An endophytic fungus Al-EF1 of shallot and its application in promoting vegetable growth

Abstract

The present invention belongs to the technical field of microorganisms, and discloses an endophytic fungus Al-EF1 of shallot and its application in promoting the growth of vegetables. The present invention separates and obtains the endophytic fungus (Apiospora rasikravindrae) Al-EF1 of shallot for the first time, and verifies through experiments that the fungus has the effect of significantly promoting the growth of vegetable plants (such as peppers, tomatoes, and cucumbers), thereby providing resource guarantee for promoting the growth of crops.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic fungus Al-EF1 of shallot and application thereof in promoting vegetable growth. Background Art

[0002] There are rich microbial communities in plants, including endophytic groups such as bacteria and fungi, which can establish mutually beneficial symbiotic relationships with host plants. Due to their taxonomic diversity, only a few species of endophytic fungi have been characterized and identified, but some scholars have found that endophytic fungi associations have beneficial effects on plants, such as regulating hormone levels in plants by increasing jasmonic acid and reducing endogenous salicylic acid levels, allowing host plants to effectively respond to external stresses. Endophytic fungi can also co-evolve with host plants, enabling them to tolerate various stresses, promote the absorption of nutrients, and effectively promote plant growth. In short, endophytic fungi benefit host plants in a variety of ways and are gradually being used as biological control agents. As an environmentally friendly alternative to chemical fertilizers and pesticides, they can solve the environmental problems currently existing in the vegetable industry caused by excessive use of chemical fertilizers and pesticides.

[0003] Shallot (Allium fistulosum L.) is a plant of the genus Allium in the Amaryllis family. It has a spicy smell and has a cultivation history of more than 3,000 years in my country. Intercropping of Allium plants with other plants can effectively alleviate the obstacles of continuous cropping, improve the growth of plants, increase yields and reduce the occurrence of diseases. Although the Apiospora genus fungi have been discovered, there is currently no clear Chinese name. Previous studies have found that this genus of fungi has been reported as endophytes or humus worldwide. In 2022, Apiospora rasikravindrae was recorded as a new species, but its function has not yet been determined. Summary of the invention

[0004] The first aspect of the present invention aims to provide an endophytic fungus of shallot (Apiospora rasikravindrae) Al-EF1.

[0005] The second aspect of the present invention aims to provide a method for preparing a culture of endophytic fungus Al-EF1 of shallot.

[0006] The third aspect of the present invention aims to provide a culture of endophytic fungus Al-EF1 of shallot.

[0007] The fourth aspect of the present invention aims to provide a microbial agent.

[0008] The purpose of the fifth aspect of the present invention is to provide the use of the shallot endophytic fungus Al-EF1 of the first aspect of the present invention, the shallot endophytic fungus Al-EF1 culture of the third aspect of the present invention or the microbial agent of the fourth aspect of the present invention in promoting plant growth / preparing products that promote plant growth.

[0009] The sixth aspect of the present invention aims to provide a method for promoting plant growth.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides an endophytic fungus Al-EF1 of shallot, the preservation name of the endophytic fungus Al-EF1 of shallot is Apiospora sp.Al-EF1, it is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO:M20242529, and the preservation date is November 11, 2024.

[0012] The present invention uses a tissue separation method to separate the endophytic fungus Al-EF1 from shallot leaves, and determines that the strain is Apiospora rasikravindrae by amplifying and detecting the ITS region of the Al-EF1 fungus. By culturing the shallot endophytic fungus Al-EF1 and treating vegetables such as tomatoes, cucumbers, and peppers with the bacterial solution through root irrigation, it is found that the growth of vegetable plants can be significantly promoted, providing a feasible regulation method for the green and efficient cultivation of vegetable crops.

[0013] The second aspect of the present invention provides a method for preparing a culture of endophytic fungus Al-EF1 of shallot, comprising:

[0014] Provide liquid culture system;

[0015] The endophytic fungus Al-EF1 of shallot according to the first aspect of the present invention is inoculated into the liquid culture system for culture to obtain a culture of the endophytic fungus Al-EF1 of shallot.

[0016] In some embodiments of the present invention, the liquid culture system contains nutrients, and the nutrients include carbon and nitrogen elements. For example, the nutrients can be provided in the form of a liquid culture medium, such as a CM liquid culture medium.

[0017] In some embodiments of the present invention, the culture conditions are 20-30° C., 100-300 rpm shaking for 3-4 days.

[0018] The third aspect of the present invention provides a culture of endophytic fungus Al-EF1 of shallot, which is prepared by the preparation method of the second aspect of the present invention.

[0019] The term "culture" refers to a general term for liquid or solid products (all substances in the culture container, fermentation products) that grow a microbial community after artificial inoculation and cultivation, that is, products obtained by growing and / or amplifying microorganisms, which can be biologically pure cultures of microorganisms, or can contain a certain amount of culture medium, metabolites or other components produced during the culture process.

[0020] The fourth aspect of the present invention provides a microbial agent, comprising the endophytic fungus Al-EF1 of the first aspect of the present invention, the spores of the endophytic fungus Al-EF1 of the first aspect of the present invention, the culture of the endophytic fungus Al-EF1 of the third aspect of the present invention or any combination thereof.

[0021] The active ingredients of the above-mentioned microbial agent may be the above-mentioned endophytic fungus Al-EF1 of shallot, the culture (such as fermentation product) of the above-mentioned endophytic fungus Al-EF1 of shallot and / or the endospores of the above-mentioned endophytic fungus Al-EF1 of shallot. The active ingredients of the above-mentioned microbial agent may also contain other biological components or non-biological components. Those skilled in the art may determine the other active ingredients of the above-mentioned microbial agent according to the effect of the microbial agent.

[0022] According to needs, a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may be added to the bacterial agent.

[0023] The fifth aspect of the present invention provides the use of the shallot endophytic fungus Al-EF1 of the first aspect of the present invention, the shallot endophytic fungus Al-EF1 culture of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention in promoting plant growth / preparing products that promote plant growth.

[0024] In some embodiments of the present invention, the promoting plant growth includes increasing the area of ​​plant leaves and / or increasing the net photosynthetic rate of plant leaves.

[0025] In some embodiments of the present invention, the plants include plants from the family Cruciferae, Solanaceae and Cucurbitaceae.

[0026] In some embodiments of the present invention, the cruciferous plant includes at least one of radish, cabbage, Brussels sprouts, cabbage, cauliflower, kale, mustard, white mustard, black mustard, rapeseed, horseradish, Lepidium, woad, iris, clematis, scutellaria, Artemisia selengensis, Arabidopsis thaliana, violet, Arabidopsis thaliana, osmanthus fragrans and sugar mustard.

[0027] In some embodiments of the present invention, the Solanaceae plant includes at least one of eggplant, pepper, potato, tomato, tobacco, Solanum nigrum, ginseng fruit, and wolfberry.

[0028] In some embodiments of the present invention, the Cucurbitaceae plant includes at least one of watermelon, pumpkin, cucumber, loofah, cantaloupe, oil-residue fruit, cracked melon, wax gourd, bitter melon, monk fruit, poisonous melon, hat melon, chayote, phoenix melon, small-fruit pumpkin, and gourd.

[0029] The sixth aspect of the present invention provides a method for promoting plant growth, comprising the step of applying the shallot endophytic fungus Al-EF1 of the first aspect of the present invention, the shallot endophytic fungus Al-EF1 culture of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention during plant growth.

[0030] In some embodiments of the present invention, the applying comprises wetting, drenching, atomizing, drizzling, spraying, or misting the plant or the place where the plant grows.

[0031] In some embodiments of the present invention, the plants include plants from the family Cruciferae, Solanaceae and Cucurbitaceae.

[0032] In some embodiments of the present invention, the cruciferous plant includes at least one of radish, cabbage, Brussels sprouts, cabbage, cauliflower, kale, mustard, white mustard, black mustard, rapeseed, horseradish, Lepidium, woad, iris, clematis, scutellaria, Artemisia selengensis, Arabidopsis thaliana, violet, Arabidopsis thaliana, osmanthus fragrans and sugar mustard.

[0033] In some embodiments of the present invention, the Solanaceae plant includes at least one of eggplant, pepper, potato, tomato, tobacco, Solanum nigrum, ginseng fruit, and wolfberry.

[0034] In some embodiments of the present invention, the Cucurbitaceae plant includes at least one of watermelon, pumpkin, cucumber, loofah, cantaloupe, oil-residue fruit, cracked melon, wax gourd, bitter melon, monk fruit, poisonous melon, hat melon, chayote, phoenix melon, small-fruit pumpkin, and gourd.

[0035] The beneficial effects of the present invention are:

[0036] The present invention isolates the endophytic fungus Apiospora rasikravindrae Al-EF1 of shallot for the first time, and verifies through experiments that the fungus has the effect of significantly promoting the growth of vegetable plants (such as peppers, tomatoes, and cucumbers), providing resource guarantee for promoting the growth of crops. Specifically, in the growth promotion experiment, the fungal bacterial liquid was infused at the taproot of the vegetable plants. The results showed that the overall growth of the plants inoculated with the fungus was better than that of the uninoculated plants. The leaf areas of peppers, tomatoes, and cucumbers increased by 48.52%, 21.29%, and 21.23%, respectively; and the net photosynthetic rates increased by 11.95%, 29.83%, and 38.47%, respectively. It shows that after the fungal strain provided by the present invention is made into a bacterial liquid and applied, it has a significant growth-promoting effect on the plant, and has good application prospects in the field of facility vegetable industry and agricultural bacterial fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The growth morphology of the endophytic fungus Al-EF1 strain of shallot on CM solid culture medium, where (a) is the front view of the culture medium and (b) is the mycelium morphology.

[0038] Figure 2 The results of identification of the endophytic fungus Al-EF1 strain of shallot, where (a) is the length of the amplified sequence of the ITS region located between the 18S, 5.8S and 28S rRNA genes of the fungus; (b) is the evolutionary tree constructed between the sequence of the endophytic fungus Al-EF1 strain of shallot and multiple strains of the genus Apiospora with extremely high similarity.

[0039] Figure 3 The effect of the inoculation of the endophytic fungus Al-EF1 strain of shallot on the growth of pepper plants; (a) is the growth diagram of pepper plants without fungus inoculation, (b) is the growth diagram of plants inoculated with Al-EF1 strain, (c) is the statistical data of leaf area size of leaves in the same leaf position of pepper control group Control and experimental group Al-EF1 plants, (d) is the statistical data of net photosynthetic rate of leaves in the same leaf position of pepper control group Control and experimental group Al-EF1 plants. The t-test was used for significance analysis, and * and *** indicate p < 0.05 and p < 0.001, respectively.

[0040] Figure 4The effect of onion endophytic fungus Al-EF1 strain inoculation on tomato plant growth, where (a) is the growth graph of tomato plants without fungus inoculation (b) is the growth graph of tomato plants after inoculation with Al-EF1 strain, (c) is the statistical data of leaf area size of leaves in the same leaf position of tomato control group Control and experimental group Al-EF1 plants, (d) is the statistical data of net photosynthetic rate of leaves in the same leaf position of tomato control group Control and experimental group Al-EF1 plants. The t-test was used for significance analysis, and * and ** respectively indicate p < 0.05 and p < 0.01.

[0041] Figure 5 The effect of the inoculation of the endophytic fungus Al-EF1 strain of shallot on the growth of cucumber plants, where (a) is the growth diagram of cucumber plants without fungus inoculation, (b) is the growth diagram of cucumber plants after inoculation with Al-EF1 strain, (c) is the statistical data of leaf area size of leaves in the same leaf position of cucumber control group Control and experimental group Al-EF1 plants, (d) is the statistical data of net photosynthetic rate of leaves in the same leaf position of cucumber control group Control and experimental group Al-EF1 plants. The t-test was used for significance analysis, and ** indicates p < 0.01. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below through specific examples.

[0043] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1 Isolation, purification and morphological observation of endophytic fungi

[0047] After the collected shallots were transplanted and grown, the shallot leaves were sterilized and separated by tissue separation method. The leaves were rinsed with clean water for 1-2 minutes, placed in a test tube containing 75% ethanol, slightly shaken for 1-2 minutes, rinsed with ddH2O 3 times, placed in a test tube containing 1.5% NaClO, slightly shaken for 1-2 minutes, and rinsed with ddH2O 3 times.

[0048] The disinfection process is carried out in a clean bench, and the instruments used must be sterilized with high-pressure steam at 121°C. Take 2.5-5 μL of sterile water used to rinse the tissue for the last time and apply it to the MEA solid culture medium as a control. Cut the leaves into small pieces of 0.5 cm × 0.5 cm and spread them flat on the MEA solid culture medium, and place them in a 28°C constant temperature incubator for culture. When hyphae grow around the tissue, use an inoculation loop to inoculate them into new MEA solid culture medium and CM solid culture medium for culture. After purification for 2-3 generations, growth statistics and strain identification can be performed.

[0049] MEA solid medium formula: 15 g malt extract, 15 g soy peptone, 15 g agar, supplemented with ddH2O to 1 L, pH adjusted to 5.6±0.2.

[0050] CM solid medium formula: 3g acid hydrolyzed casein, 3g acid hydrolyzed casein, 6g yeast extract, 10g sucrose, 15g agar, supplemented with ddH2O to 1L.

[0051] Experimental results: An endophytic fungus was obtained by isolation and cultivation, named as endophytic fungus Al-EF1. After 7-14 days of cultivation on MEA solid medium and CM solid medium, the fungus grew well, its colony was white, and the velvety hyphae were dense and developed. The density of hyphae gradually decreased from the center to the edge, and the overall shape was a standard circle ( Figure 1 ).

[0052] Example 2 Molecular Biological Identification of Endophytic Fungus Al-EF1

[0053] Use a hole puncher to obtain 1-2 pieces of culture medium with hyphae (Example 1), remove the culture medium as much as possible, put it into a grinding tube with steel balls, and immediately put it into liquid nitrogen for quick freezing before grinding. The ground sample is used to extract genomic DNA using a fungal genomic DNA rapid extraction kit (purchased from Sangon Biotech).

[0054] The above-extracted DNA solution was used as a template, and universal primers (ITS1: 5'-CTTGGCATTTAGAGGAAGTAA-3' (SEQ ID NO: 1); ITS4: 5'-GCTGCGTTCTTCATGGATGC-3' (SEQ ID NO: 2)) were used as upstream and downstream primers to perform PCR amplification on the ITS fragment of the identified strain. The PCR amplification system was 25 μL: 12.5 μL Green Taq Mix, 1 μL forward primer ITS1, 1 μL reverse primer ITS4, 2 μL DNA template, 8.5 μL ddH2O. Amplification conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, 35 cycles; 72°C thorough extension for 5 min.

[0055] The PCR products were detected by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results were compared with all ITS sequences in the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ) using the BLAST program, and the 10 strains with the highest similarity were selected for phylogenetic tree analysis.

[0056] Experimental results: The ITS gene sequence of the endophytic fungus Al-EF1 is shown in SEQ ID NO:3, with a length of 617 bp ( Figure 2 ). The ITS gene sequence of the strain was searched for homologous sequences in the GenBank database (NCBI), and the strain with the highest similarity was found using BLAST analysis. The results showed that the strain had a similarity of 99.16% with the discovered Apiospora rasikravindrae. At the same time, the phylogenetic tree was constructed using MEGA 11 software, and it was found that the endophytic fungus Al-EF1 was clustered with multiple strains of Apiospora fungi and was closest to Apiospora rasikravindrae ( Figure 2 ), the strain was considered to be Apiospora rasikravindrae. The endophytic fungus Al-EF1 (Apiospora sp. Al-EF1) was then deposited in the China Center for Type Culture Collection, address: Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Wuhan, the deposit date is November 11, 2024, and the deposit number is CCTCC NO: M20242529.

[0057] ITS sequence of endophytic fungus Al-EF1:

[0058] (SEQ ID NO:3).

[0059] Example 3 Growth-promoting effect of endophytic fungus Al-EF1 on vegetable crops

[0060] This example is used to investigate the effect of endophytic fungus Al-EF1 on the growth promotion effect of vegetable crops, as follows:

[0061] (1) Seeds of pepper variety "HJ008", tomato variety "Alisa Craig", and cucumber variety "Jinyanxin No. 7" were sown in 72-hole trays, and seedlings of the same size were selected and transplanted into seedling pots with a diameter of 10 cm after two cotyledons grew. The cultivation medium used (the medium type was a mixture of peat and vermiculite in a ratio of 3:1) was sterilized by high-pressure steam at 121℃, and each treatment was replicated at least 3 times.

[0062] (2) Use a hole punch to take 2-3 freshly cultured mushroom cakes (Example 1) and place them in 200-300 mL CM liquid culture medium. Culture at 28°C and 200 rpm for 3-4 days and collect the bacterial liquid. Filter the bacterial liquid with gauze, centrifuge the filtrate at 5000 r / min for 10 min, resuspend the bacteria with ddH2O and dilute to obtain a spore concentration of 1×10 7 Each plant was treated with two treatments: the control group was injected with ddH2O, and the treatment group was injected with the corresponding concentration of bacterial solution. The treatment method was to use a syringe to inject 20mL of ddH2O or bacterial solution into the main root of each pot.

[0063] The formula of CM liquid culture medium is: 3g acid hydrolyzed casein, 3g acid hydrolyzed casein, 6g yeast extract, 10g sucrose, supplemented with ddH2O to 1L, and sterilized by high pressure steam at 121℃ for 20min.

[0064] (3) After the plants were transplanted and allowed to grow for 2 to 3 days, the roots were irrigated with bacterial solution, and the treatment was carried out once every week for a total of 2 treatments. After the treatment, the growth of the plants was observed regularly, and the growth phenotype of the plants was photographed on the 35th day. The area of ​​the leaves at the same leaf position (the 4th leaf position) of each treatment plant was calculated using ImageJ software, and the net photosynthetic rate of the plant leaves was measured using a Li-6400 photosynthetic meter.

[0065] The results are as follows Figure 3 to Figure 5 As shown in Tables 1 and 2, compared with plants not inoculated with fungi, peppers inoculated with endophytic fungus Al-EF1 ( Figure 3 ),tomato( Figure 4 ) and cucumber( Figure 5 ) The leaf area of ​​the plants increased by 48.52%, 21.29%, and 21.23%, respectively, and the net photosynthetic rate increased by 11.95%, 29.83%, and 38.47%, respectively (Tables 1 and 2, calculated by taking the average of 3 repeated data under each treatment). This shows that the endophytic fungus Al-EF1 can effectively promote the growth of "HJ008" pepper, "AlisaCraig" tomato, and "Jinyan New No. 7" cucumber. The overall growth is better than that of uninoculated plants, and the net photosynthetic rate of each vegetable leaf is significantly increased. It has good application prospects in the field of facility vegetable industry and agricultural bacterial fertilizer.

[0066] Table 1 Statistical data of leaf area of ​​tomato, pepper and cucumber plants under each treatment

[0067]

[0068] Table 2 Statistical data of net photosynthetic rate of tomato, pepper and cucumber plants under each treatment

[0069]

[0070] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An endophytic fungus of shallot (Apiospora rasikravindrae) Al-EF1, characterized in that The shallot endophytic fungus Al-EF1 is stored under the name of Apiospora sp. Al-EF1 and is deposited in the China Center for Type Culture Collection with the storage number of CCTCC NO: M 20242529 and the storage date of November 11, 2024.

2. A method for preparing a culture of endophytic fungus Al-EF1 of shallot, comprising: Provide liquid culture system; The endophytic fungus Al-EF1 of shallot according to claim 1 is inoculated into the liquid culture system for culturing to obtain a culture of the endophytic fungus Al-EF1 of shallot.

3. The culture of endophytic fungus Al-EF1 of shallot is prepared by the preparation method described in claim 2.

4. A microbial agent, characterized in that: The method comprises the endophytic fungus Al-EF1 of shallot according to claim 1, the spores of the endophytic fungus Al-EF1 of shallot according to claim 1, the culture of the endophytic fungus Al-EF1 of shallot according to claim 3 or any combination thereof.

5. Use of the endophytic fungus Al-EF1 of shallot according to claim 1, the culture of the endophytic fungus Al-EF1 of shallot according to claim 3, or the microbial agent of claim 4 in promoting plant growth / preparing a product that promotes plant growth.

6. The use according to claim 5, characterized in that: The promoting plant growth includes promoting the area of ​​plant leaves and / or increasing the net photosynthetic rate of plant leaves.

7. The use according to claim 6, characterized in that: The plants include cruciferous plants, solanaceous plants and cucurbitaceous plants.

8. A method for promoting plant growth, comprising the step of applying the shallot endophytic fungus Al-EF1 according to claim 1, the shallot endophytic fungus Al-EF1 culture according to claim 3, or the microbial agent according to claim 4 during the plant growth process.

9. The method according to claim 8, characterized in that The application includes wetting, drenching, misting, drizzling, spraying, atomizing, and root irrigation to the plants or the place where the plants grow.

10. The method according to claim 9, characterized in that The plants include cruciferous plants, solanaceous plants and cucurbitaceous plants.

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