Pezicula ericae XA4 and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Pezicula ericae XA4 and its application. The present invention provides a strain of Pezicula ericae XA4, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 26, 2024, with a deposit number of CGMCC No.41350. The XA4 provided by the present invention was back-grafted on aseptically cultured Dahurian rhododendron seedlings, and trypan blue staining was used to detect that mycorrhizal fungi could be back-grafted successfully. Compared with the uninoculated strain, the inoculation of XA4 had the most significant growth-promoting effect on Dahurian rhododendron. Its plant height, leaf length, leaf width, root length, biomass, chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and root activity were all improved compared with the uninoculated strain.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a Pezicula ericae XA4 strain and application thereof. Background Art

[0002] Application value of Xing'an Rhododendron:

[0003] The Xing'an azalea (Rhododendron dauricum L.) boasts exceptional ornamental value in landscapes. A native of Northeast China, it blooms before its leaves appear. Its vibrant, rich colors, mostly pink or purple, with white rarely appearing, earn it the nickname "the most beautiful flower." Its flower shapes vary greatly. Zhou Ming discovered a double-petaled Xing'an azalea during field research. Its flowers, with two to six layers of petals, are highly ornamental. Its unique, "dead wood coming back to life" effect has made it a favorite of many flower arrangement enthusiasts. It offers numerous opportunities for landscape applications. In the wild, it can be planted in patches to create seas of azaleas. In urban areas, it can be widely used in landscaping areas such as roadsides, scenic areas, and parks. It can also be planted in combination with trees, shrubs, and herbaceous plants to create staggered landscapes. Ren Qinggui et al. have conducted preliminary research on its application in landscapes, suggesting that evergreen conifers such as Pinus sylvestris and Pinus tabulaeformis be placed in the front row, followed by interspersed plantings with plants such as Forsythia suspensa and Prunus cuckoo. This creates a sense of visual depth and enhances the overall landscape effect. In addition, the Xing'an rhododendron has many branches and is resistant to pruning. It can be planted as a hedge or made into bonsai, showing the morphological beauty of the Xing'an rhododendron.

[0004] Research on symbiotic fungi of Rhododendron xinganensis:

[0005] At present, there are relatively few studies on the symbiotic fungi of Daxinganling Rhododendron in China. Jia Rui conducted preliminary research on the mycorrhizal morphology, infection status and diversity of Daxinganling Rhododendron in Greater Khingan Range and found that its mycorrhizal morphology and structure are relatively rich, and different mycorrhizal morphologies are distributed to varying degrees in root samples in different months. However, it was found that the overall level of field mycorrhizal infection rate of Daxinganling Rhododendron was relatively low, with an average value of only 12.21±4.12 (%). The 28 isolated fungal colonies were inoculated into rhododendron seedlings, and 13 types were able to successfully infect and form rhododendron-like mycorrhizae, among which Ascomycota were dominant, and a small number belonged to Basidiomycetes. However, the effects of the inoculated species on Daxinganling Rhododendron were not studied.

[0006] Therefore, beneficial strains with obvious promoting effects on the nutrient absorption, growth and development of Rhododendron dahurica were screened out and inoculated in the early stage of artificial breeding of Rhododendron dahurica to promote mycorrhiza formation, which is of great significance to improving the soil environment adaptability and promotion and application of Rhododendron dahurica. Summary of the Invention

[0007] To address the above issues, the present invention provides a strain of Pezicula ericae XA4 and its use. XA4 was used to inoculate aseptically cultured Dahurian Rhododendron dahurica seedlings. Trypan blue staining revealed that mycorrhizal fungi could be successfully inoculated. Compared with uninoculated seedlings, inoculation with XA4 significantly promoted the growth of Dahurian Rhododendron dahurica. Plant height, leaf length, leaf width, root length, biomass, chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, transpiration rate, and root activity were all significantly improved compared to uninoculated seedlings.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a Pezicula ericae XA4, which was deposited in the General Microbiology Center of the China Culture Collection Administration on June 26, 2024, with a deposit number of CGMCC No.41350.

[0010] The present invention also provides the use of the XA4 described in the above technical solution in infecting the roots of Rhododendron dahurica.

[0011] The present invention also provides the use of XA4 described in the above technical solution in phosphate dissolution.

[0012] The present invention also provides the use of XA4 described in the above technical solution in producing indoleacetic acid.

[0013] The present invention also provides the use of the XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica.

[0014] The present invention also provides the use of XA4 described in the above technical solution in improving the growth rate of Rhododendron dahurica.

[0015] The present invention also provides the use of the XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica leaves.

[0016] The present invention also provides the use of the XA4 described in the above technical solution in promoting the root growth of Rhododendron dahurica.

[0017] The present invention also provides the use of the XA4 described in the above technical solution in enhancing the photosynthesis of Rhododendron dahurica.

[0018] The present invention also provides the use of the XA4 described in the above technical solution in improving the root activity of Rhododendron dahurica.

[0019] This study used wild Rhododendron dahurica roots from Huilong Mountain in Wuchang City, Heilongjiang Province, as test material. Randomly sampled and tested symbiotic mycorrhizae, using trypan blue staining to study the mycorrhizal morphology and infection characteristics. The mycorrhizal fungi were isolated and purified, and their types were determined using a combination of morphological and molecular biological identification. The Rhododendron dahurica tissue culture seedlings were then screened and inoculated to test the fungal growth-promoting effects. The main research conclusions are as follows:

[0020] (1) The mycorrhizal morphology of Rhododendron dahurica is quite rich. According to the distribution of mycelium, it can be roughly divided into three types: a. hyphae on the surface of root cells, mostly wandering hyphae; b. intracellular hyphae, that is, hyphae distributed in root cells, some thick hyphae form dense hyphal nodes, showing the typical structural characteristics of Rhododendron mycorrhizal fungi, and vesicles are also found in a few root samples; c. intercellular hyphae, that is, hyphae distributed between root cells, mostly longitudinally growing hyphae, and "microsclerotia" structures are also found. According to whether the hyphae contain septa, it can be divided into two types, namely septate hyphae and non-septate hyphae. Paraffin section observation showed that the structure of the mycorrhiza is simple, consisting of a layer of epidermal cells and a narrow stele surrounded by one or two layers of cortical cells. Mycorrhizal fungal infection generally does not change the anatomical structure of the hair root. Scanning electron microscopy observation showed that the surface of the hair root of Rhododendron dahurica sampled in the field was covered with a layer of loose or dense hyphae.

[0021] (2) The total root infection rate of Rhododendron dahurica was relatively low and the infection intensity was not high. It reached the level 3 infection standard, but the mycorrhizal infection rate reached 22.7%. The correlation study between the chemical properties of the soil at the sampling site and the mycorrhizal infection rate showed that there was a significant positive correlation between the mycorrhizal infection rate and total nitrogen and organic matter, and a significant negative correlation with total phosphorus, available phosphorus, and pH.

[0022] (3) The mycorrhizal fungi of Rhododendron dasyphylum in each sampling site were isolated and purified, and four strains with different morphologies were obtained. The molecular identification results showed that the mycorrhizal fungi of Rhododendron dasyphylum belonged to the subphylum Ascomycota, including three genera: Phialocephala, Pezicula, and Rhizodermea.

[0023] (4) The growth characteristics, phosphate solubilization capacity, and IAA secretion capacity of four mycorrhizal fungi were determined. The dominant strain, Pezicula ericae XA4, was selected and re-grafted into aseptically cultured Rhododendron dahurica seedlings. Trypan blue staining revealed that all mycorrhizal fungi could be re-grafted successfully. Compared with the uninoculated seedlings, the inoculation with XA4 had the most significant growth-promoting effect on Rhododendron dahurica. Plant height, leaf length, leaf width, root length, biomass, chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and root activity were all improved compared with the uninoculated seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0025] Figure 1 This is the technical route of the present invention;

[0026] Figure 2 Basic information of Huilong Mountain in Wuchang City. Note: a. Community species characteristics; b. Rhizosphere soil; c. Slope;

[0027] Figure 3 The root system characteristics of Rhododendron dahurica. Note: a. Rhododendron dahurica fibrous root system; b. Mature root; c. Young hairy root;

[0028] Figure 4 Observation of the anatomical structure of the hairy roots of Rhododendron dahurica. Note: a. cross section of wild mycorrhiza; b. cross section of sterile cultured hairy roots; c. longitudinal section of wild mycorrhiza; d. longitudinal section of sterile cultured hairy roots; 1: epidermis; 2: exoderm; 3: endodermis; 4: stele; hc: hyphae;

[0029] Figure 5 This is a scanning electron microscopy observation of the hairy roots of Rhododendron dahurica. Note: a, b, c, d are hairy root fragments of sterile cultured Rhododendron dahurica at 200, 600, 1500, and 3000 times magnification, respectively; e, f, g, h are hairy root fragments of wild Rhododendron dahurica at 200, 600, 1500, and 3000 times magnification, respectively;

[0030] Figure 6 Dyeing root segments of sterile seedlings of Rhododendron dahurica;

[0031] Figure 7 The morphological structure of mycelium in the mycorrhiza of Rhododendron dahurica. Note: a. Extraradical wandering hyphae; b. Intracellular hyphae; c. Mycelial network; d. Round vesicles; e. Oblong vesicles; f. Mycelia forming hyphae masses within cells; g. Ruptured intracellular vesicles; h. Mycelia running through cells; i. Thick intracellular hyphae forming compact hyphae nodes.

[0032] Figure 8 The "microsclerotia" structure of DSE. Note: a. The red arrow points to the DSE microsclerotia; b. The enlarged view of the "microsclerotia" structure;

[0033] Figure 9 The colony morphology of mycorrhizal fungi of Rhododendron dahurica in the sampling area;

[0034] Figure 10 Electrophoresis diagram of the ITS segment of mycorrhizal fungi amplified by PCR technology in the sampling area (M: Marker; 1-4: fungal rDNA ITS segment;

[0035] Figure 11Observation of the microstructure of four mycorrhizal fungi. Note: The arrows in a, c, e, and f point to septate hyphae, and the arrows in b and d point to scattered spores.

[0036] Figure 12 is the growth rate of four mycorrhizal fungi;

[0037] Figure 13 Identification of four mycorrhizal fungi by back-grafting. Note: a, c are the hyphal structures of dark septate endophytic fungi (DSE); b, d are microsclerotia structures; e is intracellular fusiliform hyphae; f is extraradical hyphae; g is the typical structure of EMF, i.e., intracellular hyphal masses; h is a thicker intracellular hyphal node;

[0038] Figure 14 The phosphate solubilizing capacity of four mycorrhizal fungi;

[0039] Figure 15 The IAA production capacity of four mycorrhizal fungi;

[0040] Figure 16 is the IAA standard curve;

[0041] Figure 17 The IAA content produced by four mycorrhizal fungi;

[0042] Figure 18 is the sequence of XA4 in the ITS segment;

[0043] Figure 19 The effect of different treatments on the height of Rhododendron dahurica.

[0044] Figure 20 The effect of inoculation on leaf length and width of Rhododendron dahurica seedlings 4 months after inoculation;

[0045] Figure 21 The effects of different treatments on the root length of Rhododendron dahurica seedlings after 4 months;

[0046] Figure 22 Effects of different treatments on the biomass of Rhododendron dahurica seedlings after 4 months;

[0047] Figure 23 The effects of different treatments on the chlorophyll content of Rhododendron dahurica leaves;

[0048] Figure 24 The effects of different treatments on FV / FM of Rhododendron dahurica leaves after 4 months;

[0049] Figure 25 The effects of different treatments on the photosynthetic characteristics of Rhododendron dahurica leaves;

[0050] Figure 26 is the standard curve of root activity;

[0051] Figure 27 Effects of different treatments on root activity of Rhododendron dahurica after 4 months.

[0052] Biological Deposit Description

[0053] XA4, Latin name Pezicula ericae, was deposited on June 26, 2024 in the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 41350. DETAILED DESCRIPTION

[0054] The present invention provides a Pezicula ericae XA4, which was deposited in the General Microbiology Center of the China Culture Collection Administration on June 26, 2024, with a deposit number of CGMCC No.41350.

[0055] The present invention also provides the use of the XA4 described in the above technical solution in infecting the roots of Rhododendron dahurica.

[0056] The present invention also provides the use of XA4 described in the above technical solution in phosphate dissolution.

[0057] The present invention also provides the use of XA4 described in the above technical solution in producing indoleacetic acid.

[0058] The present invention also provides the use of the XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica.

[0059] The present invention also provides the use of XA4 described in the above technical solution in improving the growth rate of Rhododendron dahurica.

[0060] The present invention also provides the use of the XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica leaves.

[0061] The present invention also provides the use of the XA4 described in the above technical solution in promoting the root growth of Rhododendron dahurica.

[0062] The present invention also provides the use of the XA4 described in the above technical solution in enhancing the photosynthesis of Rhododendron dahurica.

[0063] The present invention also provides the use of the XA4 described in the above technical solution in improving the root activity of Rhododendron dahurica.

[0064] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] Example 1

[0066] 1 Materials and Methods

[0067] 1.1 Experimental Materials

[0068] 1.1.1 Plant materials

[0069] Wild root samples: The roots of wild Rhododendron dahurica were collected from Huilong Mountain, Wuchang City, Heilongjiang Province, for morphological observation and fungal culture.

[0070] Sterile seedlings of Rhododendron dahurica: Collect Rhododendron dahurica branches and disinfect them to obtain asexual tissue culture seedlings for grafting.

[0071] 1.1.2 Overview of sampling sites

[0072] Root and soil samples of Rhododendron dahurica were collected from Huilong Mountain in Wuchang City, Heilongjiang Province. The specific sampling details are shown in the figure below.

[0073] Huilong Mountain in Wuchang City (127°10′E, 45°5′N) has a temperate continental monsoon climate, with an altitude of 147-1689 meters. At the time of sampling, most of the Dahurian rhododendrons had not yet bloomed. The soil type is loam, with a layer thickness of 5-30 cm and a pH of 4.4-4.8. The slope is 35-40°. The community is species-rich, with many associated tree species. The tree layer mainly includes willow, Mongolian oak, and Juglans mandshurica. Dahurian rhododendrons are the main species in the shrub layer, and the herbaceous plant layer mainly includes hosta, calendula, lily of the valley, and peony.

[0074] 1.1.3 Sample collection and storage

[0075] Experimental sample collection began in March 2023. Prior to sampling, soil debris, rocks, and other debris were removed from the soil surface, as well as the topsoil 3 cm deep. Three random sampling sites were selected within the sampling area, with the central location of the concentrated Dahurian Rhododendron growth area. Dug along the taproot to ensure that the roots dug were all Dahurian Rhododendron roots. Rootlets and fibrous roots, as well as soil from the 5-50 cm layer, were collected. Approximately 3 kg of soil and roots were retained from each plot, placed in labeled ziplock bags, and then stored in ice packs for transport back to the laboratory. Root samples were placed in a 4°C refrigerator, and the rhizosphere soil was allowed to air dry before being filtered through a 20-mesh sieve for storage.

[0076] 1.1.4 Experimental reagents and drugs

[0077] Main experimental drugs: trypan blue powder; potassium hydroxide; lactic acid; glycerol; concentrated hydrochloric acid; sodium hypochlorite; potato dextrose agar (PDA); anhydrous ethanol; xylene; concentrated sulfuric acid; succinic acid; TTC powder; ethyl acetate; sodium hyposulfite; Biospin fungal genomic DNA extraction kit, etc.

[0078] Primers: The rDNA amplification primers selected were the universal primers ITS1 and ITS4 of the fungal ITS region, which were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0079] The preparation methods of the main reagents used in the experiment are shown in Table 1.

[0080] Table 1 Preparation methods of main reagents

[0081]

[0082] 1.2 Experimental methods

[0083] 1.2.1 Observation on the morphological structure of the mycorrhizae and mycelium within the mycorrhizae of Rhododendron xinganensis

[0084] 1.2.1.1 Root system observation of Rhododendron dahurica

[0085] The roots of wild Rhododendron dahurica were randomly selected and their root characteristics were observed, including root diameter, morphology, and color. An electronic digital vernier caliper (accuracy 0.01 mm) was used to measure the root diameter. The roots were stained with trypan blue, and the root microstructure was observed under a microscope.

[0086] 1.2.1.2 Observation of mycorrhizal anatomical structure

[0087] Referring to Wang Wei's paraffin section method, the root system of Rhododendron dahurica was taken to observe the mycorrhizal anatomical structure.

[0088] 1.2.1.3 Observation of mycorrhizal microstructure

[0089] The microstructure of the mycorrhiza was observed using a scanning electron microscope. The cut root segments were fixed in 2.5% glutaraldehyde (pH 6.8) and placed in a 4°C refrigerator for more than 1.5 hours. They were then rinsed two or three times with 0.1 mol phosphate buffer (pH 6.8) for 10 minutes each time. Dehydrated with 50%, 70%, and 90% ethanol for 10-15 minutes each time; dehydrated with 100% ethanol two or three times for 10-15 minutes each time; and washed once with (100% ethanol: tert-butanol = 1:1); and twice with pure tert-butanol for 15 minutes each time. The sample was frozen in a -20°C refrigerator for 30 minutes and then dried in a desiccator for about 4 hours. With the observation side of the sample facing upward, it was attached to the scanning electron microscope sample stage with conductive tape. A 100-15 angstrom metal film was deposited on the sample surface using an ion sputtering coater, and the sample was observed and photographed using a high-precision scanning electron microscope (SU-8010HITACHI).

[0090] 1.2.1.4 Observation of the morphological structure of mycelium within mycorrhizae

[0091] Trypan blue staining was used to stain the roots of Rhododendron dahurica. The specific method is as follows:

[0092] 1. Root washing: Wash the root sample from the soil, fix it in FAA fixative, rinse it with distilled water 2-3 times, take about 0.5-1.0g of young roots, and cut them into 1cm long root segments.

[0093] 2. Transparent: Soak the root segment in 10% KOH, transparent in a 90℃ water bath for 20-30 minutes, and then wash with water in time.

[0094] 3. Acidification: Soak in 2% HCl at room temperature for 5 minutes.

[0095] 4. Staining: Pour out the HCl and directly add 0.05% trypan blue staining solution and incubate in a 70°C water bath for 30 min.

[0096] 5. Decolorization: Pour the dye into a special waste liquid bottle and add lactic acid glycerol solution for static decolorization for more than 12 hours.

[0097] 6. Preparation of slides: Randomly select 20 root segments of uniform thickness and length from the stained root segments, and arrange them on 2 slides, 10 roots on each slide. Add 1-2 drops of lactic acid glycerol solution and cover with a coverslip. Press lightly to flatten the roots and remove bubbles.

[0098] 7. Microscopic examination: Observe the mycorrhizal infection structure under a microscope.

[0099] 1.2.2 Determination and classification of mycorrhizal infection rate

[0100] The mycorrhizal infection rate was determined by referring to the conventional method of root segment frequency proposed by Linderman.

[0101] Refer to Wu Chong's calculation method for mycorrhizal infection rate and Liu Runjin's grading method for grading. The formulas for infection rate and grading are as follows:

[0102] Infection rate = ∑(0×number of root segments + 10%×number of root segments + 20%×number of root segments +…100%×number of root segments) / total number of observed root segments

[0103] Grade: Grade 1 (0 ≤ infection rate < 1%), Grade 2 (1% < infection rate < 20%), Grade 3 (20% < infection rate < 40%), Grade 4 (40% < infection rate < 60%), Grade 5 (infection rate > 60%)

[0104] 1.2.3 Determination of soil chemical properties at the sampling site

[0105] The soil chemical properties included six indicators: total nitrogen, total phosphorus, available phosphorus, organic matter, pH value and soil electrical conductivity.

[0106] Referring to Li Qiang's glass electrode method, 10 g of soil sample was weighed and the soil pH value was measured.

[0107] Referring to Zhu Dan's electrode method, 10 g of soil sample was taken to measure the electrical conductivity of the soil.

[0108] The determination was carried out with reference to the method described by Yang Jianhong in "Soil Agrochemical Analysis and Environmental Monitoring". The specific method is as follows:

[0109] Total nitrogen determination: semi-micro Kelvin method.

[0110] Total phosphorus determination: sodium hydroxide fusion-molybdenum antimony colorimetric method.

[0111] Determination of available phosphorus: NaHCO3 extraction-molybdenum antimony colorimetric method.

[0112] Determination of organic matter: Use potassium dichromate K2Cr2O7 volumetric method.

[0113] The above indicators were measured three times.

[0114] 1.2.4 Isolation, purification and preservation of mycorrhizal fungi from Rhododendron xinganensis

[0115] 1.2.4.1 Isolation and purification of strains

[0116] The separation method adopted in the present invention is a root segment direct culture method, and both the separation and purification culture media use potato agar culture media (PDA).

[0117] (1) Sample processing: Select healthy Rhododendron dahurica root samples from a 4°C refrigerator, rinse the surface soil with tap water, wrap them with sterile gauze and place them in a beaker, and rinse with running water for 1-3 hours until the surface soil is clean.

[0118] (2) Root surface sterilization: Move the root segment to a clean bench, then remove the root segment with tweezers, rinse it with sterile water 5 times, 3 minutes each time, then soak the root in 75% ethanol for 30 seconds, and then rinse it repeatedly with sterile water.

[0119] (3) Root segment isolation and culture: Cut root segments into 0.5 cm pieces and inoculate them on PDA culture medium. Place 5 root segments on each plate. Culture 200 root segments at each sampling site, for a total of 120 plates. Label each plate and place it in a 25°C mold incubator in the dark for 2-4 weeks.

[0120] (4) Purification of strains: In the early stages of strain culture, it is important to observe the growth of the strains and remove strains that may be contaminated or growing too fast. Select strains that grow well and have clear edges, inoculate them on new PDA culture medium, and culture them upside down in the dark at 25°C. During the culture period, the morphology of the colonies needs to be observed in real time and continuously purified until a single, uncontaminated strain is isolated.

[0121] 1.2.4.2 Storage of strains

[0122] The strain was preserved using the slant preservation method. On a sterile workbench, a small amount of mycelium was picked from the purified plate and inoculated into the prepared slant PDA culture medium. The culture was sealed and marked, and cultured at room temperature until obvious mycelium grew. The culture was then transferred to a 4°C refrigerator for storage.

[0123] 1.2.5 Morphological observation of mycorrhizal fungi of Rhododendron xinganensis

[0124] 1.2.5.1 Colony morphology observation

[0125] The morphological characteristics of the colonies were observed using an optical microscope, including colony color, texture, shape, radius, the presence of secretions, and spore production. The strains were preliminarily classified with reference to the Manual of Fungal Identification.

[0126] 1.2.5.2 Observation of hyphae and spore morphology

[0127] The strain was cultured using the insert culture method. First, a single uncontaminated strain was inoculated into the PDA culture medium. A sterile cover slip was inserted into the culture medium at a 45° angle using sterile tweezers. The cover slip was sealed and marked and placed in a 25°C incubator for culture for 7-10 days. The cover slip with mycelium grown was placed on a slide for microscopic observation.

[0128] 1.2.6 Molecular Biological Identification of Mycorrhizal Fungi in Rhododendron dasyphylla

[0129] 1.2.6.1 Extraction of total DNA

[0130] After culturing on a PDA plate at 25°C for 7-14 days, fresh mycelium was scraped and ground into powder by adding liquid nitrogen. DNA was extracted from the treated strain using the Biospin Fungal Genomic DNA Extraction Kit. The specific experimental steps were carried out strictly in accordance with the kit's operating instructions.

[0131] 1.2.6.2 PCR Amplification of rDNA ITS Segments

[0132] The extracted fungal genomic DNA was diluted to a concentration of 20 ng / μl and stored at -20°C until use. The fungal rDNA ITS-specific fragment was amplified using the universal primers ITS1 / ITS4 using the extracted fungal genomic DNA as a template. The PCR products were then recovered using 1% agarose gel. The PCR reaction system is shown in Table 2.

[0133] Table 2 PCR system for amplifying ITS sequences

[0134]

[0135]

[0136] Table 3 PCR reaction parameters for fungal rDNA ITS-specific segments

[0137]

[0138] 1.2.6.3 Purification, cloning and sequencing of PCR amplification products

[0139] After the PCR amplification products were detected by electrophoresis and gel imaging system, the PCR products with ideal results were sent to Shanghai Sangon Biotechnology Co., Ltd. for purification, cloning and sequencing.

[0140] 1.2.6.4 Analysis and identification of mycorrhizal fungal species

[0141] The rDNA ITS region DNA sequences of the tested fungi were compared by BLAST homology to determine the species of the fungus or its closely related species.

[0142] 1.2.7 Identification of mycorrhizal fungi backgrafting

[0143] 1.2.7.1 Mycorrhizal fungi backgrafting method

[0144] Laboratory-grown Rhododendron dahurica tissue culture seedlings were used as experimental materials. Peat soil and vermiculite were mixed in a 3:1 ratio, adjusted to an appropriate humidity, and dispensed into conical flasks, approximately 100 mL per bottle. The mixture was sterilized by autoclaving at 121°C for 60 minutes. Well-grown Rhododendron dahurica tissue culture seedlings with consistent growth were transplanted into soil substrate on a sterile laminar flow bench and incubated aseptically at 25°C. A 1-cm-diameter fungal disc was picked using a sterile punch and inoculated into the surrounding soil substrate approximately 1 cm from the root of the Rhododendron dahurica seedlings at a depth of approximately 0.5 cm. The fungus was then incubated at 25°C. Uninoculated control samples were used as controls. A total of 250 bottles of Rhododendron dahurica seedlings were collected for each inoculation treatment, with plant tissue collected regularly for testing.

[0145] 1.2.7.2 Verification of mycorrhizal fungi inoculation

[0146] The roots of Rhododendron dahurica 40 days after inoculation were used as experimental materials. The mycorrhizal types and mycelial structures were observed under a microscope using the trypan blue staining method. The staining and microscopic examination procedures were the same as those in 1.2.1.4.

[0147] 1.2.8 Preliminary determination of the growth-promoting ability of mycorrhizal fungi in vitro

[0148] 1.2.8.1 Preliminary determination of the phosphate-solubilizing capacity of mycorrhizal fungi

[0149] Referring to Yang Shun's method, the four selected mycorrhizal fungi were activated, and their phosphate solubilizing ability was preliminarily determined using the phosphate solubilization circle method.

[0150] 1.2.8.2 Preliminary determination of the ability of mycorrhizal fungi to secrete IAA

[0151] Qualitative determination: Using the Salkowski colorimetric method, the strain is first inoculated on PDA medium and cultured until the colonies cover the surface of the culture medium. Then, bacterial pieces with a diameter of about 2 mm are inoculated into King culture medium without tryptophan. Each triangular flask is filled with 40 mL of culture medium, and the process is repeated 3 times in total. Then, culture is carried out in a shaker at 28°C and a speed of 120 r / min for 8 days. After the culture is completed, take the clarified bacterial solution and the blank control, add 100 uL of colorimetric solution to each, and observe the color change after standing in the dark for 0.5 hours. By comparing the color with the standard solution, check whether the solution is colored (pink or pink). If color development occurs, it indicates that the strain can secrete indole substances. The same color indicates the ability to secrete IAA, and the darker the color, the larger the amount secreted.

[0152] Quantitative determination: Preparation involves using an auxin standard to prepare IAA standard solutions at concentrations of 0, 1, 2, 3, 4, 6, 8, and 10 μg / mL. Color development is then performed according to the method described above, and the OD value at each concentration is measured at 530. A standard curve is then plotted. Salkowski colorimetric solution is then mixed with the centrifuged fermentation supernatant, allowed to stand in the dark for 30 minutes, and the OD value at 530 is measured. Subsequently, the IAA content in the mycorrhizal fungi is calculated using the standard curve equation, with concentration as the horizontal axis and OD value at 530 as the vertical axis.

[0153] 1.2.9 Determination of the effect of mycorrhizal fungi back-grafting

[0154] 1.2.9.1 Growth index determination

[0155] Plant height was determined by measuring the distance from the root to the stem tip using an electronic digital caliper (0.01 mm accuracy). Leaf samples were placed on grid paper with a spacing of 0.5 cm, and their length and width were measured. Root length measurements were performed by washing the root tissue with tap water to remove any sediment and blotting with absorbent paper to avoid moisture interference. Uninoculated control samples served as the control group. Twenty seedlings of R. dahurica were selected for each inoculation treatment, with three biological replicates performed.

[0156] 1.2.9.2 Biomass determination

[0157] Fresh weight determination method: Remove the Dahurian Rhododendron plants, wash off the soil on the roots, and gently wipe them with a soft paper towel to remove moisture from the surface of the plants. They need to be weighed immediately (the plants contain moisture, so waiting to weigh them may cause them to dry out, resulting in inaccurate data). The uninoculated group was used as the control group. Twenty Dahurian Rhododendron seedlings were taken for each inoculation treatment and three biological replicates were performed.

[0158] Dry weight determination method: Place the sample in an oven, fix it at 100℃ for 30 minutes, then cool it to 70℃ and dry it to constant weight. Record the dry weight value. Use the uninoculated sample as the control group. Take 20 Rhododendron dahurica seedlings for each inoculation treatment and perform three biological replicates.

[0159] 1.2.9.3 Determination of photosynthetic physiological indicators

[0160] (1) Chlorophyll content determination

[0161] Soak Rhododendron dahurica leaves with different inoculation treatments in the dark for 24 hours. Extraction is complete when the leaf tissue turns white. Centrifuge at 4000 rpm for 6 minutes. Collect the supernatant and measure the absorbance of A665 and A649 using a UV spectrophotometer. Calculate using the following formula:

[0162] Ca=13.95A 665 -6.88A 649

[0163] Cb=24.96A 649 -7.32A 665

[0164] C T (mg·L -1 )=C a +C b =18.16A 649 +6.63A 665

[0165] The chlorophyll content in the leaf tissue of Rhododendron dahurica was calculated according to the following formula:

[0166] Chloroplast pigment content (mg / g.FW) = n×C×N×W -1

[0167] In the formula, n is the volume of the extract in mL.

[0168] C: Pigment mass concentration mg·L -1

[0169] N: dilution multiple

[0170] W: fresh weight of sample g

[0171] (2) Determination of photosynthetic parameters

[0172] A portable photosynthesis measurement system (LICOR-6400USA) was used to measure the four photosynthetic parameters, including net photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration, on the third fully expanded leaf of Rhododendron dahurica from the top under different inoculation treatments. The uninoculated leaves were used as controls. Five leaves were collected for each inoculation treatment, and three biological replicates were used.

[0173] (3) Chlorophyll fluorescence parameter determination

[0174] The third round of fully expanded leaves of Rhododendron dasyphylla seedlings with the same growth were taken. After dark treatment for 15 minutes, the chlorophyll fluorescence parameters of the leaves were measured using a chlorophyll fluorescence meter, mainly including initial fluorescence F0, maximum fluorescence FM, and maximum photochemical efficiency (Fv / Fm). The uninoculated leaves were used as the control. Five leaves were taken for each inoculation treatment, and three biological replicates were used.

[0175] 1.2.9.4 Root activity determination (TTC method)

[0176] Refer to Zou Qi's "Plant Physiology and Biochemistry Experiment Guide" to determine root activity using the TTC method.

[0177] 2 Results and Analysis

[0178] 2.1 Mycorrhizae of Rhododendron xing'anensis and the morphological structure of mycelium within the mycorrhizae

[0179] 2.1.1 Mycorrhiza morphology

[0180] The morphological structure of mycorrhiza is the basis for studying mycorrhiza. Since the external morphology of normal roots and mycorrhiza is very similar, before studying the morphological structure of mycorrhiza, the characteristics of the normal root system of Rhododendron dahurica collected from the sampling site were also microscopically observed and measured. Figure 3 As shown:

[0181] Depend on Figure 3It can be seen that the root system of wild Xing'an Rhododendron is a fibrous root system, which is composed of many thin hair roots. The diameter of the hair roots is about 70-100μm. The intact root system starts from the root tip and the color gradually deepens. The young hair roots are milky white (such as Figure 3 c), while more mature hair roots are darker in color, generally light brown or light brown (e.g. Figure 3 In middle b), the young hairy roots are the main objects of observation and analysis in this experiment.

[0182] 2.1.1.1 Mycorrhizal anatomy

[0183] With the sterile seedling root system as the control, 20 tender root segments of Rhododendron dahurica were taken by paraffin section method, totaling 60, and the anatomical structure of Rhododendron dahurica mycorrhiza was observed. The results are as follows Figure 4 As shown:

[0184] like Figure 4 As shown in middle a: The cross-section of the wild Rhododendron dahurica mycorrhiza is basically circular or nearly circular, and the anatomical structure of the hair roots is relatively simple, with a layer of epidermal cells and one to two layers of cortical cells, including the exoderm and endodermis, with a narrow pith in the middle. The infection of mycorrhizal fungi basically does not change the anatomical structure of the hair roots, except that there are hyphae or hyphal ring structures in the cortical cells of the mycorrhiza.

[0185] like Figure 4 (B) The cross-section of the sterile cultured hairy root segment is essentially identical to that of the wild Rhododendron dahurica mycorrhizae, also circular in shape. Both the epidermal and cortical cells of the mycorrhizae are clearly visible, with no hyphae or hyphal loops visible. This indicates that the mycorrhizal fungi have not been infected.

[0186] like Figure 4 As shown in Figure c: The anatomical structure of the longitudinal section of the wild Rhododendron dasyphylla mycorrhiza is also relatively simple. Mycelium is found in the middle of the mycorrhiza, but not in the cortical cells.

[0187] like Figure 4 As shown in middle d: The anatomical structure of the longitudinal section of the sterile cultured hairy root segment is no different from that of the mycorrhiza infected with mycorrhizal fungi, and no hyphae or hyphal ring structures were observed in the epidermal and cortical cells.

[0188] 1.1.1.2 Mycorrhiza submicroscopic structure

[0189] The anatomical structure of mycorrhiza preliminarily indicates the distribution position of hyphae, but the size and thickness of hyphae are unclear and not intuitive. Therefore, the present invention uses sterile seedling root system as a control and adopts electron microscopy scanning method to observe the submicroscopic structure of Rhododendron dahurica mycorrhiza.

[0190] like Figure 5As shown: Different from the anatomical structure of mycorrhiza, by scanning the hairy roots of Rhododendron dahurica at different magnifications, it can be seen intuitively that the root epidermal cells are wrapped with a layer of hyphae, which are either dense or loose, and the hyphae are uneven in thickness. Since the hyphae are entangled on the surface of the root, it is difficult to observe other structures on the root surface. From the electron microscope scanning images of the sterile cultured Rhododendron dahurica hairy roots at different magnifications, it can be seen that the appearance is relatively smooth, wrinkled, and has ridged protrusions, but there is no hyphae entanglement.

[0191] 1.1.2 Mycelial morphology and structure within mycorrhizae

[0192] After the fungus infects the host plant roots, the external morphology is very similar to that of normal roots. Therefore, it is necessary to use trypan blue staining and pressing to observe the morphological structure of the mycelium in the Dahurian Rhododendron mycorrhiza under an optical microscope. In this experiment, 20 young Dahurian Rhododendron root segments were taken, a total of 60, and the roots of sterile seedlings were used as controls. The staining results are shown in the figure below. Figure 6 shown.

[0193] like Figure 6 As shown: There are no hyphae entangled on the surface of the root cells of the sterile seedlings of Xing'an Rhododendron, and no mycelial structure is found inside or between the root cells, indicating that the roots of the sterile seedlings are not infected by mycorrhizal fungi.

[0194] like Figure 7 As shown in the figure: The morphological structure of mycelium in the mycorrhiza of Rhododendron dahurica is relatively rich and complex, but there are differences in morphology. The mycelium can be divided into different types according to different standards.

[0195] According to the distribution of mycelium, it can be roughly divided into three types: 1. Mycelium is distributed on the surface of root cells. On the surface of root cells, most of the mycelium is adhering to the outer wall of the root (such as Figure 7 In addition, some hyphae form mycelial networks on the surface of the root segments (e.g. Figure 7 2. The hyphae are distributed in the root cells, and their morphology changes more than that on the surface of the root cells. Some thick hyphae form dense hyphal nodes, showing the typical structural characteristics of azalea mycorrhizal fungi (such as Figure 7 i), often filling the entire cell, and in addition, forming intracellular hyphae and interpenetrating hyphae structures (e.g. Figure 7 b), in a few root samples, vesicles may also appear, and the vesicles are round or oblong (as in question 7 d, e). 3. Hyphae are distributed between root cells. Most of the intercellular hyphae grow longitudinally along the root cells (as in question 7 d, e). Figure 7 h).

[0196] According to whether the hyphae contain septa, they can be divided into two types, namely, with septa and without septa. 1. With septa. The septa divide the mycelium into many small sections, and the middle part of the section will swell to form a septum structure (such as Figure 72. No septa. The hyphae do not branch, and after entering the cell, they grow along the cell wall to form a hyphae-like structure (such as Figure 7 (a)

[0197] like Figure 8 As shown: During the staining, it was also found that a special structure, namely "microsclerotia", was formed in the mycorrhiza. It is often distributed in the epidermis, cortical cells or intercellular spaces, and is rarely seen in the vascular tissue. The "microsclerotia" structure is a typical structural feature of dark septate endophytic fungi (DSE) in the root cells of the host plant. From the magnified image of the "microsclerotia", it can be seen that its structure is composed of closely arranged granular cells with thickened and swollen cell walls. They vary in shape and size, and are mostly dark brown in color. In addition, the color depth of the same microsclerotia group is uneven.

[0198] Table 4 Morphological characteristics of mycelium in the mycorrhiza of Rhododendron dahurica at the sampling site

[0199]

[0200]

[0201] As shown in Table 4, the mycelial morphology of the Rhododendron dahurica mycorrhiza in the sampling area is rich, including hyphal nodes, vesicles, peripheral wandering hyphae, microsclerotia and other structures.

[0202] 2.2 Analysis of mycorrhizal infection characteristics of Rhododendron xinganensis

[0203] 2.2.1 Mycorrhizal infection rate and infection level

[0204] The infection rate and infection grade are important indicators for measuring the infection status and can indicate the infection characteristics of Rhododendron dahurica. In order to investigate the infection status of mycorrhizal fungi, the present invention measured 100 root segments at the sampling site. The results of microscopic examination after trypan blue staining are shown in the following table:

[0205] Table 5 Mycorrhizal infection rate and infection grade of Rhododendron xinganensis at sampling sites

[0206]

[0207] Note: The same letters indicate no significant difference, different letters indicate significant difference (p < 0.05), Duncan test.

[0208] As shown in Table 5, the roots of the Dahurian Rhododendron in the sampling area were all infected by fungi, but the natural mycorrhizal infection rate was low and the infection intensity was not high, reaching the level 3 infection standard, and the mycorrhizal infection rate was only 22.7%.

[0209] 2.2.2 Correlation analysis between mycorrhizal infection rate and soil chemical properties at sampling sites

[0210] Studies have found that the mycorrhizal infection rate is related to the rhizosphere soil. Therefore, this experiment determined the chemical properties of the rhizosphere soil of the Xing'an Rhododendron at the sampling site. The results are shown in the table below.

[0211] Table 6 Soil chemical properties of sampling sites

[0212]

[0213] It can be seen from Table 6 that the soil in the sampling area is acidic, with a pH value of about 4.46 and a soil conductivity of 374.00uS / cm. The soil contains a lot of organic matter, which is between 38.29g / kg and g / kg, and the available phosphorus content is 27.28mg / kg.

[0214] Table 7 Correlation analysis between soil chemical properties and mycorrhizal infection rate

[0215]

[0216] The data were analyzed by one-way ANOVA (Duncan's multiple comparisons (P)) and Spearman correlation analysis using IBM SPSS Statistics 26 software. The results showed that the chemical properties of the soil had an impact on the mycorrhizal infection of Rhododendron dahurica. The mycorrhizal infection rate was positively correlated with total nitrogen, organic matter, and electrical conductivity, with correlation coefficients of 0.900 and 0.917, respectively. It was negatively correlated with total phosphorus, available phosphorus, and pH, with correlation coefficients of -0.933, -0.983, and -0.608, respectively. The correlations were significant.

[0217] 2.3 Morphological observation and molecular biological identification of mycorrhizal fungi of Rhododendron xinganensis

[0218] 2.3.1 Morphological observation of mycorrhizal fungi of Rhododendron xing'anensis

[0219] Four strains with different morphologies were isolated from the hairy roots of Rhododendron dasyphylla collected from Huilong Mountain, Wuchang City. After culturing for 2-4 weeks, the growth rates were slightly different. Most of the strains were 3-4 cm in diameter. The colonies were mainly black, gray, and white. Most of the colonies were fuzzy, and some were tapered. Pigment production could be observed in some hyphae. The morphological characteristics are shown in Table 8. The fungal colony morphology is shown in Table 8. Figure 9 .

[0220] Table 8 Morphological characteristics of mycorrhizal fungi colonies of Rhododendron dahurica in the sampling area

[0221]

[0222] 2.3.2 Molecular Biological Identification of Mycorrhizal Fungi of Rhododendron xing'anensis

[0223] Because fungi have complex morphological characteristics and are unstable during growth due to environmental influences, some isolated fungi do not readily produce reproductive structures in pure culture and cannot be classified based solely on mycelial characteristics. Therefore, molecular biological identification methods have been introduced into fungal identification. This paper uses rDNA-ITS sequencing for molecular biological identification of mycorrhizal fungi.

[0224] (1) Fungal DNA extraction and ITS segment PCR amplification

[0225] Four different mycorrhizal fungi were isolated from the root tissue of Rhododendron dasyphylla at the sampling site. Fungal hyphae genomic DNA was extracted and the ITS segment of the fungal genomic DNA was amplified using primers ITS1 / ITS4. The electrophoresis results were as follows: Figure 10 .

[0226] (2) Blast alignment results of fungal ITS sequences

[0227] The sequencing results of the mycorrhizal fungi at site A were compared by sequence alignment, and the optimal alignment results for each strain are shown in the table below.

[0228] Table 9 Alignment results of ITS segment sequences of mycorrhizal fungi in the sampling area

[0229] Fungus number GenBank No. Coverage ratio (%) Sequence similarity (%) Fungi with the highest sequence similarity XA1 LC206543.1 98％ 98.45％ Phialocephala fortinii XA2 KM008626.1 93％ 99.52％ Uncultured phialocephala XA3 MT321766.1 98％ 100％ Rhizodermea veluwensis XA4 KY910226.1 93％ 99.53％ Pezicula ericae

[0230] Table 9 shows that the four mycorrhizal fungi isolated from the roots of Rhododendron dasyphyllum at the sampling site—Phialocephala fortinii, Uncultured phialocephala, Rhizodermea veluwensis, and Pezicula ericae—had the highest similarity, with sequence similarities ranging from 98% to 100%. All four belong to the subdivision Ascomycota. At the genus level, three genera were identified: Phialocephala, Pezicula, and Rhizodermea.

[0231] 2.4 Microstructure and growth characteristics of four mycorrhizal fungi

[0232] 2.4.1 Microscopic structure of four mycorrhizal fungi

[0233] In order to understand the microstructure of the four selected mycorrhizal fungi, including the mycelial morphology and spore morphology, the four mycorrhizal fungi were cultured for 7-10 days using the insert culture method and observed under an optical microscope. The results are as follows Figure 11 shown.

[0234] like Figure 11As shown: The XA1 strain has a gray front with a black edge and a black back. It has a velvety texture, branched hyphae with septa, and round spores. The XA2 strain has a brown to gray front and back with a tapety texture, thick branched hyphae with septa, and scattered spores, mostly round in shape. The XA3 strain has a gray-black front and back with a velvety texture, septate hyphae, and no conidia. The XA4 strain has a white front and back with a tapety texture, thick hyphae with septa, but no spores.

[0235] 2.4.2 Growth characteristics of four mycorrhizal fungi

[0236] In the present invention, it was found that the growth rates of the four mycorrhizal fungi were all slow. In order to better understand their growth cycle, the colony diameters were observed and measured with a cycle of 40 days until the strains grew stably in the culture medium. The growth rate curves were drawn as shown in FIG. Figure 12 As shown:

[0237] like Figure 12 As shown in the figure, the growth trends of the four mycorrhizal fungi on the culture medium went through three periods, namely, slow growth period, rapid growth period and stable growth period. The complete growth period of the four fungi was about 25-30 days, of which the slow growth period was about 7-10 days and the rapid growth period was 5-10 days. Among them, XA4 grew faster and reached the stable growth period the earliest. Finally, the colony radius stabilized at about 3-4 cm.

[0238] 2.5 Identification of four mycorrhizal fungi and preliminary determination of their growth-promoting ability in vitro

[0239] 2.5.1 Identification of four mycorrhizal fungi by back-grafting

[0240] In order to understand the inoculation of four mycorrhizal fungi in Rhododendron dahurica seedlings, trypan blue staining was used to identify the roots of Rhododendron dahurica 4 months after inoculation. After each inoculation treatment, 100 roots were tested, totaling 400 roots. The infection rate of the inoculation was also determined. Figure 13 As shown:

[0241] Depend on Figure 13 It was found that the four mycorrhizal fungi could all be recolonized in the roots of Rhododendron dahurica, i.e., the inoculation was successful. The fungal hyphae could invade the roots of Rhododendron dahurica and form mycorrhizal structures in the roots of Rhododendron dahurica. The mycorrhizal morphology of the hyphae invading plant cells was observed under an optical microscope. The strains XA1 and XA2 had dark brown intracellular mycelial masses and formed "microsclerotia" structures in the late invasion stage ( Figure 13 a, b, c, d); the typical structural characteristics of dark septate endophytic fungi (DSE), in strain XA3, intracellular hyphae and peripheral hyphae were found ( Figure 13In the middle (e, f), strain XA4 can form a typical ericoid mycorrhizal fungal structure, i.e., dense intracellular hyphae and thick hyphae nodes are formed in the epidermal cells ( Figure 13 (g,h), belongs to the ericoid mycorrhizal fungi (EMF).

[0242] 2.5.2 Analysis of the growth-promoting ability of four mycorrhizal fungi in vitro

[0243] 2.5.2.1 Phosphate-solubilizing capacity of four mycorrhizal fungi

[0244] Phosphorus is one of the three essential nutrients for plant growth and plays a vital role in plant growth, development and metabolism. Studies have shown that mycorrhizal fungi can convert insoluble phosphorus in the soil into soluble phosphorus that can be absorbed and utilized by plants, thereby promoting the absorption and utilization of phosphorus by plants. The phosphorus solubilization capacity of four mycorrhizal fungi was preliminarily tested using the phosphorus solubilization circle method. The results are as follows: Figure 14 shown.

[0245] During the growth process, the strain will dissolve the insoluble phosphate in the inorganic culture medium, thus forming a transparent phosphate-dissolving circle on the surface of the culture medium. The larger the ratio of the transparent phosphate-dissolving circle diameter (d) to the colony diameter (D), the stronger the phosphate-dissolving ability. Therefore, the present invention uses the phosphate-dissolving circle method to make a preliminary determination of the phosphate-dissolving ability of four mycorrhizal fungi. Figure 14 It can be seen that after the four mycorrhizal fungi were inoculated into inorganic phosphorus solid culture medium for 7 days, it was found that none of the four mycorrhizal fungi produced transparent phosphate-solubilizing circles, indicating that the four mycorrhizal fungi had no phosphate-solubilizing ability.

[0246] 2.5.2.2 Ability of Four Mycorrhizal Fungi to Secrete IAA

[0247] Studies have shown that plant mycorrhizal fungi can promote plant growth. One direct way is to secrete growth-promoting substances such as bioregulators and plant hormones (auxin, cytokinin, gibberellin), etc. during the symbiosis with host plants, thereby achieving the purpose of promoting plant growth and development. Therefore, this experiment conducted a preliminary test on the ability of four mycorrhizal fungi to secrete IAA.

[0248] (1) IAA qualitative determination:

[0249] Depend on Figure 15 It can be seen that: through the Salkowski colorimetric method, it was found that the four mycorrhizal fungi can all produce IAA, and the color was compared with the standard solution to observe whether the four solutions showed color (pink or pink). It was found that the four solutions XA1, XA2, XA3, and XA4 all showed color, indicating that the four mycorrhizal fungi can secrete indole substances. The darker the color, the more secretion, among which XA1 and XA4 have darker colors.

[0250] (2) Quantitative determination of IAA:

[0251] A standard curve was drawn based on the OD=530 of the IAA standard solution at 0, 1, 2, 3, 4, 6, 8, and 10 μg / mL, and the formula for calculating the IAA concentration was determined to be y=0.0556x+0.0834, R 2 =0.9911

[0252] Figure 16 is the IAA standard curve, such as Figure 17 As shown: The ability of four mycorrhizal fungi to produce IAA was quantitatively determined, and it was found that the IAA secreted by XA1 was significantly higher than that of other strains, with a concentration of 20.89 μg / mL, followed by XA4, which secreted IAA at a concentration of 19.77 ug / mL. The IAA content produced by XA2 and XA3 was relatively low, with concentrations of 7.08 μg / mL and 9.80 μg / mL, respectively.

[0253] 2.6 Study on the effect of mycorrhizal fungi back-grafting

[0254] The back-grafting effect is an important part of studying mycorrhizal fungi and is the ultimate goal of strain isolation and identification. At the same time, it is also the basis for screening excellent strains and applying mycorrhizal technology to garden and horticultural production practices. The mycorrhizal fungi isolated from the root system of Dahurain Rhododendron in the present invention, but whether these mycorrhizal fungi have a promoting effect on the growth of Dahurain Rhododendron still needs to be verified by back-grafting the strains into Dahurain Rhododendron tissue culture seedlings. Through the above experiments, the present invention found that XA1 and XA4 have strong abilities to secrete IAA, and it is speculated that they will promote root growth. However, XA1 grows slowly and is easily inactivated and difficult to preserve. However, XA4 grows fast, is not easily inactivated, is easy to preserve, and can be applied in practical production processes. Therefore, the present invention selected XA4 for back-grafting to observe the growth status of Dahurain Rhododendron. The sequence of XA4 in the ITS segment is as follows: Figure 18 shown.

[0255] 2.6.1 Effects of mycorrhizal fungi inoculation on growth parameters of Rhododendron dahurica

[0256] 2.6.1.1 Effects of mycorrhizal fungi inoculation on plant height of Rhododendron dahurica

[0257] The seedlings of Rhododendron dahurica were taken from the control group without inoculation and the treatment with XA4. Five seedlings were taken from each treatment. Three biological replicates were used. The plant heights of Rhododendron dahurica were measured at 1 month, 2 months, 3 months and 4 months. The results are as follows: Figure 19 As shown:

[0258] like Figure 19 As shown in the results, 4 months after inoculation, the height of Rhododendron dahurica seedlings inoculated with XA4 increased significantly compared with the control group, reaching 7.9-8.2 cm, a 41.87% increase compared with the CK. This indicates that artificial inoculation of mycorrhizal fungus XA4 can promote the growth of Rhododendron dahurica seedlings.

[0259] 2.6.1.2 Effects of mycorrhizal fungi inoculation on leaf length and width of Rhododendron dahurica

[0260] Four months after artificial inoculation of mycorrhizal fungi, five seedlings were taken from each treatment and three biological replicates were used to measure the leaf length and leaf width of the Rhododendron dasyphylla plants under different treatments. The results are as follows: Figure 20 As shown:

[0261] like Figure 20 As shown: 4 months after inoculation, the leaf length of the plants inoculated with XA4 mycorrhizal fungus was greater than that of the uninoculated plants, and the leaf width of the inoculated plants was greater than that of the uninoculated plants, indicating that the presence of the mycorrhizal fungus XA4 better promoted the growth of the leaves of the Xing'an Rhododendron plants.

[0262] 2.6.1.3 Effects of mycorrhizal fungi inoculation on root growth of Rhododendron dahurica

[0263] Four months after artificial inoculation with XA4 mycorrhizal fungi, five seedlings were taken from each treatment and three biological replicates were used to measure the root lengths of the Rhododendron dasyphylla plants under different treatments. Figure 21 As shown:

[0264] like Figure 21 As shown, 4 months after inoculation, the root length of the plant inoculated with XA4 mycorrhizal fungus increased to 41.3 mm, indicating that the mycorrhizal fungus XA4 better promoted the growth of the underground part of the Rhododendron dahurica plant.

[0265] 2.6.1.4 Effects of mycorrhizal fungi inoculation on Rhododendron biomass

[0266] Plant biomass is one of the important indicators of plant growth and development, which can reflect the growth status of the aboveground and underground parts respectively. In this study, the biomass of Rhododendron dasyphylla seedlings was measured 4 months after artificial inoculation with XA4 mycorrhizal fungus. Five seedlings were selected for each treatment, and three biological replicates were performed. The results are shown in Figure 22 .

[0267] like Figure 22 As shown in the figure, compared with the control group, the biomass of both aboveground and belowground parts of the Dahurain Rhododendron inoculated with XA4 mycorrhizal fungus increased. In terms of biomass distribution, the belowground biomass of the inoculated Dahurain Rhododendron plants was greater than the aboveground biomass. This indicates that inoculation with XA4 mycorrhizal fungus promotes the vegetative growth of Dahurain Rhododendron to varying degrees at different locations, with a more significant effect on the belowground parts.

[0268] 2.6.2 Effects of mycorrhizal fungi inoculation on photosynthetic parameters of Rhododendron dahurica

[0269] 2.6.2.1 Effects of different treatments on chlorophyll content in Rhododendron dahurica leaves

[0270] Plant growth and development are inseparable from photosynthesis, and chlorophyll is a type of green pigment used by plants for photosynthesis. Chlorophyll content has a direct impact on plant growth and can, to a certain extent, reflect the potential of a plant for photosynthesis. Four months after inoculation with XA4 mycorrhizal fungi, the chlorophyll content in the leaves of Rhododendron dahurica plants was measured. The results are as follows: Figure 23 As shown:

[0271] like Figure 23 It can be seen that 4 months after inoculation with XA4 mycorrhizal fungi, the chlorophyll content exceeded that of the control group. After inoculation with XA4 strain, the total chlorophyll content was 2.84 mg / g, which was 1.29 times that of the CK group. It can be seen that inoculation with XA4 mycorrhizal fungi can increase the chlorophyll content of Dahurian Rhododendron, and the increase in chlorophyll content will enhance the photosynthesis of Dahurian Rhododendron, thereby promoting the growth of Dahurian Rhododendron.

[0272] 2.6.2.2 Effects of different treatments on chlorophyll fluorescence parameters of Rhododendron dahurica leaves

[0273] Chlorophyll fluorescence technology is a rapid and non-destructive method for studying photosynthesis. The chlorophyll fluorescence parameter Fv / Fm is an important indicator of the degree of photoinhibition. This parameter can reflect the overall efficiency of plant photosynthesis and the efficiency of light energy utilization. As Fv / Fm increases, it indicates that the photosynthetic efficiency and light energy utilization efficiency of the plant have been improved. This experiment measured the FV / FM of the leaves of Rhododendron dahurica after 4 months of different treatments. The results are as follows: Figure 24 As shown:

[0274] Depend on Figure 24 It can be seen that 4 months after artificial inoculation with XA4 mycorrhizal fungus, the FV / FM value of the leaves of Rhododendron dasyphylla in the XA4 group was higher than that in the CK group, and Fv / Fm reached 0.79. This shows that inoculation with mycorrhizal fungus XA4 can improve the photosynthetic efficiency of plant leaves.

[0275] 2.6.2.3 Effects of different treatments on photosynthetic indices of Rhododendron dahurica leaves

[0276] Plant photosynthetic characteristic indices can not only reflect the growth characteristics and physiological characteristics of plants, but also understand the plant's needs for light, water, temperature and CO2 concentration by analyzing the photosynthetic parameters of plants, and clarify its ecological adaptability to the environment. Therefore, this experiment measured four photosynthetic characteristic indices of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate under different treatments. The measurement results are as follows: Figure 25 As shown:

[0277] Depend on Figure 25As shown in Figure a, the net photosynthetic rate of Rhododendron dahurica leaves after inoculation with XA4 mycorrhizal fungi was significantly different from that without inoculation, indicating that inoculation with XA4 can significantly increase the net photosynthetic rate of Rhododendron dahurica seedlings.

[0278] Depend on Figure 25 As shown in Figure b, inoculation with XA4 mycorrhizal fungi had a significant effect on the intercellular CO2 concentration in the leaves of Rhododendron dahurica seedlings. Compared with the uninoculated leaves, the intercellular CO2 concentration in the leaves increased after inoculation.

[0279] Depend on Figure 25 As shown in Figure c, the stomatal conductance of the fungus was affected by inoculation with XA4, and the stomatal conductance was 0.91mmol·m -2 ·s -1 .

[0280] Depend on Figure 25 As shown in Figure d, the transpiration rate of Rhododendron dahurica leaves increased significantly after inoculation with XA4, reaching 1.94 mmol·m -2 ·s -1 .

[0281] In summary, inoculation with mycorrhizal fungus XA4 significantly improved the photosynthetic indexes of Rhododendron dahurica, which was beneficial to its growth and development.

[0282] 2.6.3 Effects of mycorrhizal fungi inoculation on root activity of Rhododendron dahurica

[0283] 2.6.3.1 Drawing of the root activity standard curve

[0284] A standard curve was drawn based on the absorbance at OD = 485 nm using standard colorimetric solutions of 0 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, and 150 μg of tritium. Figure 26 is the standard curve of root activity,

[0285] The standard curve formula for calculating root activity is y = 0.0045x + 0.0019, R 2 =0.9976.

[0286] 2.6.3.2 Effects of different treatments on root activity of Rhododendron dahurica seedlings

[0287] The root activity of Rhododendron dahurica can directly reflect the growth and nutrient absorption of the root system. Therefore, this experiment measured the root activity of different treatments. The results are as follows: Figure 27 As shown:

[0288] like Figure 27 As shown in the figure, the root activity of Rhododendron dahurica inoculated with XA4 was significantly improved, reaching 1617.9 μg·g -1 ·h -1It can be seen that inoculation with mycorrhizal fungus XA4 can improve the root activity of Rhododendron dahurica seedlings, thereby promoting the plant's absorption of water and nutrients.

[0289] 3 Discussion

[0290] 3.1 Mycorrhizal infection characteristics of Rhododendron dahurica

[0291] Research has shown that most Ericaceae plants inhabit soils low in mineral nutrients and high in organic matter. Furthermore, the infection rate of ericoid fungi is high in these soils. The present invention also reached the same conclusion, demonstrating that soil nutrient content and pH significantly influence the mycorrhizal infection rate of Dahurian rhododendron. Therefore, when planting Dahurian rhododendron, attention should be paid to soil nutrient content and pH to increase the mycorrhizal infection rate, thereby promoting plant growth and development. Furthermore, during sampling, the present invention discovered that microhabitats varied across different sampling locations, suggesting that mycorrhizal infection rates are significantly correlated with ecological factors such as plant density, soil thickness, slope, sunlight, associated tree species, and community type. Studies have shown that tree age influences mycorrhizal infection rates. A study of the mycorrhizal infection rate of Vaccinium vulgaris found that trees between 8 and 18 years old had the highest mycorrhizal infection rate, while seedlings aged 2 to 4 years had a relatively low infection rate. Therefore, in the process of introduction, cultivation, protection and development and utilization of Xing'an Rhododendron, the present invention must pay attention to protecting various ecological factors related to the infection rate in the ecosystem, improving the mycorrhizal infection rate, and thus promoting it to fully play its role in a good environment.

[0292] 3.2 Isolation and purification of mycorrhizal fungi from the roots of Rhododendron xinganensis

[0293] A large number of mycorrhizal fungi were isolated in the mycorrhizal isolation and purification experiments of other Ericaceae plants. Among them, Zhang Chunying used a variety of culture media including Martin-Bengal red medium, PDA medium, MMN medium, etc. for cultivation, and isolated 280 strains from Yunjin Rhododendron, which were divided into 17 types. Wu Jiayu used a variety of culture media including MMN medium, PDA medium, MEA medium, etc. for cultivation, and isolated a total of 68 fungi from Vaccinium myrtillus, and 23 types of fungi were obtained after identification. However, the separation and purification culture medium in the present invention only used potato agar medium (PDA), which is more widely used in China. Finally, the number of fungi isolated and purified from the hairy roots of Xing'an Rhododendron was small, and only 20 mycorrhizal fungi with different morphologies were obtained. This may be due to the single culture conditions and the small number of culture medium types, which makes it difficult to pure culture some strains. The strains that can be isolated in the laboratory are limited, and may only be a small part of them.

[0294] 3.3 Morphological and molecular biological identification of mycorrhizal fungi of Rhododendron xinganensis

[0295] The identification results of the present invention show that the Phialocephala fortinii fungus occupies a higher proportion in the Xing'an Rhododendron. This genus is a dark septate endophytic fungus (DSE). In the experiment of the present invention, the isolated Phialocephala fungal colonies are mainly black or gray-black. Although belonging to the same type of fungi, there are large differences in their colony morphology. Some Phialocephala fungi are darker in overall color, black or gray-black, while other colonies are black in the middle and have obvious gray-white edges on the periphery. However, the molecular identification results show that the two fungi with different morphologies are the same species. Therefore, in the identification of mycorrhizal fungi, it is more accurate to adopt a method combining morphological identification with molecular biological identification.

[0296] Research has shown that the fungi that can form ericoid mycorrhizal fungi are primarily members of the Ascomycotina and Basidiomycetes classes, with a small number of Zygomycotina members also present. Morphological and molecular identification of the fungi isolated from the Xing'an rhododendron species in this study revealed that they all belong to the Ascomycota, consistent with Jia Rui's results from his work isolating and identifying Xing'an rhododendron species in the Greater Khingan Range. However, the study found that a very typical ericoid mycorrhizal fungus, the genus Oidiodendron, was not isolated. This is speculated to be related to the location and time of sample collection, possibly due to different habitats or host plants. This difference is reflected in the species of symbiotic fungi: some fungi may be more prone to growing in specific locations or times, while others may be better adapted to different environmental conditions. Therefore, the differences found in the collected samples provide valuable information for studying and understanding the distribution and ecological functions of fungi.

[0297] 3.4 Inoculation effect of mycorrhizal fungi of Rhododendron xinganensis

[0298] In the present invention, since the inoculation experiments were conducted using tissue culture seedlings, it was found that inoculation with Pezicula ericae XA4 promoted the growth of Rhododendron dahurica tissue culture seedlings compared to uninoculated seedlings. However, further verification is needed on seedlings. After inoculation, strains that can promote the growth and development of Rhododendron dahurica were screened. However, no experiments were conducted on the stress resistance, seed germination rate, and cutting survival rate of Rhododendron dahurica, and further research is needed. When the growth-promoting ability of four mycorrhizal fungi was tested, it was found that the four fungi had no phosphate solubilization ability, but all could produce IAA, which may be due to the fungi promoting the growth and development of Rhododendron dahurica plants by producing plant hormones.

[0299] 4 Conclusion

[0300] This study randomly sampled and tested symbiotic mycorrhizae from concentrated distribution sites of wild Rhododendron dahurica. Trypan blue staining was used to investigate the mycorrhizal morphology and infection characteristics. Mycorrhizal fungi were isolated and purified, and their types were determined using a combination of morphological and molecular biological methods. Furthermore, tissue culture seedlings of Rhododendron dahurica were screened and regrafted to test their growth-promoting effects. The main conclusions of the study are as follows:

[0301] (1) Trypan blue staining results showed that the mycorrhizal morphology of Rhododendron dahurica was relatively rich, but there were differences in morphology. According to the distribution of mycelium, it can be roughly divided into three types: a. hyphae on the surface of root cells. Most of them are wandering hyphae; b. intracellular hyphae, some of which are dense hyphae formed by thick hyphae, showing the typical structural characteristics of Rhododendron mycorrhizal fungi, and vesicles can also be found in a few root samples; c. intercellular hyphae. Most of them are longitudinally growing hyphae, and "microsclerotia" structures are also found. According to whether the hyphae contain septa, it can be divided into two types, namely septate hyphae and non-septate hyphae. Observation through paraffin sections showed that the mycorrhizal structure is very simple, consisting of a layer of epidermal cells and a narrow pith surrounded by one or two layers of cortical cells. Mycorrhizal fungal infection generally does not change the anatomical structure of the hair roots. Scanning electron microscopy observations showed that the surface of the hair roots of Rhododendron dahurica sampled in the field was covered with a layer of loose or dense hyphae.

[0302] (2) The roots of Rhododendron chinense in the sampling site were infected by fungi, but the total root infection rate was low and the infection intensity was not high, reaching the level 3 infection standard, and the infection rate was only 22.7%. Through the correlation study of the soil chemical properties and mycorrhizal infection rate of the sample site, it was found that there was a significant positive correlation between the mycorrhizal infection rate and total nitrogen, organic matter, and electrical conductivity, and a significant negative correlation between the mycorrhizal infection rate and total phosphorus, available phosphorus, and pH value.

[0303] (3) Preliminary morphological observation and classification showed that four strains with different morphologies were obtained. Molecular identification results showed that the mycorrhizal fungi all belonged to the subphylum Ascomycota. At the genus level, they included three genera: Phialocephala, Pezicula, and Rhizodermea.

[0304] (4) The growth characteristics, phosphate solubilization capacity, and IAA secretion capacity of four mycorrhizal fungi were determined. The dominant fungus, Pezicula ericae XA4, was selected and re-grafted into aseptically cultured Rhododendron dahurica seedlings. Trypan blue staining was used to detect that all mycorrhizal fungi could be re-grafted successfully. Compared with the uninoculated seedlings, the inoculation with XA4 had the most significant growth-promoting effect on Rhododendron dahurica. Its plant height, leaf length, leaf width, root length, biomass, chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and root activity were all improved compared with the uninoculated seedlings.

[0305] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. One plant Pezicula ericae XA4, characterized by It was deposited in the General Microbiology Center of China Culture Collection Administration on June 26, 2024, with the deposit number CGMCC No.41350.

2. Use of XA4 according to claim 1 in producing indoleacetic acid.

3. Use of the XA4 described in claim 1 in promoting the growth of Dahurian rhododendron.

4. Use of XA4 according to claim 1 in improving the growth rate of Rhododendron dahurica.

5. Use of the XA4 according to claim 1 in promoting the growth of Rhododendron dahurica leaves.

6. Use of the XA4 according to claim 1 in promoting the root growth of Rhododendron dahurica.

7. Use of the XA4 according to claim 1 in enhancing photosynthesis of Rhododendron dahurica.

8. Use of the XA4 according to claim 1 in improving the root activity of Rhododendron dahurica.

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