Smoke tube fungus MR-8 with drought resisting effect and application of smoke tube fungus MR-8

Abstract

The invention discloses a smoketube fungus MR-8 with a drought resisting effect and application thereof. 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 MR-8 separated from K326 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 research field of tobacco endophytic fungi, and in particular to drought-resistant tobacco pipe fungus MR-8 and application 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 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 drought-resistant tobacco pipe fungus MR-8 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] The drought-resistant tobacco pipe fungus MR-8, the deposit number of which is: CGMCC N0.41402. It was deposited on July 5, 2024 in the General Microbiological Center of the China Microbiological Culture Collection Administration, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The tobacco pipe fungus MR-8 was obtained by the following method:

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

[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, the functional endophytic fungus Nicotiana tabacum MR-8 with drought resistance was obtained.

[0014] Identification of Bjerkanderas MR-8: The rDNA ITS of the strain was amplified by PCR using the universal primers ITS1 / ITS4 for fungi, and the ITS PCR amplification products were sequenced. The obtained sequences were searched by BLAST in the GenBank nuclear sequence database. It was found that the ITS regions of the tested strain of Bjerkanderas MR-8 had the highest homology with those reported previously. Combined with morphological identification, the strain was identified.

[0015] The Bjerkandera sp. MR-8 has a semicircular cap, 2-7 cm wide, 0.1-0.6 cm thick, and a light yellow, gray to light brown surface with villi, which will be detached later, and the surface is nearly smooth or slightly rough, with inconspicuous rings; the edge is thin, wavy, blackened, and there is no hymenium underneath. The flesh is soft and leathery, crisp after drying, fibrous, and white to gray.

[0016] The preparation method of the MR-8 bacterial liquid with drought resistance is as follows: the strain is inoculated into 100 ml of liquid culture medium, placed in a 250 ml conical flask, the bottle mouth is sealed with a film, and cultured on a shaker 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: potato glucose broth (25 g / L), with streptomycin (10 mg / L) added.

[0017] 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.

[0018] 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 effect of the tobacco tube fungus MR-8 liquid on the tobacco seedlings was further confirmed, providing basic support for its further application in production.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] The POD concentration and activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, treated with MR-8 were all improved. The POD concentration of K326 under the action of MR-8 was higher than that of other tobacco varieties; the POD concentrations of Yunyan 97 and K326 under the action of MR-8 were higher than those of other strains.

[0021] MR-8 tobacco pipe fungus can increase the SOD activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 tobacco pipe fungus 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 MR-8 tobacco pipe fungus is higher than that of other varieties. MR-8 tobacco pipe fungus has the strongest effect on Yunyan 97 and Honghua Dajinyuan.

[0022] MR-8 can increase the GSH concentration of four tobacco varieties: Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 can significantly increase the GSH concentration of Yunyan 97 and Honghua Dajinyuan. MR-8 has the strongest effect on Yunyan 97 and Honghua Dajinyuan.

[0023] The CAT concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were all increased after being treated with MR-8. MR-8 could significantly increase the CAT concentration of Honghua Dajinyuan, but had no significant effect on the CAT concentrations of the other three tobacco varieties.

[0024] The MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were significantly reduced after being treated with MR-8. MR-8 can significantly reduce the MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 can significantly reduce the MDA concentration of Yunyan 87, and the strongest effect on Yunyan 97, K326 and Honghua Dajinyuan is MR-8. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The tobacco seedling raising tray and sowing method of the present invention;

[0026] Figure 2 The morphology of the tobacco endophytic fungus MR-8 of the present invention;

[0027] Figure 3 The drought resistance effect of tobacco endophytic fungus MR-8 on the growth of tobacco seedlings of different varieties is shown in the figure, wherein Figure 3 a- Figure 3 f are: relative water content of tobacco seedlings, POD concentration of tobacco seedlings, SOD concentration of tobacco seedlings, GSH concentration of tobacco seedlings, CAT concentration of tobacco seedlings, and MDA concentration of tobacco seedlings;

[0028] Figure 4 The fermentation diagram of the tobacco endophytic fungus MR-8 of the present invention;

[0029] Figure 5 The growth of tobacco seedlings inoculated with the endophytic fungus of the present invention was compared with that of tobacco seedlings not inoculated with the endophytic fungus. 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 As shown, the drought-resistant tobacco pipe fungus Bjerkandera adusta, the deposit number of the strain is: CGMCC N0.41402. On July 5, 2024, it was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The tobacco pipe fungus MR-8 was obtained by the following method:

[0032] A. Materials: Tobacco K326 plants harvested from the field were divided into six parts: upper leaves, middle and lower leaves, tobacco stems, thick roots, and fibrous roots;

[0033] 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;

[0034] 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.

[0035] 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;

[0036] 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, the functional endophytic fungus Nicotiana tabacum MR-8 with drought resistance was obtained.

[0037] Identification of Bjerkanderas MR-8: The rDNA ITS of the strain was amplified by PCR using the universal primers ITS1 / ITS4 for fungi, and the ITS PCR amplification products were sequenced. The obtained sequences were searched by BLAST in the GenBank nuclear sequence database. It was found that the ITS regions of the tested strain of Bjerkanderas MR-8 had the highest homology with those reported previously. Combined with morphological identification, the strain was identified.

[0038] The Bjerkandera sp. MR-8 has a semicircular cap, 2-7 cm wide, 0.1-0.6 cm thick, and a light yellow, gray to light brown surface with villi, which will be detached later, and the surface is nearly smooth or slightly rough, with inconspicuous rings; the edge is thin, wavy, blackened, and there is no hymenium underneath. The flesh is soft and leathery, crisp after drying, fibrous, and white to gray.

[0039] The preparation method of the MR-8 bacterial liquid with drought resistance is as follows: the strain is inoculated into 100 ml of liquid culture medium, placed in a 250 ml conical flask, the bottle mouth is sealed with a film, and cultured on a shaker 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: potato glucose broth (25 g / L), with streptomycin (10 mg / L) added.

[0040] 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.

[0041] 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 effect of the tobacco tube fungus MR-8 liquid on the tobacco seedlings was further confirmed, providing basic support for its further application in production.

[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 irrigation

[0045] Take 1.5 cm of the isolated and purified endophytic fungi 2The bacterial 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. The culture temperature was 30 ° C, the speed was 220 rpm, and the culture time was 48 to 72 hours. When the tobacco seedlings grew to 5 to 6 true leaves, the bacterial solution was taken to pour the aboveground part and root soil of the tobacco seedlings. Before pouring, one tobacco seedling was kept 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 pouring, 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 poured with the same volume of liquid culture medium without pouring bacterial solution. When the tobacco seedlings grew to 7 true leaves, the relative water content, POD, SOD, GSH, CAT, MDA and other indicators of the tobacco seedlings were measured.

[0046] Effect of Bjerkandera sp. MR-8 on relative moisture content of different tobacco seedling varieties.

[0047] The results showed that the relative moisture content of tobacco K326 treated with Bjerkandera sp. MR-8 was 14.26% higher than the control; Yunyan 97 treated with Bjerkandera sp. MR-8 was 15.86% higher than the control; K326 treated with Bjerkandera sp. MR-8 was 16.28% higher; and Honghua Dajinyuan treated with Bjerkandera sp. MR-8 was 24.62%; among them, the relative moisture content of Honghua Dajinyuan increased the most.

[0048] Effects of Bjerkandera sp. MR-8 on the activities of POD, SOD, GSH, CAT, MDA and other enzymes in different tobacco seedling varieties.

[0049] The results showed that the POD concentration and activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, treated with MR-8 were all increased. The POD concentration of K326 under the action of MR-8 was higher than that of other tobacco varieties; the POD concentrations of Yunyan 97 and K326 under the action of MR-8 were higher than those of other strains.

[0050] MR-8 tobacco pipe fungus can increase the SOD activity of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 tobacco pipe fungus 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 MR-8 tobacco pipe fungus is higher than that of other varieties. MR-8 tobacco pipe fungus has the strongest effect on Yunyan 97 and Honghua Dajinyuan.

[0051] MR-8 can increase the GSH concentration of four tobacco varieties: Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 can significantly increase the GSH concentration of Yunyan 97 and Honghua Dajinyuan. MR-8 has the strongest effect on Yunyan 97 and Honghua Dajinyuan.

[0052] The CAT concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were all increased after being treated with MR-8. MR-8 could significantly increase the CAT concentration of Honghua Dajinyuan, but had no significant effect on the CAT concentrations of the other three tobacco varieties.

[0053] The MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan, were significantly reduced after being treated with MR-8. MR-8 can significantly reduce the MDA concentrations of four tobacco varieties, Yunyan 87, Yunyan 97, K326 and Honghua Dajinyuan. MR-8 can significantly reduce the MDA concentration of Yunyan 87, and the strongest effect on Yunyan 97, K326 and Honghua Dajinyuan is MR-8.

[0054] 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. Drought-resistant tobacco pipe fungus MR-8, characterized by: The deposit number of the strain is: CGMCC No. 41402. The tobacco pipe fungus MR-8 is obtained by the following method: 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 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 can be separated and purified first, and the slower growing strains can be separated and purified after they are stable.

2. The tobacco pipe fungus MR-8 and bacterial liquid as claimed in claim 1 are used to promote the drought resistance of tobacco seedlings.

3. The use according to claim 2, characterized in that: The preparation method of the bacterial liquid is as follows: the tobacco pipe fungus MR-8 described in claim 1 is inoculated into a liquid culture medium, and the culture is carried out on a shaker at a rotation speed of 220 rpm, a culture temperature of 30° C., and a culture time of 48 to 72 hours, and then set aside; the liquid culture medium is potato glucose broth, wherein 5 g / L potato extract powder, 20 g / L glucose, and 10 mg / L streptomycin.

4. The use according to claim 3, characterized in that: The bacterial liquid is used for pouring into the aboveground parts and root matrix soil of tobacco seedlings of different varieties to promote the drought resistance of the tobacco seedlings.

5. 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.

6. 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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