Tobacco endophytic fungus YUAN-001H-10 for promoting growth of tobacco seedlings

Abstract

The invention discloses a tobacco endophytic fungus YUAN-001H-10 capable of promoting the growth of tobacco seedlings. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) N0.41399, tobacco is a leaf commercial crop and is a main source of national financial income and common people economic income, 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 a seedling stage or a field stage, and the yield and quality of tobacco leaves are seriously threatened. By applying the seven endophytic fungus solutions separated from Yunyan 87 to soil of the overground parts and roots of different varieties of tobacco seedlings, the biomass and agronomic traits of the tobacco seedlings can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the research field of tobacco endophytes, and in particular to a tobacco endophytic fungus YUAN-001H-10 for promoting the growth of tobacco seedlings. 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 fungi flora of the dominant tobacco variety Yunyan 87 in Yunnan, a total of 103 endophytic fungi, 52 species, belonging to 29 genera, were isolated from Yunyan 87 tobacco plants picked in the field. Among them, there were 11 endophytic fungi of 10 genera in the upper leaves, 8 endophytic fungi of 7 genera in the middle leaves, 8 endophytic fungi of 6 genera in the lower leaves, 11 endophytic fungi of 9 genera in the stems, 21 endophytic fungi of 12 genera in the thick roots, and 7 endophytic fungi of 5 genera in the fibrous roots. After screening, a total of 7 endophytic fungi significantly promoted the biomass and trait indicators of tobacco seedlings of different tobacco varieties. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide tobacco endophytic fungus YUAN-001H-10 that promotes the growth of tobacco seedlings, 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 tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth has a deposit number of CGMCCN0.41399. It was deposited in the General Microbiological Center of the 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 Aspergillus terreus.

[0009] The bacteria are obtained by the following steps:

[0010] A. Materials: Yunyan 87 plants collected from the field were divided into three parts: roots, stems and leaves;

[0011] B. Disinfection: Upper leaves, middle leaves, and lower leaves: soak in 75% alcohol for 30 seconds, soak in 0.1% mercuric chloride for 15 seconds, and rinse with sterile water for 3 times; Tobacco stems: wipe the epidermis with 75% alcohol, and keep the cortex and pith; Thick roots and fibrous roots: soak in 75% alcohol for 60 seconds, soak in 0.1% mercuric chloride for 20 seconds, and rinse with sterile water for 3 times. The sterile water after the last rinse is streaked on the plate as a control to check whether the sample is thoroughly disinfected;

[0012] C. Inoculation and separation: Grind the tobacco leaf part with sterile water, and evenly spread the grinding liquid and residue on the surface of the culture medium; cut the tobacco stem part into 5-8 mm pieces with an inoculation knife, and spread one side of the cut surface downward on the surface of the culture medium; cut the thick root part into 3-5 mm small pieces with an inoculation knife, and spread the cut surface downward on the surface of the culture medium; mix the fibrous root part with sterile water and grind it, and evenly spread the grinding liquid and residue on the surface of the culture medium. The treated materials are cultured in a constant temperature incubator at 28°C for 5-7 days, and the growth of the colony is observed.

[0013] D. Solid culture: Use potato dextrose agar (PDA) medium (46 g / L) with streptomycin (10 mg / L) added to prevent bacterial contamination, culture temperature 28°C, culture time 5 to 7 days;

[0014] The different types of endophytic fungi separated were inoculated into new sterile PDA culture medium using an inoculation loop for purification, and the inoculation purification was repeated 2 to 3 times until the colonies were single. After screening, the tobacco endophytic fungus YUAN-001H-10 that promoted the growth of tobacco seedlings was obtained.

[0015] Furthermore, the tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth is Aspergillus terreus 001YUAN-001H-10, the surface of the colony is flat or has shallow radial wrinkles, velvety or occasionally flocculent, cinnamon to sandy brown, and the back of the colony and the matrix are dull yellow to brown. The liquid culture ball is small in size and aggregates to form a flocculent bacterial colony.

[0016] Furthermore, the identification operation of the tobacco endophytic fungus YUAN-001H-10 that promotes the growth of tobacco seedlings is as follows: using the universal fungal primers ITS1 / ITS4 to perform PCR amplification of the rDNA ITS of the strain, and sequencing the ITS PCR amplification product, performing a BLAST search on the obtained sequence in the GenBank nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported Aspergillus terreus, and combining with morphological identification, the species of the endophytic fungus strain is determined.

[0017] Furthermore, the method for preparing the fungal application bacterial liquid is to inoculate the endophytic fungus described in claim 1 from a solid culture medium into a liquid culture medium using an inoculation loop, and culture it on a shaker at a speed of 220 rpm, a culture time of 48 to 72 hours, and a culture temperature of 30°C for standby use; the liquid culture medium: potato glucose broth, which contains 5 g / L potato extract powder, 20 g / L glucose, and 10 mg / L streptomycin.

[0018] Furthermore, the bacterial liquid is poured into the aboveground part and root matrix soil of different varieties of tobacco seedlings to promote the growth of the tobacco seedlings.

[0019] Furthermore, the bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings were tested two weeks later.

[0020] Furthermore, the biomass and agronomic traits to be tested include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The tobacco endophytic fungus YUAN-001H-10, which promotes the growth of tobacco seedlings, was inoculated into four different varieties of tobacco seedlings, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97. Based on the evaluation of seven indicators such as tobacco seedling biomass and agronomic traits, it was further confirmed that these seven endophytic fungi have a role in promoting the growth of tobacco seedlings, providing basic support for their further application in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Tobacco seedling trays and sowing methods;

[0024] Figure 2 Morphology of endophytic fungi of 1 strain Yunyan 87;

[0025] Figure 3 1 Endophytic fungal fermentation diagram of Yunyan 87;

[0026] Figure 4 The tobacco endophytic fungus promotes the growth of tobacco seedlings of different varieties;

[0027] Figure 5 Comparison of the growth of tobacco seedlings inoculated with endophytic fungi and those not inoculated with endophytic fungi. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0029] like Figure 1-5 As shown: Tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth, the deposit number of the strain is: CGMCC N0.41399. It was deposited in the General Microbiological Center of the 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 Aspergillus terreus.

[0030] The bacteria are obtained by the following steps:

[0031] A. Materials: Yunyan 87 plants collected from the field were divided into three parts: roots, stems and leaves;

[0032] 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;

[0033] C. Inoculation and separation: Grind the tobacco leaf part with sterile water, and evenly spread the grinding liquid and residue on the surface of the culture medium; cut the tobacco stem part into 5-8 mm pieces with an inoculation knife, and spread one side of the cut surface downward on the surface of the culture medium; cut the thick root part into 3-5 mm small pieces with an inoculation knife, and spread the cut surface downward on the surface of the culture medium; mix the fibrous root part with sterile water and grind it, and evenly spread the grinding liquid and residue on the surface of the culture medium. The treated materials are cultured in a constant temperature incubator at 28°C for 5-7 days, and the growth of the colony is observed.

[0034] D. Solid culture: Use potato dextrose agar (PDA) medium (46 g / L) with streptomycin (10 mg / L) added to prevent bacterial contamination, culture temperature 28°C, culture time 5 to 7 days;

[0035] The different types of endophytic fungi separated were inoculated into new sterile PDA culture medium using an inoculation loop for purification, and the inoculation purification was repeated 2 to 3 times until the colonies were single. After screening, the tobacco endophytic fungus YUAN-001H-10 that promoted the growth of tobacco seedlings was obtained.

[0036] Furthermore, the tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth is Aspergillus terreus 001YUAN-001H-10, the surface of the colony is flat or has shallow radial wrinkles, velvety or occasionally flocculent, cinnamon to sandy brown, and the back of the colony and the matrix are dull yellow to brown. The liquid culture ball is small in size and aggregates to form a flocculent bacterial colony.

[0037] Furthermore, the identification operation of the tobacco endophytic fungus YUAN-001H-10 that promotes the growth of tobacco seedlings is as follows: using the universal fungal primers ITS1 / ITS4 to perform PCR amplification of the rDNA ITS of the strain, and sequencing the ITS PCR amplification product, performing a BLAST search on the obtained sequence in the GenBank nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported Aspergillus terreus, and combining with morphological identification, the species of the endophytic fungus strain is determined.

[0038] Furthermore, the method for preparing the fungal application bacterial liquid is to inoculate the endophytic fungus described in claim 1 from a solid culture medium into a liquid culture medium using an inoculation loop, and culture it on a shaker at a speed of 220 rpm, a culture time of 48 to 72 hours, and a culture temperature of 30°C for standby use; the liquid culture medium: potato glucose broth, which contains 5 g / L potato extract powder, 20 g / L glucose, and 10 mg / L streptomycin.

[0039] Furthermore, the bacterial liquid is poured into the aboveground part and root matrix soil of different varieties of tobacco seedlings to promote the growth of the tobacco seedlings.

[0040] Furthermore, the bacterial liquid was applied when the tobacco seedlings grew 5 to 6 true leaves, and the biomass and agronomic traits of the tobacco seedlings were tested two weeks later.

[0041] Furthermore, the biomass and agronomic traits to be tested include: fresh weight, root weight, aboveground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length.

[0042] Example: Cultivation of plant material;

[0043] The invention selects four different varieties of tobacco coated seeds, namely Honghua Dajinyuan, Yunyan 87, K326 and Yunyan 97, and sows them on a 3*4 small seedling tray (such as Figure 2 ), 3 seeds were sown in each hole, the nutrient soil used for seedling cultivation was sterilized by high pressure (121℃, 20min), and light and dark alternating cultivation was carried out in the incubator, light cultivation for 16h, light intensity 18000LX, 28℃; dark cultivation for 8h, 16℃; nutrient solution was added to the bottom of the seedling tray, and the nutrient solution needed to be replenished every two days.

[0044] Application of endophytic fungi liquid

[0045] Take 1.5 cm 2 A fungus block about the size of about 1 ml was inoculated into a 100 ml liquid culture medium, which was placed in a 250 ml conical flask and cultured in a shaking incubator at a temperature of 30 ° C, a rotation speed of 220 rpm, and a culture time of 48 to 72 h. When the tobacco seedlings grew to 5 to 6 true leaves, the bacterial solution was taken for watering of the above-ground part and root soil of the tobacco seedlings. Before watering, one tobacco seedling was retained in each hole in the seedling tray to ensure that the growth of each tray of tobacco seedlings was consistent. 3 ml of bacterial solution was taken from each hole and watered on the tobacco seedlings, and the bacterial solution was shaken evenly before taking. After watering, a transparent isolation cover was placed on the top of the seedling tray and the tray was placed in an incubator for continued cultivation. A control tobacco seedling was also set up, which was cultured normally without watering with bacterial solution. When the tobacco seedlings grew to 7 true leaves, a total of 7 indicators including fresh weight, root weight, above-ground fresh weight, plant height, maximum leaf length, maximum leaf width, and root length of the tobacco seedlings were measured.

[0046] Effect of Aspergillus terreus 001YUAN-001H-10 on the growth of different tobacco varieties

[0047] The results showed that 001YUAN-001H-10 had a significant growth-promoting effect. Compared with the control, the plant height growth rates of Yunyan 97, Yunyan 87, K326 and Honghua Dajinyuan seedlings inoculated with 001YUAN-001H-10 were 13.78%, 17.03%, 25.66% and 6.95% respectively; the maximum leaf length growth rates were 34.59%, 30.92%, 34.09% and 6.89% respectively; the maximum leaf width growth rates were 39.05%, 56. 87%, 49.98% and 11.54% respectively; the growth rates of root length were 23.98%, 28.27%, 36.69% and 31.97% respectively; the growth rates of fresh weight were 98.56%, 172.85%, 108.53% and 30.89% respectively; the growth rates of root fresh weight were 169.21%, 209.57%, 102.89% and 121.47% respectively; the growth rates of aboveground fresh weight were 90.26%, 169.18%, 109.52% and 28.83% respectively.

[0048] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. Tobacco endophytic fungus YUAN-001H-10 for promoting tobacco seedling growth, characterized in that: The deposit number of this strain is: CGMCC N0.41399.

2. Tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth, characterized in that: The bacteria are 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 in the above material treatment is streaked on the plate as a control to observe whether there is fungal growth to check whether the sample is thoroughly disinfected; C. Inoculation and separation: Mix the tobacco leaf part with sterile water and grind it, and evenly spread the grinding liquid and residue on the surface of the culture medium; cut the tobacco stem part into 5-8 mm with an inoculation knife, and spread one side of the cut surface downward on the surface of the culture medium; cut the thick root part into 3-5 mm small sections with an inoculation knife, and spread the cut surface downward on the surface of the culture medium; mix the fibrous root part with sterile water and grind it, and evenly spread the grinding liquid and residue on the surface of the culture medium. The treated materials are placed in a constant temperature incubator at 28°C for 5-7 days to observe the growth of the colony; D. Solid culture: Use potato dextrose agar (PDA) medium (46 g / L) with streptomycin (10 mg / L) added to prevent bacterial contamination, culture temperature 28°C, culture time 5 to 7 days; The different types of endophytic fungi separated were inoculated into new sterile PDA culture medium using an inoculation loop for purification, and the inoculation purification was repeated 2 to 3 times until the colonies were single. After screening, the tobacco endophytic fungus YUAN-001H-10 that promoted the growth of tobacco seedlings was obtained.

3. The tobacco endophytic fungus YUAN-001H-10 for promoting tobacco seedling growth according to claim 2, characterized in that: The tobacco endophytic fungus YUAN-001H-10 that promotes tobacco seedling growth is Aspergillus terreus 001YUAN-001H-10, the surface of the colony is flat or has shallow radial wrinkles, velvety or occasionally flocculent, cinnamon to sandy brown, the back of the colony and the matrix are dull yellow to brown, the liquid culture ball is small in size, and aggregation will form a flocculent bacterial colony.

4. The tobacco endophytic fungus YUAN-001H-10 for promoting tobacco seedling growth according to claim 2, characterized in that: The identification operation of the tobacco endophytic fungus YUAN-001H-10 that promotes the growth of tobacco seedlings is 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 nuclear sequence database, and finding that the ITS region of the test strain has the highest homology with the reported Aspergillus terreus, and combining morphological identification to determine the species of the endophytic fungus strain.

5. The tobacco endophytic fungus YUAN-001H-10 for promoting tobacco seedling growth according to claim 2, characterized in that: The preparation method of the fungus application bacterial liquid comprises the following steps: inoculating the endophytic fungus described in claim 1 into a liquid culture medium, culturing on a shaking table, rotating at 220 rpm, culturing at 30°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.

6. The use of tobacco endophytic fungus YUAN-001H-10 for promoting tobacco seedling growth according to claim 2, characterized in that: The bacterial solution described in claim 4 was poured into the aboveground part and root matrix soil of different varieties of tobacco seedlings to observe the effect of promoting the growth of tobacco seedlings.

7. The use according to claim 6, 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.

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

Images