Tobacco endophytic fungi for promoting tobacco seedling growth and application thereof

Abstract

The invention discloses a tobacco endophytic fungus for promoting growth of tobacco seedlings and application of the tobacco endophytic fungus. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) Tobacco is a leaf commercial crop, tobacco planting and production are often limited by multiple factors such as diseases and insect pests, heavy metal stress and drought stress in recent years, tobacco plants grow slowly in the seedling stage or the field stage, and the yield and quality of tobacco leaves are seriously threatened. The endophytic fungus liquid separated from K326 is applied to soil at the upper parts and roots of different varieties of tobacco seedlings, so that the biomass and agronomic characters of the tobacco seedlings can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the field of tobacco endophytic fungi, in particular to tobacco endophytic fungi for promoting tobacco seedling growth and applications 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 yield and quality.

[0005] In our study on the endophytic fungal flora of Yunnan tobacco dominant varieties K326 and Yunyan 87. A total of 154 endophytic fungi were isolated from tobacco K326 under different culture conditions, and identified as 40 genera and 72 species. Among them, 106 endophytic fungi were isolated from tobacco K326 under field growth conditions, identified as 37 genera and 62 species, 23 endophytic fungi were isolated from K326 cultured in water, identified as 8 genera and 11 species, and 25 endophytic fungi were isolated from K326 cultured in sterile soil, identified as 4 genera and 7 species. After screening, a total of 8 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 above problems existing in the prior art, the object of the present invention is to provide endophytic fungi that promote the growth of tobacco seedlings.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] The purpose of the present invention is to provide endophytic fungi for promoting the growth of tobacco seedlings, and to provide a new method for improving the yield and quality of tobacco leaves.

[0009] The deposit number of this strain is: CGMCC N0.41395. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 5, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as Geotrichum candidum.

[0010] Isolation of the above mentioned fungi includes:

[0011] A. Materials: K326 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, taproots, and fibrous roots;

[0012] 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;

[0013] 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.

[0014] Solid culture: Use potato dextrose agar (PDA) (46 g / L), add streptomycin (10 mg / L) to prevent bacterial growth, culture temperature 28 ° C, culture time 5 to 7 days;

[0015] The isolated different types of endophytic fungi were inoculated into PDA culture medium for purification, and the purification was repeated 2 to 3 times until the colonies were single. After screening, 8 functional endophytic fungi that promoted the growth of tobacco seedlings were obtained.

[0016] Identification of the above-mentioned endophytic fungi: rDNA ITS of the strains was amplified by PCR using the universal fungal primers ITS1 / ITS4, and the ITS PCR amplification products were sequenced. The obtained sequences were searched by BLAST in the GenBank nuclear sequence database, and it was found that the ITS region of the test strain had the highest homology with the reported Geotrichum candidum. Combined with morphological identification, the species of the three endophytic fungi were determined.

[0017] The tobacco endophytic fungi:

[0018] Geotrichum candidum W11, hyphae are septate hyphae, colonies spread out in a plane, grow fast, flat, milky white, short velvet or nearly powdery, with a central protrusion. In liquid culture, white mold, velvet or powdery.

[0019] The preparation method of the above-mentioned endophytic fungus application liquid that can promote the growth of tobacco seedlings is to inoculate the above-mentioned endophytic fungus strain into 100ml liquid culture medium, put it into a 250ml 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 220rpm, and a culture time of 48 to 72h; liquid culture medium: potato glucose broth (25g / L), added with streptomycin (10mg / L).

[0020] The method for applying the endophytic fungus solution that can promote the growth of tobacco seedlings is to evenly pour 15 ml of the endophytic fungus solution (with hyphae and spores) into the soil matrix of the above-ground part and the root part of the tobacco seedlings.

[0021] The above-mentioned fungus was inoculated into tobacco seedlings of four different varieties, 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 effect of this endophytic fungus on promoting the growth of tobacco seedlings was further confirmed, providing basic support for its further application in production.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The endophytic fungus W11 for promoting the growth of tobacco seedlings of the present invention has obvious growth-promoting effect. Compared with the control, the growth rates of plant heights of tobacco seedlings of four varieties, namely, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, inoculated with W11 are 6.13%, 1.43%, 1.30% and 1.00% respectively; the growth rates of maximum leaf lengths are 1.47%, 12.07%, 14.27% and 14.30% respectively; the growth rates of maximum leaf widths are 4.60%, 18.27%, 14.40% and 19.50% respectively; the growth rates of root lengths are 13.17%, 2.07%, 75.90% and 19.33% respectively; the growth rates of underground fresh weights are 192.17%, 66.67%, 176.53% and 23.9% respectively; and the growth rates of aboveground fresh weights are 19.23%, 31.63%, 31.03% and 22.70% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Tobacco seedling trays and sowing methods;

[0025] Figure 2 The morphology of endophytic fungi in Yunyan 87;

[0026] Figure 3 Yunyan 87 Endophytic Fungal Fermentation Diagram;

[0027] Figure 4 The promoting effect of tobacco endophytes on the growth of tobacco seedlings of different varieties;

[0028] Figure 5 Comparison of the growth of tobacco seedlings inoculated with endophytic fungi and those not inoculated with endophytic fungi. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0030] like Figure 1-5 As shown,

[0031] An endophytic fungus that promotes the growth of tobacco seedlings.

[0032] The deposit number of this strain is: CGMCC N0.41395. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 5, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as Geotrichum candidum.

[0033] Isolation of the above mentioned fungi includes:

[0034] A. Materials: K326 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, taproots, and fibrous roots;

[0035] 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;

[0036] 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.

[0037] Solid culture: Use potato dextrose agar (PDA) (46 g / L), add streptomycin (10 mg / L) to prevent bacterial growth, culture temperature 28 ° C, culture time 5 to 7 days;

[0038] The isolated different types of endophytic fungi were inoculated into PDA culture medium for purification, and the purification was repeated 2 to 3 times until the colonies were single. After screening, 8 functional endophytic fungi that promoted the growth of tobacco seedlings were obtained.

[0039] Identification of the above-mentioned endophytic fungi: rDNA ITS of the strains was amplified by PCR using the universal fungal primers ITS1 / ITS4, and the ITS PCR amplification products were sequenced. The obtained sequences were searched by BLAST in the GenBank nuclear sequence database, and it was found that the ITS region of the test strain had the highest homology with the reported Geotrichum candidum. Combined with morphological identification, the species of the three endophytic fungi were determined.

[0040] The tobacco endophytic fungi:

[0041] Geotrichum candidum W11, hyphae are septate hyphae, colonies spread out in a plane, grow fast, flat, milky white, short velvet or nearly powdery, with a central protrusion. In liquid culture, white mold, velvet or powdery.

[0042] The preparation method of the above-mentioned endophytic fungus application liquid that can promote the growth of tobacco seedlings is to inoculate the above-mentioned endophytic fungus strain into 100ml liquid culture medium, put it into a 250ml 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 220rpm, and a culture time of 48 to 72h; liquid culture medium: potato glucose broth (25g / L), added with streptomycin (10mg / L).

[0043] The method for applying the endophytic fungus solution that can promote the growth of tobacco seedlings is to evenly pour 15 ml of the endophytic fungus solution (with hyphae and spores) into the soil matrix of the above-ground part and the root part of the tobacco seedlings.

[0044] The above-mentioned fungus was inoculated into tobacco seedlings of four different varieties, 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 effect of this endophytic fungus on promoting the growth of tobacco seedlings was further confirmed, providing basic support for its further application in production.

[0045] Example: Cultivation of plant material;

[0046] 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.

[0047] Application of endophytic fungi liquid irrigation

[0048] Take a fungus block of about 1.5 cm2 obtained by separation and purification and inoculate it into 100 ml liquid culture medium. The culture medium is placed in a 250 ml conical flask and cultured in a shaking incubator. The culture temperature is 30°C, the rotation speed is 220 rpm, and the culture time is 48 to 72 hours. When the tobacco seedlings grow to 5 to 6 true leaves, take the bacterial solution to water the aboveground part and root soil of the tobacco seedlings. Before watering, keep one tobacco seedling in each hole in the seedling tray to ensure that the growth of each tobacco seedling is consistent. Take 3 ml of bacterial solution from each hole and water the tobacco seedlings. Shake the bacterial solution evenly before taking it. After watering, cover the top of the seedling tray with a transparent isolation cover and continue to culture in an incubator. Set up a control tobacco seedling, culture the control tobacco seedling normally without watering the bacterial solution. When the tobacco seedlings grow to 7 true leaves, measure 6 indicators of the tobacco seedlings, including the fresh weight of the underground part, the fresh weight of the aboveground part, the plant height, the maximum leaf length, the maximum leaf width, and the root length.

[0049] The promoting effect of Geotrichum candidum W11 on the growth of different tobacco varieties; Figure 4 .

[0050] The results showed that W11 had a significant growth-promoting effect. Figure 5 As shown, compared with the control, the plant height growth rates of the four tobacco varieties Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan inoculated with W11 were 6.13%, 1.43%, 1.30% and 1.00%, respectively; the maximum leaf length growth rates were 1.47%, 12.07%, 14.27% and 14.30%, respectively; the maximum leaf width growth rates were 4.60%, 18.27%, 14.40% and 19.50%, respectively; the root length growth rates were 13.17%, 2.07%, 75.90% and 19.33%, respectively; the underground fresh weight growth rates were 192.17%, 66.67%, 176.53% and 23.9%, respectively; the aboveground fresh weight growth rates were 19.23%, 31.63%, 31.03% and 22.70%, respectively.

[0051] 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. Promoting the growth of endophytic fungi in tobacco seedlings, characterized by: The deposit number of this strain is: CGMCC N0.41395.

2. Promoting the growth of endophytic fungi in tobacco seedlings, characterized by: Isolations of this fungus include: A. Materials: K326 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, taproots, 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 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 them on the surface of the culture medium with the cut surface facing down; 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 constant temperature incubator at 28℃ for 5-7 days to observe the growth of the colony; due to the different reproduction rates of different fungi, first separate and purify the faster growing strains, and wait for the slower growing strains to stabilize before separation and purification.

3. The endophytic fungus and bacterial liquid according to claim 1 are used to promote the growth of tobacco seedlings.

4. The use according to claim 3, characterized in that: The preparation method of the bacterial liquid of the endophytic fungus comprises the following steps: inoculating the endophytic fungus into a liquid culture medium, culturing on a shaking table, rotating at 220 rpm, culturing at 30 DEG C, culturing for 48 to 72 hours, and setting aside; the liquid culture medium comprises potato glucose broth, wherein the content of potato extract powder is 5 g / L, glucose is 20 g / L, and streptomycin is 10 mg / L.

5. The use according to claim 4, characterized in that: The bacterial liquid is used to pour into the above-ground part and root matrix soil of different varieties of tobacco seedlings to promote the growth of tobacco seedlings.

6. The use according to claim 4, characterized in that: The bacterial solution 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.

7. The use according to claim 6, characterized in that: The biomass and agronomic traits to be tested include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.

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