing methods;
[0024] Figure 2This is the morphology of the endophytic fungus of strain Yunyan 87;
[0025] Figure 3 This is a fermentation diagram of endophytic fungi of Yunyan 87;
[0026] Figure 4 The endophytic fungus of tobacco promotes the growth of tobacco seedlings of different varieties;
[0027] Figure 5 To compare the growth of tobacco seedlings inoculated with endophytic fungi and those not inoculated with endophytic fungi. DETAILED DESCRIPTION
[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:
[0029] like Figure 1-5 As shown, an endophytic fungus I-26 that promotes the growth of tobacco seedlings, the endophytic fungus is: Talaromyces minioluteus 001I-26; the preservation number of the strain is: CGMCC N0.41397; on July 5, 2024, it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0030] The strain was obtained by the following method:
[0031] D. Materials: The Yunyan 87 plants collected from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;
[0032] E. Disinfection: Upper leaves, middle leaves, and lower 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; Thick roots and fibrous 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 streaked on the plate as a control to check whether the sample is thoroughly disinfected;
[0033] F. Inoculation and separation: 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 downward 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, and culture all the materials in a 28℃ constant temperature incubator for 5-7 days to observe the growth of the colony. 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.
[0034] Furthermore, the culture is specifically as follows: using potato dextrose agar medium (PDA) (46 g / L), adding streptomycin (10 mg / L) to prevent bacterial growth, the culture temperature is 28° C., and the culture time is 5 to 7 days;
[0035] The isolated endophytic fungi are inoculated into PDA culture medium for purification, and the purification is repeated 2 to 3 times until the colonies are single, and the functional endophytic fungi that promote the growth of tobacco seedlings are obtained after screening.
[0036] Furthermore, the endophytic fungus identification step is: using fungal universal primers ITS1 / ITS4 to perform PCR amplification of rDNA ITS of the strain, and sequencing the ITS PCR amplification product, performing BLAST search on the obtained sequence in the GenBank nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported Talaromyces minioluteus, and combined with morphological identification, the species of the endophytic fungus of the strain is determined.
[0037] Furthermore, the physical and chemical properties of the Talaromyces minioluteus 001I-26 are that the colonies are similar to those of Penicillium, the surface is discolored, brown and slightly concave, the hyphae are white to yellow, some of the hyphae are sparse to dense, and there are dark green spores on the surface.
[0038] Furthermore, the preparation method of the endophytic fungus I-26 bacterial liquid that promotes the growth of tobacco seedlings is to inoculate the endophytic fungus I-26 strain into 100 ml of liquid culture medium, put it into a 250 ml conical flask, seal the bottle mouth with a film, and culture it on a shaker with a culture temperature of 30° C., a rotation speed of 220 rpm, and a culture time of 48 to 72 hours; the liquid culture medium: potato glucose broth (25 g / L), added with streptomycin (10 mg / L) for culture.
[0039] The endophytic fungus I-26 and bacterial solution for promoting the growth of tobacco seedlings are used for pouring into the above-ground part and root substrate soil of different varieties of tobacco seedlings to promote the growth of tobacco seedlings.
[0040] Furthermore, the bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings were tested two weeks later.
[0041] Furthermore, the biomass and agronomic traits to be detected include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.
[0042] Example: Cultivation of plant material;
[0043] The invention selects four different varieties of tobacco coated seeds, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97, and sows them on a 3*4 small seedling tray (such as Figure 2 ), 3 seeds were sown in each hole, the nutrient soil used for seedling cultivation was sterilized by high pressure, and the cells were cultured in an incubator under alternating light and darkness, with 16 hours of light cultivation, 18000LX of light intensity, and 28°C; and 8 hours of dark cultivation, at 16°C; nutrient solution was added to the bottom of the seedling tray, and the nutrient solution needed to be replenished every two days.
[0044] Application of endophytic fungi liquid
[0045] Take 1.5 cm 2 A fungus block about the size of about 1 ml was inoculated into a 100 ml liquid culture medium, which was placed in a 250 ml conical flask and cultured in a shaking incubator at a temperature of 30 ° C, a rotation speed of 220 rpm, and a culture time of 48 to 72 h. When the tobacco seedlings grew to 5 to 6 true leaves, the bacterial solution was taken for watering of the above-ground part and root soil of the tobacco seedlings. Before watering, one tobacco seedling was retained in each hole in the seedling tray to ensure that the growth of each tray of tobacco seedlings was consistent. 3 ml of bacterial solution was taken from each hole and watered on the tobacco seedlings, and the bacterial solution was shaken evenly before taking. After watering, a transparent isolation cover was placed on the top of the seedling tray and the tray was placed in an incubator for continued cultivation. A control tobacco seedling was also set up, which was cultured normally without watering with bacterial solution. When the tobacco seedlings grew to 7 true leaves, a total of 7 indicators including fresh weight, root weight, above-ground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length of the tobacco seedlings were measured.
[0046] Effect of Talaromyces minioluteus 001I-26 on the growth of tobacco seedlings of different varieties
[0047] The results showed that 001I-26 had a significant growth-promoting effect. Compared with the control, the plant height growth rates of the four tobacco varieties Yunyan 97, Yunyan 87, K326 and Honghua Dajinyuan inoculated with 001I-26 were 42.65%, 71.66%, 50.55% and 40.31% respectively; the maximum leaf length growth rates were 36.11%, 30.13%, 24.00% and 24.21% respectively; the maximum leaf width growth rates were 24.36%, 26.70% and 26.71% respectively. %, 7.70% and 28.17% respectively; the growth rates of root length were 25.83%, 19.69%, 16.28% and 5.77% respectively; the growth rates of fresh weight were 39.03%, 53.45%, 56.03% and 28.41% respectively; the growth rates of root fresh weight were 18.36%, 18.16%, 26.64% and 16.19% respectively; the growth rates of aboveground fresh weight were 42.78%, 59.00%, 60.55% and 31.10% respectively.
[0048] 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 endophytic fungus I-26 for promoting tobacco seedling growth, characterized in that: The endophytic fungi are: Talaromyces vermilionii 001I-26; The deposit number of this strain is: CGMCC N0.41397; it was deposited on July 5, 2024 at the General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The strain was obtained by the following method: A. Materials: The Yunyan 87 plants collected from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots; B. Disinfection: Upper leaves, middle leaves, and lower 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; Thick roots and fibrous 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 streaked on the plate as a control to check whether the sample is thoroughly disinfected; C. Inoculation and separation: add sterile water to the tobacco leaf part and grind it, 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 them on the surface of the culture medium with the cut surface facing down; add sterile water to the fibrous root part and grind it, and evenly spread the grinding liquid and residue on the surface of the culture medium. Culture all the materials in a constant temperature incubator at 28℃ for 5-7 days, and observe the growth of the colony. 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.
2. The endophytic fungus I-26 for promoting tobacco seedling growth according to claim 1, characterized in that: The culture is specifically as follows: using potato dextrose agar medium (PDA) (46 g / L), adding streptomycin (10 mg / L) to prevent bacterial growth, the culture temperature is 28° C., and the culture time is 5 to 7 days; The isolated endophytic fungi are inoculated into PDA culture medium for purification, and the purification is repeated 2 to 3 times until the colonies are single, and the functional endophytic fungi that promote the growth of tobacco seedlings are obtained after screening.
3. The endophytic fungus I-26 for promoting tobacco seedling growth according to claim 1, characterized in that: The endophytic fungus identification step is: using fungal universal primers ITS1 / ITS4 to perform PCR amplification of rDNA ITS of the strain, and sequencing the ITS PCR amplification product, performing BLAST search on the obtained sequence in the GenBank nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported red-yellow tularensis, combined with morphological identification, to determine the species of the endophytic fungus of the strain.
4. The endophytic fungus I-26 for promoting tobacco seedling growth according to claim 1, characterized in that: The physical and chemical properties of the Talaromyces minioluteus 001I-26 are that the colonies are similar to those of Penicillium, the surface is discolored, brown and slightly concave, the hyphae are white to yellow, some hyphae are sparsely to densely formed, and there are dark green spores on the surface.
5. The endophytic fungus I-26 for promoting tobacco seedling growth according to claim 1, characterized in that: The preparation method of the endophytic fungus I-26 bacterial liquid for promoting tobacco seedling growth comprises the following steps: inoculating the endophytic fungus I-26 strain into 100 ml of liquid culture medium, placing the liquid culture medium into a 250 ml conical flask, sealing the flask mouth with a film, and culturing the liquid culture medium on a shaker at a temperature of 30° C., a rotation speed of 220 rpm, and a culturing time of 48 to 72 hours; the liquid culture medium is prepared by adding potato glucose broth (25 g / L) and streptomycin (10 mg / L) for culturing.
6. The endophytic fungus I-26 and bacterial solution for promoting the growth of tobacco seedlings according to any one of claims 1 to 5 are used for pouring into the soil matrix of the aboveground part and root part of tobacco seedlings of different varieties to promote the growth of tobacco seedlings.
7. The use according to claim 6, characterized in that: The bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings were tested two weeks later.
8. The use according to claim 7, characterized in that: The biomass and agronomic traits to be detected include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.