Tobacco endophytic fungus X-5 with remarkable drought resisting effect and application of tobacco endophytic fungus X-5

Abstract

The invention discloses a tobacco endophytic fungus X-5 with a remarkable drought-resistant effect and application of the tobacco endophytic fungus X-5. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) 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 Yunyan 87 is applied to soil at the upper 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 endophytic fungi with drought resistance, and in particular to tobacco endophytic fungi X-5 with significant drought resistance 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 leaves' yield and quality.

[0005] In our study of 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 1 drought-resistant 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 tobacco endophytic fungus X-5 with significant drought resistance and its application; and 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] Tobacco endophytic fungus X-5 with significant drought resistance

[0009] The tobacco endophytic fungi are: Schizophyllum commune X-5;

[0010] The deposit number of this strain is: CGMCC N0.41401; 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;

[0011] The strain is obtained by the following steps:

[0012] A. Materials: The Yunyan 87 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;

[0013] 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;

[0014] 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, the faster growing strains are separated and purified first, and the slower growing strains are separated and purified after they are stable.

[0015] Furthermore, in the culture medium, 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;

[0016] 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 colony was single. After screening, a functional endophytic fungus with drought resistance was obtained.

[0017] Furthermore, the endophytic fungus identification is specifically as follows: 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 nucleic acid sequence database, and finding that the ITS region of the test strain of Schizophyllum commune X-5 has the highest homology with the reported ones, and combining morphological identification to determine the species of one endophytic fungus;

[0018] The Schizophyllum commune X-5 has a cap diameter of 0.6-4.2 cm, is white to off-white, has villi or coarse hairs, is fan-shaped or kidney-shaped, has many lobes, has thin white flesh, has narrow gills radiating from the base, is white or off-white, sometimes lavender, has longitudinal cracks along the edges and curls back, and has a short or absent stipe.

[0019] Furthermore, the preparation method of the tobacco endophytic fungus X-5 bacterial liquid is as follows: the Schizophyllum fungus X-5 is inoculated into a liquid culture medium, cultured on a shaker, with a rotation speed of 220 rpm, a culture temperature of 30° C., a culture time of 48 to 72 hours, and set aside; the liquid culture medium: potato glucose broth, wherein potato extract powder 5 g / L, glucose 20 g / L, and streptomycin 10 mg / L.

[0020] Application of tobacco endophytic fungus X-5 with significant drought resistance; its bacterial liquid is applied to the aboveground part and root matrix soil of different varieties of tobacco seedlings to improve their drought resistance.

[0021] Furthermore, the bacterial liquid is applied when the tobacco seedlings grow 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings are tested two weeks later.

[0022] Furthermore, the biomass and agronomic traits to be detected include: relative water content and agronomic traits.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] Tobacco endophytic fungus X-5 with significant drought resistance and its application. The 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, its drought resistance effect on tobacco seedlings was further confirmed, providing basic support for further application in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Tobacco seedling trays and sowing methods;

[0026] Figure 2 The morphology of endophytic fungi in Yunyan 87;

[0027] Figure 3 Yunyan 87 Endophytic Fungal Fermentation Diagram;

[0028] Figure 4 The drought resistance effect of the tobacco endophytic fungi of the present invention on the growth of tobacco seedlings of different varieties;

[0029] Figure 5 Comparison of the growth of tobacco seedlings inoculated with endophytic fungi and those not inoculated with endophytic fungi. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0031] like Figure 1-5 Shown: Tobacco endophytic fungus X-5 with significant drought resistance

[0032] The tobacco endophytic fungi are: Schizophyllum commune X-5;

[0033] The deposit number of this strain is: CGMCC N0.41401; 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;

[0034] The strain is obtained by the following steps:

[0035] A. Materials: The Yunyan 87 plants harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, thick roots, and fibrous roots;

[0036] 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;

[0037] 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, the faster growing strains are separated and purified first, and the slower growing strains are separated and purified after they are stable.

[0038] Furthermore, in the culture medium, 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;

[0039] 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 colony was single. After screening, a functional endophytic fungus with drought resistance was obtained.

[0040] Furthermore, the endophytic fungus identification is specifically as follows: 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 nucleic acid sequence database, and finding that the ITS region of the test strain of Schizophyllum commune X-5 has the highest homology with the reported ones, and combining morphological identification to determine the species of one endophytic fungus;

[0041] The Schizophyllum commune X-5 has a cap diameter of 0.6-4.2 cm, is white to off-white, has villi or coarse hairs, is fan-shaped or kidney-shaped, has many lobes, has thin white flesh, has narrow gills radiating from the base, is white or off-white, sometimes lavender, has longitudinal cracks along the edges and curls back, and has a short or absent stipe.

[0042] Furthermore, the preparation method of the tobacco endophytic fungus X-5 bacterial liquid is as follows: the Schizophyllum fungus X-5 is inoculated into a liquid culture medium, cultured on a shaker, with a rotation speed of 220 rpm, a culture temperature of 30° C., a culture time of 48 to 72 hours, and set aside; the liquid culture medium: potato glucose broth, wherein potato extract powder 5 g / L, glucose 20 g / L, and streptomycin 10 mg / L.

[0043] Application of tobacco endophytic fungus X-5 with significant drought resistance; its bacterial liquid is applied to the aboveground part and root matrix soil of different varieties of tobacco seedlings to improve their drought resistance.

[0044] Furthermore, the bacterial liquid is applied when the tobacco seedlings grow 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings are tested two weeks later.

[0045] Furthermore, the biomass and agronomic traits to be detected include: relative water content and agronomic traits.

[0046] Example: Cultivation of plant material

[0047] 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.

[0048] Application of endophytic fungi liquid

[0049] Take 1.5 cm 2The fungus block of about 100 ml was inoculated into 100 ml liquid culture medium, which was placed in a 250 ml conical bottle and cultured in a shaking incubator at a temperature of 30 ° C, a speed of 220 rpm, and a culture time of 48 to 72 hours. When the tobacco seedlings grew to 5 to 6 true leaves, the bacterial solution was taken to water the aboveground 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 poured on the tobacco seedlings. The bacterial solution was shaken evenly before taking. After watering, a transparent isolation cover was covered on the top of the seedling tray. After three days, the transparent isolation cover was removed and continued to be cultured in the incubator, that is, no culture solution and water were added during the whole process. A control tobacco seedling was set up, and the control tobacco seedlings were only watered with the same volume of liquid culture medium without water. When the tobacco seedlings grew to 7 true leaves, the relative water content, agronomic traits and other indicators of the tobacco seedlings were measured.

[0050] Effect of Schizophyllum commune X-5 strain on relative moisture content of different tobacco seedling varieties.

[0051] The results showed that the relative water contents of the Yunyan 87 control seedlings and the Yunyan 87 seedlings irrigated with S. commune were 33.49% and 44.65%, respectively. The relative water contents of the Yunyan 87 seedlings irrigated with endophytic fungi were significantly higher than those of the control seedlings, 11.16% higher than those of the control seedlings.

[0052] The relative water content of Yunyan 97 seedlings after irrigating with S. commune was 32.73% and 46.64% respectively. The relative water content of Yunyan 87 seedlings after irrigating with endophytic fungi was significantly higher than that of the control seedlings, 13.91% higher than that of the control seedlings.

[0053] The relative moisture content of tobacco K326 seedlings treated with S. commune was 40.12% and 58.28% respectively. The relative moisture content of tobacco K326 seedlings treated with endophytic fungi was significantly higher than that of the control seedlings, 18.17% higher than that of the control seedlings.

[0054] The relative water content of the control seedlings of Honghua Dajinyuan and the seedlings irrigated with S. commune were 34.37% and 51.10% respectively. The relative water content of the seedlings irrigated with endophytic fungi was significantly higher than that of the control seedlings, 16.73% higher than that of the control seedlings.

[0055] Effects of Schizophyllum commune X-5 strain on agronomic traits of different tobacco seedling varieties.

[0056] The results showed that the plant heights of the Yunyan 87 control seedlings and the Yunyan 87 seedlings irrigated with S. commune fungus were 1.25 cm and 3.91 cm, respectively, 2.66 cm taller than the control seedlings; the maximum leaf lengths were 6.92 cm and 7.95 cm, respectively; the maximum leaf widths were 3.48 cm and 4.33 cm, respectively; the root lengths were 8.28 cm and 9.06 cm, respectively, and there were no significant differences between the two differently treated seedlings.

[0057] The plant heights of the Yunyan 97 control seedlings and the Yunyan 97 seedlings irrigated with S. commune fungus were 1.25 cm and 2.10 cm, respectively; the maximum leaf lengths were 7.34 cm and 8.52 cm, respectively; the maximum leaf widths were 3.20 cm and 4.18 cm, respectively; and the root lengths were 8.06 cm and 9.22 cm, respectively. There were no significant differences between the two different treatments.

[0058] The plant heights of the tobacco K326 control seedlings and the tobacco K326 seedlings watered with S. commune fungus were 1.60 cm and 5.69 cm, respectively, and the maximum leaf lengths were 7.58 cm and 7.45 cm, respectively, with no significant differences between the two treatments; the maximum leaf widths were 3.61 cm and 4.43 cm, respectively, with no significant differences between the two treatments; the root lengths were 8.93 cm and 10.80 cm, respectively, with no significant differences between the two treatments.

[0059] The plant heights of the control Honghua Dajinyuan seedlings and the Honghua Dajinyuan seedlings irrigated with S. commune fungus were 1.60cm and 2.84cm, respectively. There was no significant difference in the plant height between the Honghua Dajinyuan seedlings irrigated with S. commune and the control seedlings; the maximum leaf lengths were 7.34cm and 7.45cm, respectively, and there was no significant difference between the two different treatments; the maximum leaf widths were 3.52cm and 3.80cm, respectively, and there was no significant difference between the two different treatments; the root lengths of Honghua Dajinyuan were 7.61cm and 9.34cm, respectively, and there was no significant difference between the two different treatments.

[0060] 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. Tobacco endophytic fungus X-5 with significant drought resistance, characterized in that: The tobacco endophytic fungi are: Schizophyllum commune X-5; The deposit number of this strain is: CGMCC N0.41401; 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 is obtained by the following steps: 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 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, the faster growing strains are separated and purified first, and the slower growing strains are separated and purified after they are stable.

2. The tobacco endophytic fungus X-5 with significant drought resistance according to claim 1, characterized in that: In the culture medium, 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; 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 colony was single. After screening, a functional endophytic fungus with drought resistance was obtained.

3. The tobacco endophytic fungus X-5 with significant drought resistance according to claim 1, characterized in that: The identification of the endophytic fungi is specifically as follows: using universal primers ITS1 / ITS4 for fungi to perform PCR amplification of rDNA ITS of the strain, and sequencing the ITS PCR amplification product, and performing BLAST search on the obtained sequence in the GenBank nucleic acid sequence database, and it is found that the ITS region of the test strain of Schizophyllum commune X-5 has the highest homology with the reported ones, and combined with morphological identification, the species of one endophytic fungus is determined; The Schizophyllum commune X-5 has a cap diameter of 0.6-4.2 cm, is white to off-white, has villi or coarse hairs, is fan-shaped or kidney-shaped, has many lobes, has thin white flesh, has narrow gills radiating from the base, is white or off-white, sometimes lavender, has longitudinal cracks along the edges and curls back, and has a short or absent stipe.

4. The tobacco endophytic fungus X-5 with significant drought resistance according to claim 1, characterized in that: The preparation method of the tobacco endophytic fungus X-5 bacterial liquid is as follows: the Schizophyllum fungus X-5 is inoculated into a liquid culture medium, and the culture is carried out on a shaking table at a rotation speed of 220 rpm, a culture temperature of 30° C., and a culture time of 48 to 72 hours for standby use; the liquid culture medium is potato glucose broth, wherein the potato extract powder is 5 g / L, the glucose is 20 g / L, and the streptomycin is 10 mg / L.

5. Application of tobacco endophytic fungus X-5 with significant drought resistance; characterized in that: The bacterial solution is applied to the above-ground part and root matrix soil of different varieties of tobacco seedlings to improve their drought resistance.

6. The application according to claim 5, characterized in that: The bacterial liquids are applied when the tobacco seedlings grow 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings are tested two weeks later.

7. The application according to claim 6, characterized in that: The biomass and agronomic traits to be detected as described in claim 5 include: relative water content and agronomic traits.

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