A ericoid mycorrhizal fungus wf17-2-1 and its application and product for promoting growth of blueberries

Abstract

The application relates to a rhododendron ericoid mycorrhizal fungus WF17-2-1 and its application and product in the growth promotion of blueberries. The strain WF17-2-1 can form a mycelium cluster on the epidermal cells of the blueberry roots, significantly improves the root growth, increases the nutrient absorption capacity of the blueberries, promotes the synthesis of plant hormones, adjusts the growth metabolism of the blueberries, enhances the photosynthesis of the plants, helps the accumulation of mineral elements and photosynthetic products and other nutrients of the blueberries, improves the nutritional value of the blueberries, and is beneficial to the development of a biological fertilizer or a growth promoter based on the strain WF17-2-1 and the green and efficient development of the blueberry industry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a rhododendron mycorrhizal fungus WF17-2-1 and its application and products in promoting blueberry growth. Background Technology

[0002] blueberry( Vaccinium Blueberries (Vaccinium spp.) are a shrub belonging to the genus Vaccinium in the Ericaceae family. Their fruits are blue, sweet and sour, and rich in various vitamins, minerals, and antioxidants, making them highly nutritious. Therefore, the blueberry industry has developed rapidly worldwide, becoming a high-value economic product. However, blueberry growth has high requirements for soil conditions and is difficult to cultivate, resulting in low levels of commercialization and large-scale production, hindering the development of the blueberry industry. Existing blueberry growth-promoting technologies heavily rely on improved variety selection and fertilizer application. Improved variety selection is time-consuming and results in a limited number of varieties, while fertilizer application easily leads to food residues and environmental pollution. Therefore, the development of the blueberry industry requires greener and more efficient technological support.

[0003] Ericoid mycorrhizal fungi (EMF) are a class of soil fungi that can form symbiotic relationships with plant roots. Their relationships with plants can be categorized into three types: mutualistic symbiosis, commensal symbiosis, and parasitic symbiosis. Among these, mutualistic symbiotic fungi enhance a plant's ability to absorb nutrients and decompose insoluble complexes and organic matter after infection, maintaining the fungi's own needs while simultaneously promoting plant growth. Therefore, mutualistic symbiotic strains are often developed for use as bio-fertilizers or humus to improve crop growth.

[0004] The screening of symbiotic fungi is of great significance to blueberry cultivation and development. On the one hand, the rational development of symbiotic fungi can alleviate the current technical difficulties in blueberry cultivation, improve the quality and yield of blueberries, and promote the diversification of the blueberry industry. On the other hand, the compounding of symbiotic fungi into bio-fertilizers can reduce the use of chemical fertilizers and help solve problems such as soil acidification and salinization that occur during blueberry cultivation. This is crucial for the sustainable development of the blueberry industry and the protection of the ecological environment. Summary of the Invention

[0005] To address the above problems, this invention provides a rhododendron mycorrhizal fungus WF17-2-1 and its applications. (From *Rhododendron molle* (…)) Vaccinium A rhodomycorrhizal fungus was obtained through root screening of *Bracteatum Thunb.*. This strain can enhance the blueberry's ability to absorb nutrients and promote its growth after infecting the blueberry root system, which is of great significance for reducing costs and increasing efficiency in the blueberry industry.

[0006] Application of a rhododendron mycorrhizal fungus WF17-2-1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 1, 2024, with accession number GDMCC No. 64931.

[0007] Based on morphological identification using pure fungal culture, strain WF17-2-1 was identified as a filamentous fungus, exhibiting an irregular yellow colony morphology on solid culture medium with a raised center and filamentous edges.

[0008] Molecular biological identification revealed that strain WF17-2-1 belongs to the phylum Ascomycota, class Leotiomycetes, order Helotiales, Hyaloscyphaceae, and Hyaloscypha.sp.

[0009] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in increasing blueberry biomass, when co-cultured with blueberries, can increase the fresh and dry weight of the entire blueberry plant.

[0010] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in improving blueberry root systems involves co-culturing the strain with blueberries, which can form mycelial clusters within the epidermal cells of blueberry roots, thereby increasing the root length, root surface area, and volume of blueberries.

[0011] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in regulating blueberry growth metabolism, whereby co-culturing this strain with blueberries can increase the accumulation of zeatin nucleosides in blueberries.

[0012] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in promoting blueberry photosynthesis involves co-culturing the strain with blueberries, which can enhance the photosynthetic enzyme activity of blueberries.

[0013] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in improving the nutritional value of blueberries, when co-cultured with blueberries, can increase the total phosphorus and total potassium content of blueberries.

[0014] Preferably, the rhododendron mycorrhizal fungus WF17-2-1 and its applications include the application of strain WF17-2-1 in blueberry cultivation.

[0015] A product that promotes blueberry growth, said product comprising azadirachtic mycorrhizal fungus WF17-2-1 or its metabolites.

[0016] Preferably, the product includes a growth promoter or a growth-promoting fertilizer.

[0017] Compared with the prior art, the beneficial effects of this invention are that the rhododendron mycorrhizal fungus WF17-2-1 can form a symbiotic relationship with blueberry roots, forming mycelial clusters in the epidermal cells of blueberry roots. This significantly improves root growth, increases nutrient absorption, promotes the synthesis of plant hormones, regulates plant growth and metabolism, enhances plant photosynthesis, helps blueberry accumulate nutrients, improves the nutritional value of blueberries, and is conducive to the development of bio-fertilizers or growth promoters based on strain WF17-2-1, thus contributing to the green and efficient development of the blueberry cultivation industry. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] In the attached image:

[0021] Figure 1 : Mycelial masses formed by the rhododendron mycorrhizal fungus WF17-2-1 infecting the epidermal cells of blueberry roots;

[0022] Figure 2 Colony morphology of rhododendron mycorrhizal fungus WF17-2-1;

[0023] Figure 3 Phylogenetic tree constructed based on the ITS sequencing results of the rhododendron mycorrhizal fungus WF17-2-1;

[0024] Figure 4 Phylogenetic tree constructed based on the 18S rDNA sequencing results of the rhododendron mycorrhizal fungus WF17-2-1

[0025] Figure 5 Biomass test results of blueberries after inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0026] Figure 6 Results of root length test on blueberries after inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0027] Figure 7 Results of root surface area test after blueberry inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0028] Figure 8Results of root volume test on blueberries after inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0029] Figure 9 Results of zeatin nucleoside test after blueberry inoculation with rhodomycorrhizal fungus WF17-2-1;

[0030] Figure 10 Results of total phosphorus test after blueberry inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0031] Figure 11 Total potassium test results after blueberry inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0032] Figure 12 Results of maximum quantum yield test of photosystem II after blueberry inoculation with rhododendron mycorrhizal fungus WF17-2-1;

[0033] Figure 13 Results of actual photonic efficiency test after blueberry inoculation with rhododendron mycorrhizal fungus WF17-2-1. Detailed Implementation

[0034] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are all commercially available; unless specifically specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0035] A rhododendron mycorrhizal fungus WF17-2-1 and its applications, wherein the rhododendron mycorrhizal fungus WF17-2-1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 1, 2024, with accession number GDMCC No. 64931.

[0036] Preferably, the rhododendron mycorrhizal fungus WF17-2-1 and its applications include the application of strain WF17-2-1 in blueberry cultivation.

[0037] Preferably, the application of the rhododendron mycorrhizal fungus WF17-2-1 in blueberry cultivation includes any of the following applications:

[0038] Application of rhododendron mycorrhizal fungus WF17-2-1 in increasing blueberry biomass, including increasing the total fresh weight and total dry weight of the whole blueberry plant;

[0039] The application of rhododendron mycorrhizal fungus WF17-2-1 in improving blueberry root systems includes the formation of hyphal clusters within blueberry root epidermal cells, resulting in increased blueberry root length, root surface area, and volume.

[0040] Application of rhodomycorrhizal fungus WF17-2-1 in regulating blueberry growth metabolism, including increased accumulation of zeatin nucleosides in blueberries;

[0041] Application of rhododendron mycorrhizal fungus WF17-2-1 in promoting blueberry photosynthesis, including enhanced photosynthetic enzyme activity in blueberries;

[0042] Application of rhodomycorrhizal fungus WF17-2-1 in improving the nutritional value of blueberries, including increasing the total phosphorus and total potassium content of blueberries.

[0043] A product that promotes blueberry growth, said product comprising azadirachtic mycorrhizal fungus WF17-2-1 or its metabolites.

[0044] Preferably, the product includes a growth promoter or a growth-promoting fertilizer.

[0045] Example 1: Strain Isolation

[0046] Samples of *Rhizophora stricta* were collected from the root system of *Rhizophora spp.* in Shangzhen Village, Daba Town, Heping County, Heyuan City, Guangdong Province (coordinates: 24º31'N, 114º55'E; altitude: 207 m). Strains were isolated from the root system of the samples according to the following steps:

[0047] (1) Cleaning: Clean the soil and impurities from the root system of the sample. Select young roots with a diameter of 0.5-1.0 mm from the cleaned root system as separation material. Soak the separation material in sterile water to remove impurities.

[0048] (2) Disinfection: In a sterile environment, soak the separation material in 75% alcohol for 2 minutes and then wash it with sterile water. This is one alcohol disinfection. Repeat the alcohol disinfection step 3 times. Soak the separation material in 5% sodium hypochlorite for 15 minutes and then wash it with sterile water.

[0049] (3) Isolation and culture: Under sterile conditions, the isolated material is divided into root segments of 0.3-0.5 cm and transferred to potato dextrose agar (PDA) medium. Five root segments are placed in each medium and the medium is placed in the dark at 25°C for 2-4 weeks until colonies grow from the root segments.

[0050] (4) Purification: Under sterile conditions, use a picking needle to pick up a small amount of hyphae from the edge of each colony and transfer them to a new PDA medium. Incubate in the dark at 25°C. Repeat this step until a single colony is obtained.

[0051] Example 2: Strain Screening

[0052] From all the single colonies obtained in Example 1, strains capable of infecting the roots of blueberries (tested plant: blueberry tissue culture seedling ZY01) were selected and screened according to the following steps:

[0053] (1) Preparation of bacterial culture: Mycelia were picked from all the single colonies obtained in Example 1 and transferred to potato dextrose (PDB) medium. The PDB medium was placed on a shaker and cultured at 25°C and 150 rpm for 2 weeks to obtain single colony bacterial culture.

[0054] (2) Strains were reintroduced: Two blueberry tissue culture seedlings were used as a group. Under sterile conditions, 10 mL of single colony bacterial solution was inoculated onto the culture medium of the tissue culture seedlings. Each single colony bacterial solution was used to treat 5 groups separately. At the same time, 5 groups of tissue culture seedlings were treated with an equal amount of sterile PDB culture medium as a control. The treated tissue culture seedlings were placed in an environment of 25℃ and cultured for 20 days.

[0055] (3) Preparation of mycorrhizal sections by trypan blue staining:

[0056] Fixation: After the culture is completed, remove the blueberry roots, select young roots with a diameter of 0.5-1.0 mm, wash them and cut them into 1 cm root segments, and soak them in the freshly prepared standard fixative solution (38% formaldehyde: acetic acid: glycerol: 50% alcohol = 1:1:1:18) for 24 hours.

[0057] Acidification: After fixation, remove the root segments, wash them with water, soak them in 2% hydrochloric acid for 5 minutes, and wash them again until clean.

[0058] Staining: Immerse the acidified root segments in 0.05% trypan blue staining solution and heat at 90℃ for 30 minutes. After removing, wash with water until clean, transfer to decolorizing solution (lactic acid: glycerol = 1:1) and soak for one day, then remove and wash clean.

[0059] Slide preparation: Place a drop of water in the center of a glass slide, place the stained root segment in glycerol, cover with a coverslip, and you will get a blueberry root segment slice.

[0060] Microscopic observation of the sections revealed that strain WF17-2-1 exhibited the strongest infectivity, forming mycelial clusters within the epidermal cells of blueberry roots and establishing a beneficial symbiotic relationship with the blueberry roots. Figure 1 As shown.

[0061] Example 3: Strain Identification

[0062] Morphological identification: Under sterile conditions, a small amount of marginal hyphae were picked from a single purified colony of strain WF17-2-1 in Example 1, placed in a new PDA medium, and cultured in the dark at 25°C for 15 days to observe the biological morphology of the colony.

[0063] The identification results showed that strain WF17-2-1 was a filamentous fungus, and its colony morphology on solid culture medium was an irregular yellow colony with a raised center and filamentous edges.

[0064] Molecular biological identification: DNA was extracted from a small amount of single colony culture of strain WF17-2-1, and the endogenous ribosomal spacer region (ITS) and the small ribosomal subunit (18S rDNA) of the strain were amplified and sequenced. The sequencing primers are as follows:

[0065] ITS sequencing primers:

[0066] ITS1: 5´-TCCGTAGGTGAACCTGCGG-3´

[0067] ITS4: 5´-TCCTCCGCTTATTGATATGC-3´

[0068] 18S rDNA sequencing primers:

[0069] NS1: 5´-GTAGTCATATGCTTGTCTC-3´

[0070] NS8: 5´-TCCGCAGGTTCACCTACGGA-3´

[0071] The ITS and 18S rDNA sequencing results are shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the sequence listing. Phylogenetic analysis of the sequencing sequences was performed using the MEGA11 Kimura 2-parameter model, the NJ algorithm (bootstrap repeated 1000 times), and the NCBI database.

[0072] The phylogenetic tree results, obtained from 18S rDNA sequencing, are as follows: Figure 3 , Figure 4 As shown.

[0073] According to the ITS phylogenetic tree analysis, strain WF17-2-1 belongs to Hyaloscyphaceae, and according to the 18S rDNA phylogenetic tree analysis, strain WF17-2-1 is Hyaloscypha.sp.

[0074] Therefore, strain WF17-2-1 was identified as Ascomycota, Leotiomycetes, Helotiales, Hyaloscyphaceae, Hyaloscypha.sp.

[0075] Example 4: Co-culture of strain with blueberries

[0076] The strain WF17-2-1 was inoculated onto blueberry tissue culture seedlings and co-cultured. After co-culture, various physiological indicators of blueberries were detected to evaluate the growth-promoting effect of the strain on blueberries.

[0077] Perform co-cultivation according to the following steps:

[0078] (1) Preparation of bacterial agent: Under sterile conditions, a small amount of edge hyphae were picked from a single colony purified from strain WF17-2-1 in Example 1 and placed in a new PDA medium. The colony was cultured in the dark at 25°C for 15 days. One single colony obtained is one bacterial agent.

[0079] (2) Inoculation with bacterial strains: Under aseptic conditions, rooted blueberry tissue culture seedlings (variety ZY01) were transferred to culture bottles containing sterilized substrate and blueberry-specific culture medium. After hardening off, four portions of bacterial agent were inoculated into the substrate of each tissue culture seedling, for a total of 15 seedlings, serving as the experimental group. At the same time, another 15 new tissue culture seedlings were taken, and four portions of PDA medium were inoculated into the substrate of each seedling, serving as the control group. The experimental group and the control group were placed in an incubator at 25℃ for two months.

[0080] (3) Index detection: After the co-culture was completed, samples from the experimental group and the control group were collected respectively, and various physiological indicators of blueberries were detected.

[0081] Example 5: Blueberry Biomass Detection

[0082] Remove the blueberry tissue culture seedlings from the culture medium, wash away impurities, wipe off surface moisture, and weigh them to obtain the total fresh weight. Then, place the blueberries in an oven at 105℃ for 15 minutes to blanch them, lower the temperature and maintain it at 80℃ to continue drying until the weight of the tissue culture seedlings no longer changes. Weigh them again to obtain the total dry weight.

[0083] like Figure 5 As shown, compared with the control group (CK), the blueberries in the experimental group, after being inoculated with WF17-2-1 inoculant, had a total fresh weight exceeding 0.4 g, an increase of 18.3%, and a total dry weight increase of 16.8%. Total fresh weight and total dry weight are indicators for evaluating plant biomass, indicating that the accumulation of water content and organic matter increased (* indicates p < 0.1, indicating a difference between the two), thus increasing the biomass of the whole plant and contributing to increased blueberry yield.

[0084] Example 6: Detection of Blueberry Root Growth Indicators

[0085] After cleaning the roots of the tissue culture seedlings, the roots were scanned using a WINRHIZO root scanner. Based on the scanned images, relevant indicators of root growth, such as root length, root volume, and root surface area, were calculated.

[0086] like Figure 6 , Figure 7 and Figure 8As shown, compared with the control group (CK), the blueberries in the experimental group, after being inoculated with WF17-2-1 inoculant, showed an increase in root length from less than 35 cm to nearly 60 cm, and an increase in root surface area from less than 3 cm². 2 Increased to nearly 5.5 cm 2 The root volume is less than 0.02 cm. 3 Increased to over 0.04 cm 3 The root length, root surface area, and root volume increased by 82.3%, 104.4%, and 130.9%, respectively, indicating that after strain WF17-2-1 and blueberry roots were symbiotic, the cell growth and proliferation rate of the roots were significantly improved (* indicates p < 0.1, and ** indicates p < 0.05 ...

[0087] Example 7: Detection of zeatin content in blueberries

[0088] Whole samples of tissue culture seedlings were taken, washed, and thoroughly ground. The zeatin nucleoside content was detected by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using a double antibody method.

[0089] like Figure 9 As shown, compared with the control group (CK), the blueberries in the experimental group had a zeatin nucleotide (ZR) content as high as 4.6 ng / mL after inoculation with WF17-2-1 fungicide, which increased by 18.1% (* indicates p < 0.1, indicating a difference between the two groups). Zeatin nucleotide, as a plant growth regulator, can promote cell division, stimulate the differentiation of lateral buds and the formation of callus tissue. The accumulation of zeatin nucleotide can regulate the growth metabolism of blueberries and accelerate their growth and development.

[0090] Example 8: Detection of total potassium and total phosphorus content in blueberries

[0091] Total phosphorus content was determined by the phosphomolybdic blue colorimetric method, and total potassium content was determined by the sodium tetraphenylborate turbidimetric method.

[0092] like Figure 10 and Figure 11 As shown, compared with the control group (CK), the total phosphorus content of blueberries in the experimental group increased by more than 0.5 g / kg (an increase of 19.5%) and the total potassium content increased by 18.2% after inoculation with WF17-2-1 inoculant. This indicates that after the symbiotic relationship between strain WF17-2-1 and the root system, the nutrient absorption capacity of the root system was significantly improved, resulting in increased phosphorus and potassium accumulation in the whole blueberry plant (** indicates p < 0.05, indicating a significant difference; *** indicates p < 0.001, indicating a highly significant difference), thus improving the nutritional value of blueberries.

[0093] Example 9: Blueberry photosynthetic capacity detection

[0094] After washing the leaf samples from tissue culture seedlings, the chlorophyll fluorescence characteristics were measured using the IMAGING-PAM chlorophyll fluorescence imaging system, and the maximum quantum yield and actual photon efficiency of PSII were calculated.

[0095] like Figure 12 and Figure 13 As shown, compared with the control group (CK), the experimental group of blueberries, after inoculation with WF17-2-1 fungicide, showed a higher PS ratio. The maximum quantum yield (Fv / Fm) increased significantly by 1.6% (** indicates p < 0.05, indicating a significant difference between the two), and the actual photometric efficiency (Y(II)) increased from less than 0.23% to nearly 0.33%, a significant increase of 53.8% (*** indicates p < 0.001, indicating a highly significant difference between the two). The maximum quantum yield refers to the highest light energy conversion efficiency per unit area of ​​the plant's functional leaves when they are in optimal condition, representing the limit of the leaves' utilization of absorbable radiation. The actual photometric efficiency refers to the light energy conversion efficiency of the leaves under actual conditions, representing the light energy absorbed by the leaves per unit area per unit time. These chlorophyll fluorescence characteristic parameters can reflect the activity of photosynthetic enzymes in the leaves. The experimental results fully demonstrate that the light energy conversion efficiency of blueberry leaves is significantly improved when they are in optimal condition. Under the same conditions, inoculation with strain WF17-2-1 can enhance the activity of photosynthetic enzymes in the leaves, promote photosynthesis in blueberries, increase the accumulation of photosynthetic products, and help increase blueberry yield.

[0096] Based on the above experimental results, among all the strains isolated from *Ulmus pumila*, strain WF17-2-1 showed the best infectivity. Therefore, strain WF17-2-1 was selected as the symbiotic fungus for blueberry roots. After inoculation with strain WF17-2-1, the strain can form mycelial clusters in the epidermal cells of blueberry roots, significantly improving root growth, increasing nutrient absorption, promoting the synthesis of plant hormones, regulating plant growth metabolism, enhancing plant photosynthesis, helping blueberry accumulate nutrients, improving the nutritional value of blueberries, and facilitating the development of bio-fertilizers or growth promoters based on strain WF17-2-1, thus contributing to the green and efficient development of the blueberry industry.

[0097] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1, characterized in that, The rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 was deposited on August 1, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 64931), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1 in increasing blueberry biomass.

3. The application of the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1 in improving blueberry root systems.

4. The application of the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1 in regulating blueberry growth metabolism.

5. The application of the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1 in promoting blueberry photosynthesis.

6. The application of the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1 in improving the nutritional value of blueberries.

7. A product that can promote blueberry growth, characterized in that, Contains the rhododendron mycorrhizal fungus Hyaloscypha.sp WF17-2-1 as described in claim 1.

8. The product according to claim 7, characterized in that, The products include growth promoters or growth-promoting fertilizers.

Images