Endophytic fungus of tea tree and its application in promoting growth of Dendrobium officinale

Abstract

The present invention discloses an endophytic fungus of tea tree and its application in promoting the growth of Dendrobium officinale, belonging to the field of microbial technology. The abnormal Wickerham yeast strain (Wickerhamomyces p.) involved in the present invention was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 15, 2022. The strain preservation name is: KLBMP0506, and the preservation number is: CGMCC No. 25719. It has a significant effect on promoting the growth of Dendrobium officinale and the accumulation of secondary metabolites, provides excellent strain resources for the development of rare medicinal Dendrobium plant growth-promoting fertilizers, promotes the industrialization development of Dendrobium, and has good application development prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an abnormal Wickerhamomyces strain of endophytic fungi of tea trees and application thereof in promoting the growth of Dendrobium officinale. Background Art

[0002] Dendrobium officinale Kimura et Migo, also known as black knot grass, belongs to the genus Dendrobium of the Orchidaceae family. It is a rare and endangered medicinal plant unique to my country. It has the effects of nourishing yin and promoting body fluid, protecting the liver and gallbladder, nourishing the stomach and intestines, lowering blood sugar and improving eyesight, and preventing cancer and aging. It has been included in the "State Pharmacopoeia Commission of P.R. China, 2010" and the catalog of medicine and food homology. Due to its extremely high medicinal and economic value, the market demand for Dendrobium officinale has continued to expand, leading to serious over-exploitation and over-digging. In addition, its slow growth rate in the natural environment, low seed germination rate, and strict requirements on habitats have almost exhausted the wild resources of Dendrobium officinale. Artificial cultivation is an effective way to solve the problem of its wild resource shortage. However, artificial cultivation of Dendrobium officinale still faces problems such as long growth cycle and low average yield. In order to pursue high yield and shorten the growth cycle, the current Dendrobium officinale cultivation industry has a serious problem of excessive use of plant growth regulators and chemical fertilizers, resulting in poor quality of artificially cultivated Dendrobium officinale. These problems have restricted the industrial development of Dendrobium officinale to a certain extent.

[0003] Plant endophytes refer to a type of microorganisms that live in healthy plant tissues or organs at a certain stage or throughout their life cycle and do not cause obvious disease symptoms to the host plant. They mainly include endophytic fungi, endophytic bacteria, and endophytic actinomycetes. As an important type of microbial resource, plant endophytes play an important role in promoting plant growth and development, the accumulation of secondary metabolites, and the improvement of stress resistance. In recent years, exploring the interaction between endophytes and medicinal plants and using endophytes to improve the yield and quality of medicinal plants have become research hotspots in many fields. High yield and high quality have always been the goals pursued in the research of traditional Chinese medicine. Using endophytic resources to improve the yield and quality of medicinal plants has important ecological significance for the protection and development of medicinal plant resources.

[0004] At present, there have been some studies on the use of endophytes to promote the growth of Dendrobium officinale, but the results are not ideal. The functional strains currently used are difficult to achieve both the ability to promote the growth of Dendrobium officinale and to increase the content of its secondary metabolites. Hu Xiufang et al. used Sphingomonas sp. to promote the growth of Dendrobium officinale tissue culture seedlings, but the fresh weight and dry weight only increased by about 10%. Summary of the invention

[0005] In view of the shortcomings of the existing technology, an abnormal Wickerham yeast strain (Wickerhamomyces p.) is provided which has a significant growth-promoting effect on Dendrobium officinale. The strain can promote the growth of Dendrobium officinale and the accumulation of secondary metabolites, which is beneficial to solving technical problems such as slow growth rate, long cycle and poor quality in the cultivation and planting process of Dendrobium officinale.

[0006] In order to achieve the above-mentioned technical objectives, the present invention provides an endophytic fungus for tea trees, characterized in that: the classification of the endophytic fungus for tea trees is clearly named as an abnormal Wickham yeast strain (Wickerhamomyces p.), the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain preservation name is: KLBMP0506, the preservation number is: CGMCC No. 25719, and the preservation date is September 15, 2022.

[0007] An application of the endophytic fungi in tea trees can promote the growth of Dendrobium officinale and the accumulation of secondary metabolites.

[0008] A method for preparing an endophytic fungus agent for tea trees, using the abnormal Wickerhamomyces strain (Wickerhamomyces p.) to perform the following treatment:

[0009] The strain KLBMP0506 was inoculated into fresh YPD liquid medium and cultured at 28°C in a shaking incubator at 200 rpm / min for 16 h to activate the strain;

[0010] Transfer 2 mL of activated bacterial solution to 200 mL of YPD liquid culture medium at a volume ratio of 1:100 and culture at 28°C, 200 rpm / min in a shaking incubator until OD 600 =0.6;

[0011] The culture product was centrifuged at 6000 rpm / min for 10 min, the supernatant was discarded to collect the bacteria, and the bacteria were resuspended in sterile water to adjust the bacterial solution concentration to OD 600 =0.5.

[0012] Furthermore, the KLBMP0506 bacterial solution was inoculated into the Dendrobium officinale tissue culture seedlings, and each bottle of tissue culture seedlings was inoculated with 200 μL of bacterial solution, and the bacterial solution concentration was OD 600 =0.5, the bacterial solution was inoculated near the base of the tissue culture seedlings, thereby promoting the growth of Dendrobium officinale and the accumulation of secondary metabolites.

[0013] Furthermore, the YPD liquid culture medium is: 10 g yeast extract, 20 g peptone, 20 g glucose, fixed to 1000 mL distilled water, PH natural.

[0014] Furthermore, the bacterial agent is a microbial fertilizer.

[0015] A composition comprises the endophytic fungus of tea tree.

[0016] The tea tree endophytic fungus of the present invention is an abnormal Wickerhamomyces strain (Wickerhamomyces p.), and the strain KLBMP0506 is separated from tea tree fruits by using endophytic fungus separation and purification technology. The specific separation and purification method is as follows:

[0017] 1) Take tea fruit collected from Gao'an, Jiangxi, rinse with running water for 10 minutes, and ultrasonically clean the rinsed samples for 10 minutes to avoid the difficult-to-rinse substances attached to the tissue surface affecting the separation results. Then, in the clean bench, soak the rinsed samples in 75% ethanol for 1 minute, rinse with sterile water once, and then soak and disinfect with 2% sodium hypochlorite solution for 6 minutes, and rinse with sterile water 3 times. The surface-sterilized samples were cut into small tissues with scissors and spread on YPD solid culture medium, and cultured at 28°C for 2-5 days until a single colony grows;

[0018] 2) Pick a single colony and purify it by streaking on the same YPD plate as in step (1) above, incubate it at 28°C, and perform streak purification and separation to obtain a pure culture.

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

[0020] The strain KLBMP0506 of the present invention has a significant growth-promoting effect on Dendrobium officinale, and can effectively promote the accumulation of secondary metabolites of Dendrobium officinale. The strain can be used as a microbial fertilizer for seedling cultivation and artificial planting of Dendrobium officinale, improve the quality of its medicinal materials, ensure the effectiveness and safety of the use of medicinal materials, and reduce the environmental pollution caused by the abuse of chemical fertilizers.

[0021] The present invention uses strain KLBMP0506 to significantly promote the growth of Dendrobium officinale from the perspective of plant endophytes, and effectively increases the content of secondary metabolites of Dendrobium officinale. The strain provided by the present invention can be used as a microbial fertilizer for seedling cultivation and artificial cultivation of Dendrobium officinale, improve the quality of Dendrobium officinale medicinal materials, ensure the effectiveness and safety of its medicinal materials, and also reduce the environmental pollution caused by the large-scale use of chemical fertilizers.

[0022] The strain KLBMP0506 of the present invention has simple culture conditions, is easy to preserve, is genetically stable, is easy to industrialize and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a morphological diagram of the strain KLBMP0506 described in the present invention on YPD solid culture medium, wherein A is the front side of the plate and B is the back side of the plate.

[0024] Figure 2 The figure shows the effect of the strain KLBMP0506 described in the present invention on the growth of Dendrobium officinale (CK is the control group, 3w, 6w, and 9w represent the co-cultivation of strain KLBMP0506 and Dendrobium officinale for 3 weeks, 6 weeks, and 9 weeks, respectively). In the figure, A is the fresh weight; B is the dry weight.

[0025] Figure 3 This is the effect of the strain KLBMP0506 described in the present invention on the accumulation of polysaccharides in Dendrobium officinale. In the figure, A is the standard curve of total polysaccharides; and B is the total polysaccharide content of each group.

[0026] Figure 4 The figure shows the effect of the strain KLBMP0506 described in the present invention on the accumulation of dendrobium alkaloids in Dendrobium officinale. In the figure, A is the standard curve of dendrobium alkaloids; and B is the content of dendrobium alkaloids in each group. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments to help better understand the present invention, but the protection scope of the present invention is not limited thereto:

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Isolation, purification and identification of strain KLBMP0506

[0030] 1. Isolation and purification of strains

[0031] (1) The strain was isolated from healthy tea fruit picked from Gao'an, Jiangxi Province. The fresh tea fruit was sealed in a ziplock bag and brought back to the laboratory for storage at 4°C for subsequent isolation of endophytic fungi.

[0032] (2) The tea fruit was rinsed with running water for 10 min, and the rinsed samples were ultrasonically cleaned for 10 min to prevent the difficult-to-rinse substances attached to the tissue surface from affecting the separation results.

[0033] (3) The cleaned samples were surface disinfected in a clean bench. They were first treated with 75% ethanol solution for 1 min, rinsed once with sterile water, and then treated with 2% sodium hypochlorite solution for 6 min. The sterile water from the last rinse was retained for later use. The surface disinfected samples were placed on sterile filter paper to absorb the moisture for later use.

[0034] (4) Cut the disinfected sample into small pieces with sterilized scissors, then spread the small pieces on a YPD plate and culture at 28°C for 2-5 days. At the same time, perform routine sterility testing on the sterile water used for the last rinse in step (3) above to ensure that the surface of the sample is thoroughly disinfected.

[0035] (5) Pick a single colony grown on the YPD plate and inoculate it onto the same medium (YPD) for streak purification and isolation. Incubate at 28°C for 2-5 days. Continue to pick a single colony and repeat the isolation, purification and culture process several times until a pure culture is obtained.

[0036] 2. Identification of strains

[0037] The colony, morphological characteristics, and physiological and biochemical characteristics of the abnormal Wickerhamomyces strain of tea tree endophytic fungi are as follows:

[0038] (1) Morphological characteristics of strains

[0039] The purified strain grew well on YPD solid medium. The colonies were round, milky white, smooth and moist, with neat edges and the same color on both sides (such as Figure 1 ).

[0040] (2) Physiological and biochemical characteristics of strains

[0041] Strain KLBMP0506 can utilize glucose, hydrolyze urea, mannitol, xylose, and gelatin, but not hydrolyze starch. Its oxidase reaction and VP reaction are negative. It can dissolve phosphorus and produce tryptophan. It grows in 6% NaCl but not in 8% NaCl. Its optimum growth temperature is 28°C.

[0042] (3) Strain ITS sequence sequencing

[0043] The purified strain was amplified by PCR and sequenced (amplified by conventional PCR method). The ITS sequence sequencing result is shown in SEQ ID NO.1. The sequencing result was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed that the similarity between strain KLBMP0506 and Wickerhamomyces p. strain LP8 was 99.82%. Combining morphological characteristics and molecular methods, strain KLBMP0506 was identified as abnormal Wickerhamomyces genus (Wickerhamomyces p.)

[0044] Example 2: Effect of strain KLBMP0506 on the growth of Dendrobium officinale, Figure 2 As shown,

[0045] (1) The strain KLBMP0506 was inoculated into fresh YPD liquid medium and cultured at 28°C, 200 rpm for about 16 h to activate the strain; then the strain was transferred to 200 mL YPD liquid medium at a ratio of 1:100 and cultured at 28°C, 200 rpm until the OD 600 =0.6, centrifuge at 6000 rpm for 10 min, discard the supernatant to collect the bacteria, and resuspend the bacteria with sterile water to adjust the concentration of the bacterial solution to OD 600 =0.5.

[0046] (2) Select Dendrobium officinale tissue culture seedlings with consistent growth in the clean bench, weigh them and transfer them to new culture medium (about 4.5 g per bottle of tissue culture seedlings), inoculate three clusters of plants in each bottle in a triangle, and transfer a total of 50 bottles of seedlings and place them in the tissue culture room for culture at 25°C, light intensity of 1500 lx, and light time of 12 h / d.

[0047] (3) In the 0th, 3rd and 6th weeks of culture, 200 μL of the bacterial solution in step (1) was respectively inoculated in the middle of the three clusters of tissue culture seedlings in each bottle of the transferred seedlings in step (2), and 10 bottles were repeated for each treatment time. In addition, a blank control group was set up, i.e., Dendrobium officinale tissue culture seedlings that were not inoculated with bacteria, and the control group was also repeated for 10 bottles.

[0048] (4) All groups were harvested after being transferred to the new culture medium for 9 weeks, namely, the control group, co-cultured with bacteria for 3 weeks, co-cultured with bacteria for 6 weeks, and co-cultured with bacteria for 9 weeks, a total of 4 groups of materials.

[0049] Weigh the fresh weight and dry weight of the four groups of materials. Figure 2As shown, the fresh weight of Dendrobium officinale tissue culture seedlings co-cultured with strain KLBMP0506 for 3, 6 and 9 weeks increased by 5.8%, 9.8% and 16.1% respectively compared with the control group, and the dry weight also showed an increasing trend compared with the control group, increasing by 30.4%, 42.0% and 47.3% respectively.

[0050] Example 3: Effect of strain KLBMP0506 on the accumulation of polysaccharides in Dendrobium officinale Figure 3 As shown,

[0051] The polysaccharide content of the four groups of materials in Example 2 was measured. The specific measurement method is as follows: 1. Dry the sample to be tested, weigh about 0.05g of the sample, add 1mL of distilled water and grind evenly; then heat in a 100℃ boiling water bath for 2h, let stand to cool, and centrifuge at 10000rcf for 10min; aspirate 200μL of the supernatant, slowly add 800μL of anhydrous ethanol, mix upside down, and place in a 4℃ refrigerator overnight to settle; aspirate 200μL of the above solution, add 100μL of phenol and 500μL of concentrated sulfuric acid, mix evenly, and react in a 90℃ water bath for 20min, and cool to room temperature with running water; take 200μL and add to the ELISA plate, measure the absorbance at 490nm, and record it as A490; draw a standard curve: prepare the glucose standard into a 1mg / mL solution, and dilute it in a gradient, and then detect it according to the above method. Calculation of total polysaccharide content of the sample: Total polysaccharide content (mg / g) The sample volume was diluted 5 times, y was the value obtained by inserting into the standard curve, V was the volume of the supernatant (0.2 mL), and W was the mass of the sample (g).

[0052] Based on the standard curve drawn ( Figure 3 -A), and the polysaccharide content of each group was calculated ( Figure 3 -B), the polysaccharide content of the control group was 53.799 mg / g, while the polysaccharide contents of the three groups of materials co-existing with KLBMP0506 for 3 weeks, 6 weeks, and 9 weeks were 93.911 mg / g, 117.542 mg / g, and 144.365 mg / g, respectively, which were increased by 74.6%, 118.5%, and 168.3% compared with the control group.

[0053] Example 4: Effect of strain KLBMP0506 on the accumulation of dendrobium alkaloids in Dendrobium officinale, such as Figure 4 As shown,

[0054] The dendrobium alkaloid content of the four groups of materials in Example 2 was determined. The determination of dendrobium alkaloid was based on the 2020 edition of the Chinese Pharmacopoeia and detected by gas chromatography.

[0055] After the plant sample to be tested is dried at a constant temperature, it is ground into powder with a mortar. Accurately weigh 0.1g of the powder, add 10mL of methanol (containing 0.05% formic acid), heat and reflux for about 3h, cool to room temperature, transfer the extract to a new beaker and dilute to 10mL, mix well, and filter the extract for standby use. Detection conditions: chromatographic column DB-1: length 30m, inner diameter width 0.25mm, membrane thickness 0.25μm, column temperature 80℃, injection volume 5μL, detection temperature 250℃. Draw a standard curve: dilute the dendrobium alkaloid standard to 0.1μg / mL, 0.25μg / mL, 0.5μg / mL, 1μg / mL and 2.5μg / mL, and then detect according to the above method.

[0056] The standard curve of dendrobium alkaloids was obtained by testing the standard product of dendrobium alkaloids: y=7.7016x-0.1856, R 2 =0.9993( Figure 4 -A). The results of the four groups obtained by gas chromatography were put into the standard curve to calculate the dendrobium alkaloid content of the four groups of materials ( Figure 4 -B), the dendrobium content of the control group was 2.045 μg / g, and the dendrobium content of the three groups of materials that had been symbiotic with strain KLBMP0506 for 3 weeks, 6 weeks, and 9 weeks were 2.443 μg / g, 4.792 μg / g, and 5.271 μg / g, respectively, which increased by 19.5%, 134.3%, and 157.8% compared with the control group.

[0057] Based on the results of the above embodiments, the strain KLBMP0506 provided by the present invention can effectively promote the growth of Dendrobium officinale and the accumulation of secondary metabolites. The strain can be used as a microbial fertilizer for the seedling cultivation and artificial cultivation of Dendrobium officinale, thereby improving the quality of the medicinal material, which is of great significance for ensuring the effectiveness and safety of the use of Dendrobium officinale medicinal materials.

[0058] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. An endophytic fungus of tea tree, characterized in that: The tea tree endophytic fungus was named as Wickham's yeast strain ( Wickerhamomyces sp.), the depository unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit name is: KLBMP0506, the deposit number is: CGMCC No. 25719, and the deposit date is September 15, 2022.

2. A use of the endophytic fungus of tea tree as claimed in claim 1, characterized in that: Promote the growth of Dendrobium officinale and the accumulation of secondary metabolites.

3. A method for preparing an endophytic fungus agent for tea trees, characterized in that: Utilizing the Wickham yeast strain of claim 1 ( Wickerhamomyces sp.), and process as follows: The strain KLBMP0506 was inoculated into fresh YPD liquid medium and cultured at 28°C in a shaking incubator at 200 rpm / min for 16 h to activate the strain; Transfer 2 mL of activated bacterial solution to 200 mL YPD liquid medium at a volume ratio of 1:100, and culture at 28°C, 200 rpm / min in a shaking incubator until OD 600 = 0.6; The culture product was centrifuged at 6000 rpm / min for 10 min, the supernatant was discarded to collect the bacteria, and the bacteria were resuspended in sterile water to adjust the bacterial solution concentration to OD 600 =0.

5.

4. The use of endophytic fungi in tea trees according to claim 3, characterized in that: KLBMP0506 bacterial solution was inoculated into Dendrobium officinale tissue culture seedlings. Each bottle of tissue culture seedlings was inoculated with 200 μL of bacterial solution. The bacterial solution concentration was OD 600 =0.5, the bacterial solution was inoculated near the base of the tissue culture seedlings, thereby promoting the growth of Dendrobium officinale and the accumulation of secondary metabolites.

5. The method for preparing the tea tree endophytic fungus agent according to claim 3, characterized in that: The YPD liquid culture medium is composed of 10 g yeast extract, 20 g peptone, and 20 g glucose, which are fixed to 1000 mL distilled water and have a natural pH.

6. The method for preparing the tea tree endophytic fungus agent according to claim 3, characterized in that: The bacterial agent is a microbial fertilizer.

7. A composition comprising the endophytic fungus of tea plant according to claim 1.

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