Tobacco seedling drought-resistant endophytic fungus W10 and application thereof

Abstract

The invention discloses a tobacco seedling drought-resistant endophytic fungus W10 and application thereof. 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; tobacco is one of important economic crops in China and plays a crucial role in economic development in China, but in recent years, tobacco planting and production are often limited by multiple factors such as diseases and insect pests, heavy metal stress and drought stress, so that 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 of the overground parts and roots of different varieties of tobacco seedlings in a watering manner, 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 K326 endophytic fungi, and in particular to tobacco seedling drought-resistant endophytic fungi W10 and applications thereof. Background Art

[0002] Plant endophytic fungi are a group of microorganisms that can promote plant growth, assist their host plants in resisting harsh environmental stresses, produce secondary metabolites similar to those of the host plants, and induce the accumulation of plant secondary metabolites. They generally include endophytic bacteria, endophytic fungi, and endophytic actinomycetes.

[0003] At present, more than 130 endophytic bacteria covering 60 genera have been found in a large number of crops, medicinal plants, fruit trees and other economic crops. Common endophytic bacteria include Agrobacterium, Enterobacter, Bacillus, Pseudomonas, Methylobacterium, Pantoea and Agrobacterium. In the study of endophytic actinomycetes, Frankia is the most studied, while the study of endophytic fungi in plants 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 Hui Feiqiong et al. found that under drought stress, inoculation with Indian pear-shaped spores can reduce the degree of damage to tobacco cell membranes, accumulate more cell osmotic regulating substances, and increase the expression of related drought-resistant genes in tobacco, thereby enhancing tobacco's drought resistance. Hamilton et al. believed that endophytic fungi can alleviate abiotic stress by changing the host's antioxidant capacity. Under drought stress, endophytic fungi colonize in the host and coexist with the host plant. The fungi interact with the plant to produce alkaloids, improve the plant's antioxidant capacity, and alleviate biological and abiotic stresses from the environment. Sun et al. believed that under drought stress, the thylakoid membrane-related Ca2+ receptors, CASmRNA levels, and CAS protein numbers of plants colonized by the endophytic fungus P. indica increased, increasing the plant's tolerance to drought stress. Shukla et al. also found that the colonization of plants by the endophytic fungus T. harzianum not only reduced the content of proline, MDA, and H2O2 in the plants, but also increased the content of phenols and MSI in the plants. Therefore, tobacco endophytic fungi are a new way to enhance tobacco leaves' resistance to stress and improve tobacco leaf production quality.

[0005] In our study on the endophytic fungi flora of K326, a dominant tobacco variety in Yunnan, a total of 154 endophytic fungi were isolated from tobacco K326 under different culture conditions, and identified as 72 species of 40 genera. Among them, 106 endophytic fungi were isolated from tobacco K326 under field growth conditions, identified as 62 species of 37 genera, 23 endophytic fungi were isolated from K326 cultured in water, identified as 11 species of 8 genera, and 25 endophytic fungi were isolated from K326 cultured in sterile soil, identified as 7 species of 4 genera. After screening, a total of 1 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 purpose of the present invention is to provide a tobacco seedling drought-resistant endophytic fungus W10 and its application, so as to provide a new method for improving the yield and quality of tobacco leaves.

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

[0008] A tobacco seedling drought-resistant endophytic fungus W10, the deposit number of which is: CGMCC N0.41404; deposited on July 5, 2024 in the General Microbiological Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; obtained through the following channels:

[0009] A. Materials: Tobacco K326 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;

[0010] 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;

[0011] 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.

[0012] Further, solid culture: 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;

[0013] 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, 3 functional endophytic fungi with drought resistance were obtained.

[0014] Furthermore, the above-mentioned endophytic fungi were identified as follows: the rDNA ITS of the strain 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 Chaetomium sp. W10 was found to have the highest homology with the reported ITS regions. Combined with morphological identification, the species of one endophytic fungus was determined.

[0015] The 1 tobacco endophytic fungus:

[0016] Chaetomium sp. W10,

[0017] The method for preparing the above-mentioned endophytic fungus application bacterial liquid with drought resistance function is to inoculate the above-mentioned endophytic fungus strain into 100 ml 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; liquid culture medium: potato glucose broth (25 g / L), add streptomycin (10 mg / L).

[0018] The method for applying the above-mentioned endophytic fungus solution with drought resistance function is to evenly pour 15 ml of the endophytic fungus solution (with hyphae and spores) into the soil matrix of the aboveground part and the root part of the tobacco seedlings.

[0019] The above-mentioned fungi were inoculated into tobacco seedlings of four different varieties, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97. Based on the evaluation of indicators such as the relative water content and enzyme activity of the tobacco seedlings, the drought-resistant effects of these three endophytic fungi on the tobacco seedlings were further confirmed, providing basic support for their further application in production.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The POD concentration and activity of four tobacco strains, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, treated with the sphaerocephala W10 of the present invention are all improved, and the sphaerocephala can significantly increase the POD concentration of K326; the POD concentration of Yunyan 87 under the action of the sphaerocephala W10 is higher than that of other drought-resistant strains.

[0022] Chaetomium globosum W10 can increase the SOD activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. Chaetomium globosum can significantly increase the SOD concentration of Yunyan 87. Among the four tobacco varieties, the SOD concentration of Honghua Dajinyuan under the action of the drought-resistant strain of Chaetomium globosum is higher than that of other varieties. Chaetomium globosum has the strongest drought-resistant effect on Yunyan 87, and Chaetomium globosum also has the strongest effect on K326.

[0023] Chaetomium globosum W10 can increase the GSH concentration of four tobacco varieties: Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. Chaetomium globosum can significantly increase the GSH concentration of Yunyan 87 and Honghua Dajinyuan. Chaetomium globosum has the strongest effect on Yunyan 87 and K326.

[0024] The CAT concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were all increased after treatment with Chaetomium globosum W10. Among them, Chaetomium globosum could significantly increase the CAT concentrations of Yunyan 97, K326 and Honghua Dajinyuan, and had the strongest effect on each tobacco variety.

[0025] The MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were significantly reduced after treatment with Chaetomium globosum W10. Among them, Chaetomium globosum could significantly reduce the MDA concentrations of Yunyan 87 and Yunyan 97, but had no significant effect on K326 and Honghua Dajinyuan. Chaetomium globosum could significantly reduce the MDA concentration of Yunyan 87, and had the strongest effect on Yunyan 87. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Tobacco seedling trays and sowing methods;

[0027] Figure 21 Morphology of endophytic fungi in tobacco strain K326;

[0028] Figure 31: Endophytic fungal fermentation diagram of tobacco strain K326;

[0029] Figure 41 Effects of tobacco endophytic fungi on the drought resistance of tobacco seedlings of different varieties;

[0030] Figure 5 Comparison of the growth of tobacco seedlings inoculated with endophytic fungi and those not inoculated with endophytic fungi. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0032] like Figure 1-5As shown, a tobacco seedling drought-resistant endophytic fungus W10, the deposit number of the strain is: CGMCCN0.41404; on July 5, 2024, it was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; it was obtained through the following channels:

[0033] A. Materials: Tobacco K326 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;

[0034] 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;

[0035] 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.

[0036] Further, solid culture: 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;

[0037] 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, 3 functional endophytic fungi with drought resistance were obtained.

[0038] Furthermore, the above-mentioned endophytic fungi were identified as follows: the rDNA ITS of the strain 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 Chaetomium sp. W10 was found to have the highest homology with the reported ITS regions. Combined with morphological identification, the species of one endophytic fungus was determined.

[0039] The 1 tobacco endophytic fungus:

[0040] Chaetomium sp. W10,

[0041] The method for preparing the above-mentioned endophytic fungus application bacterial liquid with drought resistance function is to inoculate the above-mentioned endophytic fungus strain into 100 ml 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; liquid culture medium: potato glucose broth (25 g / L), add streptomycin (10 mg / L).

[0042] The method for applying the above-mentioned endophytic fungus solution with drought resistance function is to evenly pour 15 ml of the endophytic fungus solution (with hyphae and spores) into the soil matrix of the aboveground part and the root part of the tobacco seedlings.

[0043] The above-mentioned fungi were inoculated into tobacco seedlings of four different varieties, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97. Based on the evaluation of indicators such as the relative water content and enzyme activity of the tobacco seedlings, the drought-resistant effects of these three endophytic fungi on the tobacco seedlings were further confirmed, providing basic support for their further application in production.

[0044] Example: Cultivation of plant material;

[0045] The present 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.

[0046] Application of endophytic fungi liquid

[0047] 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 and water the above-ground 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 tray of tobacco seedlings 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. After three days, remove the transparent isolation cover and continue to culture in an incubator, that is, no culture solution and water are added during the whole process. Set up a control tobacco seedling, and only water the same volume of liquid culture medium is applied to the control tobacco seedlings without watering the bacterial solution. When the tobacco seedlings grow to 7 true leaves, the relative water content, POD, SOD, GSH, CAT, MDA and other indicators of the tobacco seedlings are measured.

[0048] Effect of Chaetomium sp. W10 strain on relative moisture content of different tobacco seedling varieties.

[0049] The results showed that the relative moisture content of tobacco K326 treated with Chaetomium sp. W10 was 16.70% higher than that of the control; Yunyan 97 treated with Chaetomium sp. W10 was 17.79% higher than that of the control; K326 treated with Chaetomium sp. W10 was 14.37% higher; and Honghua Dajinyuan treated with Chaetomium sp. W10 was 20.46% higher; among them, the relative moisture content of Honghua Dajinyuan increased the most.

[0050] Effects of Chaetomium sp. W10 strain on the activities of POD, SOD, GSH, CAT, MDA and other enzymes in different tobacco seedling varieties.

[0051] The results showed that the POD concentration and activity of four tobacco strains, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, treated with Chaetomium globosum were all increased, and Chaetomium globosum could significantly increase the POD concentration of K326; the POD concentration of Yunyan 87 under the action of Chaetomium globosum was higher than that of other drought-resistant strains.

[0052] Chaetomium globosum can increase the SOD activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. Chaetomium globosum can significantly increase the SOD concentration of Yunyan 87. Among the four tobacco varieties, the SOD concentration of Honghua Dajinyuan under the action of the drought-resistant strain of Chaetomium globosum is higher than that of other varieties. Chaetomium globosum has the strongest drought-resistant effect on Yunyan 87, and also has the strongest effect on K326.

[0053] Chaetomium globosum can increase the GSH concentration of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, and Chaetomium globosum can significantly increase the GSH concentration of Yunyan 87 and Honghua Dajinyuan. Chaetomium globosum has the strongest effect on Yunyan 87 and K326.

[0054] The CAT concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were all increased after treatment with Chaetomium globosum. Among them, Chaetomium globosum could significantly increase the CAT concentrations of Yunyan 97, K326 and Honghua Dajinyuan, and had the strongest effect on each tobacco variety.

[0055] The MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were significantly reduced after treatment with Chaetomium globosum. Among them, Chaetomium globosum could significantly reduce the MDA concentrations of Yunyan 87 and Yunyan 97, but had no significant effect on K326 and Honghua Dajinyuan. Chaetomium globosum could significantly reduce the MDA concentration of Yunyan 87, and Chaetomium globosum had the strongest effect on Yunyan 87.

[0056] 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. A tobacco seedling drought-resistant endophytic fungus W10, characterized in that: The deposit number of the strain is: CGMCC N0.41404; it was deposited on July 5, 2024 at the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the tobacco seedling drought-resistant endophytic fungus W10 was obtained by the following means: A. Materials: Tobacco K326 plants harvested 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 tobacco seedling drought-resistant endophytic fungus W10 according to claim 1, characterized in that: The tobacco seedling drought-resistant endophytic fungus W10 is: Chaetomium globosum W10.

3. The Chaetomium globosum W10 and its bacterial liquid as described in claim 2 are used to promote the drought-resistant growth of tobacco seedlings.

4. The application according to claim 3, characterized in that: The preparation method of the bacterial liquid is as follows: the W10 strain of Chaetomium globosum is inoculated into 100 ml of liquid culture medium, which is then placed in a 250 ml conical flask, the mouth of which is sealed with a film, and the culture is carried out on a shaking table at a temperature of 30° C., a rotation speed of 220 rpm, and a culture time of 48 to 72 hours; the liquid culture medium includes 25 g / L of potato glucose broth and 10 mg / L of streptomycin.

5. The application according to claim 4, characterized in that: The bacterial liquid is evenly poured into the above-ground part and the root matrix soil of the tobacco seedlings to promote the drought resistance of the tobacco seedlings.

6. The use according to claim 4, characterized in that: The bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the relative water content and enzyme activity of the tobacco seedlings were tested two weeks later.

7. The use according to claim 4, characterized in that: The enzyme activities to be tested include: POD, SOD, GSH, MDA, and CAT.

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