Pezicula ericae XA4 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a strain (Pezicula ericae) XA4 and application of the strain (Pezicula ericae) XA4. The invention provides a strain (Pezicula ericae) XA4 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on June 26, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.41350. The invention also provides a method for preparing the strain (Pezicula ericae) XA4. The XA4 provided by the invention is used for carrying out tie-back on sterile cultured rhododendron dauricum seedlings, and trypan blue dyeing detection shows that mycorrhizal fungi can be successfully subjected to tie-back. Compared with the non-inoculated strain, the inoculated XA4 has the most obvious growth promoting effect on the rhododendron dauricum. The plant height, the leaf length, the leaf width, the root length, the biomass, the chlorophyll content, the net photosynthetic rate, the stomatal conductance, the intercellular CO2 concentration, the transpiration rate and the root activity of the strain are all improved compared with those of the strain without inoculation.

Description

Technical Field

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

[0002] Application value of Rhododendron dasyphylla:

[0003] Rhododendron dauricum L. has a very high ornamental value in gardens. It is a plant in Northeast China that blooms before leaves. It blooms in early spring with bright and rich colors, mostly pink or purple, and rarely white. It is known as the "most beautiful flower". Its flower shape varies. Zhou Ming once found a double-petaled Rhododendron dauricum L. in a field survey. It has double petals and 2-6 layers of petals, which is very ornamental. Rhododendron dauricum L. has a special scene of "dead wood coming back to life in spring" and is loved by many flower arrangement enthusiasts. There are multiple choices for garden applications. In the wild, it can be planted in patches to form a sea of ​​rhododendrons. In cities, it can be widely used in roads, scenic spots, parks and other landscaping. At the same time, it can be planted in combination with trees, shrubs, and herbaceous plants to create a community landscape with different heights. Ren Qinggui and others made a preliminary discussion on its application in gardens and proposed to plant evergreen conifers such as Scots pine and Pinus tabulaeformis in the front row, and then match them with Forsythia suspensa and Prunus mume to increase the visual layering and achieve a better greening effect. In addition, the Dahurian Rhododendron has many branches and is resistant to pruning. It can be planted as a hedge or made into a bonsai, showing the morphological beauty of the Dahurian 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 a preliminary study 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 mycorrhizal infection rate of Daxinganling Rhododendron in the wild 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 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 that have 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] In order to solve the above problems, the present invention provides a Pezicula ericae XA4 and its application. The XA4 provided by the present invention is used to inoculate aseptically cultured Dahurian rhododendron seedlings, and trypan blue staining is used to detect that mycorrhizal fungi can be successfully inoculated. Compared with the uninoculated bacteria, the inoculated XA4 has 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 are all improved compared with the uninoculated bacteria.

[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 China Microorganism Culture Collection Administration on June 26, 2024, with a deposit number of CGMCC No.41350.

[0010] The present invention also provides the use of 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 phosphorus 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 XA4 described in the above technical solution in promoting the growth of Rhododendron dasyphylla.

[0014] The present invention also provides the application 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 XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica leaves.

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

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

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

[0019] The present invention selects the root system of wild Rhododendron dahurica from Huilong Mountain, Wuchang City, Heilongjiang Province as the material, randomly samples and detects the symbiotic mycorrhizae, uses trypan blue staining to study the morphological structure and infection characteristics of the mycorrhizae; separates and purifies the mycorrhizal fungi and uses a combination of morphological identification and molecular biological identification to determine the type of mycorrhizal fungi; screens and inoculates Rhododendron dahurica tissue culture seedlings again to detect the fungal growth-promoting effect. 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 hyphae nodes, showing the typical structural characteristics of Rhododendron mycorrhizal fungi, and vesicles appear 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 found that the structure of the mycorrhiza is 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 hairy roots. Scanning electron microscopy observation found that the surface of the hairy roots 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 third level infection standard, but the mycorrhizal infection rate reached 22.7%. The correlation study between the soil chemical properties and mycorrhizal infection rate in the sampling area showed that there was a significant positive correlation between the mycorrhizal infection rate and total nitrogen and organic matter, and a significant negative correlation between the mycorrhizal infection rate and total phosphorus, available phosphorus, and pH value.

[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 ability and IAA secretion ability of four mycorrhizal fungi were measured, and the dominant fungus Pezicula ericae XA4 was selected for back-grafting on sterile cultured Dahurian rhododendron seedlings. Trypan blue staining was used to detect that all mycorrhizal fungi could be successfully back-grafted. Compared with the uninoculated ones, 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 ones. 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 to be used in the embodiments are briefly introduced below.

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

[0026] Figure 2 This is the basic situation of Huilong Mountain in Wuchang City. Note: a. Community species characteristics; b. Rhizosphere soil; c. Slope;

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

[0028] Figure 4 This is the observation of the anatomical structure of the hairy roots of Rhododendron dasyphylla. 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: exodermis; 3: endodermis; 4: pith; hc: hyphae;

[0029] Figure 5 This is a scanning electron microscope observation of the hairy roots of Rhododendron dahurica. Note: a, b, c, d are the hairy root fragments of sterile cultured Rhododendron dahurica at 200, 600, 1500, and 3000 times magnification, respectively. e, f, g, h are the 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 xing'anensis. Note: a. wandering hyphae outside the root; b. hyphae inside the cell; c. hyphal network; d. round vesicle; e. oblong vesicle; f. hyphae forming hyphal masses inside the cell; g. ruptured vesicles inside the cell; h. hyphae running through the cells; i. thick hyphae inside the cell forming compact hyphal 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] Fig. 9 The colony morphology of mycorrhizal fungi of Rhododendron dasyphylla in the sampling area;

[0034] Fig.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 fragment;

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

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

[0037] Fig.13 Identification of four mycorrhizal fungi. Note: a, c are the hyphae structures of dark septate endophytic fungi (DSE); b, d are microsclerotia structures; e are intracellular shuttle hyphae; f are extraradical hyphae; g is a typical structure of EMF, i.e., intracellular hyphae mass; h is a thicker intracellular hyphae node;

[0038] Fig.14 The phosphorus solubilizing capacity of four mycorrhizal fungi;

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

[0040] Fig.16 This is the IAA standard curve;

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

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

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

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

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

[0046] Fig. 22 The effects of different treatments on the biomass of Rhododendron dahurica seedlings after 4 months;

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

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

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

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

[0051] Fig. 27 This is the effect of different treatments on the 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 No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNo.41350. DETAILED DESCRIPTION

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

[0055] The present invention also provides the use of 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 phosphorus 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 XA4 described in the above technical solution in promoting the growth of Rhododendron dasyphylla.

[0059] The present invention also provides the application 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 XA4 described in the above technical solution in promoting the growth of Rhododendron dahurica leaves.

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

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

[0063] The present invention also provides the use of 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 in conjunction with 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 dasyphylla were collected from Huilong Mountain in 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 dasyphylla were collected from Huilong Mountain in Wuchang City, Heilongjiang Province. The specific sampling situation is shown in the figure below.

[0073] Huilong Mountain in Wuchang City (127°10′E, 45°5′N) has a mid-temperate continental monsoon climate, with an altitude of 147-1689m. Most of the Rhododendron dasyphyllum had not yet bloomed when the samples were collected. The soil type is loam, the soil layer thickness is 5-30cm, the soil pH is 4.4-4.8, the slope is 35-40°, the community species are relatively rich, and there are many associated tree species. The main tree layer is willow, Mongolian oak, Juglans mandshurica, etc. Rhododendron dasyphyllum is the main tree species in the shrub layer, and the main herbaceous plant layer is Hosta, Calendula officinalis, Lily of the valley, Peony, etc.

[0074] 1.1.3 Sample collection and storage

[0075] The experimental samples were collected in March 2023. Before sampling, the soil surface was cleared of fallen leaves, stones and other debris as well as the 3 cm deep surface soil. Three sampling points were randomly selected in the sampling site, and the concentrated growth area of ​​Dahurian Rhododendron was selected as the central area. The roots dug were dug along the main roots to ensure that the roots dug were all Dahurian Rhododendron roots. The small roots and fibrous roots of Dahurian Rhododendron and the soil in the 5-50 cm soil layer were collected. About 3 kg of soil and roots were retained in each sample site, put into self-sealing bags and marked with labels, and then stored in ice bags and brought back to the laboratory. The root samples were placed in a 4°C refrigerator, and the rhizosphere soil was naturally air-dried and stored through a 20-mesh sieve.

[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 in the fungal ITS region, which were synthesized by Shanghai Shenggong 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 mycorrhiza of Rhododendron xing'anensis and its mycelium

[0084] 1.2.1.1 Observation of the root system of Rhododendron dahurica

[0085] The roots of wild Rhododendron dahurica were randomly selected, and the characteristics of their roots were observed, including their diameter, morphology, and color. An electronic digital vernier caliper (accuracy 0.01 mm) was used to measure the diameter of the roots. The roots were stained with trypan blue, and the microscopic structure of the roots 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 dasyphylla was taken to observe the anatomical structure of mycorrhiza.

[0088] 1.2.1.3 Observation of mycorrhizal microstructure

[0089] The microstructure of mycorrhizae was observed by scanning electron microscopy. The cut root segments were fixed in 2.5% glutaraldehyde with pH=6.8, and placed in a 4℃ refrigerator for more than 1.5h. They were rinsed twice or three times with 0.1mol pH=6.8 phosphate buffer for 10min each time, and dehydrated with 50%, 70%, and 90% ethanol for 10-15min each time; dehydrated with 100% ethanol twice or three times for 10-15min each time, and (100% ethanol: tert-butanol = 1:1) once; pure tert-butanol twice for 15min each time. The samples were placed in a -20℃ refrigerator and frozen for 30min, and placed in a dryer to dry the samples for about 4h. The observation side of the sample was facing up and adhered to the scanning electron microscope sample stage with conductive tape. A 100-15 angstrom metal film was deposited on the surface of the sample using an ion sputtering coater, and finally observed and photographed using a high-precision scanning electron microscope (SU-8010HITACHI).

[0090] 1.2.1.4 Observation of the morphological structure of mycelium in mycorrhiza

[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 segments in 10% KOH, transparentize in a 90℃ water bath for 20-30 minutes, 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, 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: Randomly pick 20 root segments of uniform thickness and length from the stained root segments, arrange them on 2 slides, 10 roots on each slide, add 1-2 drops of lactic acid glycerol solution, cover with a coverslip, and gently press 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 of mycorrhizal infection rate and Liu Runjin's classification method for classification. The formulas for infection rate and classification 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] Classification: Level 1 (0 ≤ infection rate < 1%), Level 2 (1% < infection rate < 20%), Level 3 (20% < infection rate < 40%), Level 4 (40% < infection rate < 60%), Level 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 conductivity.

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

[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 medium use potato agar medium (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 them with running water for 1-3 hours until the surface soil is clean.

[0118] (2) Root surface disinfection: Move the root segment to a clean bench, then remove the root segment with tweezers, rinse it with sterile water five 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 the root segments into 0.5 cm pieces and inoculate them on PDA culture medium. Place 5 root segments on each plate, and culture 200 root segments in each sampling site, for a total of 120 plates. Mark them separately, place them in a 25°C mold constant temperature incubator, and culture them in the dark for 2-4 weeks.

[0120] (4) Purification of strains: In the early stage of strain culture, it is important to observe the growth of the strains and remove strains that may be contaminated or grow too fast. Select strains with good growth and clear edges, inoculate them on new PDA 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 Preservation of strains

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

[0123] 1.2.5 Morphological observation of mycorrhizal fungi of Rhododendron xing'anensis

[0124] 1.2.5.1 Colony morphology observation

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

[0126] 1.2.5.2 Observation of mycelium 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 45° using sterile tweezers. After sealing and marking, it was placed in a 25°C incubator 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 xinganensis

[0129] 1.2.6.1 Extraction of total DNA

[0130] After culturing at 25°C on a PDA plate for 7-14 days, fresh mycelium was scraped and ground into powder by adding liquid nitrogen. The Biospin fungal genomic DNA extraction kit was used to extract DNA from the treated strains. The specific experimental steps were strictly carried out in accordance with the kit operating instructions.

[0131] 1.2.6.2 PCR amplification of rDNA ITS segment

[0132] The extracted fungal genomic DNA was diluted to a concentration of 20 ng / μ1 and stored at -20°C for future use. The extracted fungal genomic DNA was used as a template and the fungal rDNA ITS-specific fragment was amplified using universal primers ITS1 / ITS4. The PCR product was 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 regions

[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 Shenggong Bioengineering Co., Ltd. for purification, cloning and sequencing.

[0140] 1.2.6.4 Analysis and identification of mycorrhizal fungi species

[0141] The DNA sequences of the tested fungi in the rDNA ITS region 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 inoculation

[0143] 1.2.7.1 Methods of inoculation of mycorrhizal fungi

[0144] The tissue culture seedlings of Rhododendron dahurica cultivated in the laboratory were used as experimental materials. Peat soil and vermiculite were mixed evenly in a ratio of 3:1, and the humidity was adjusted appropriately. The mixture was divided into conical bottles, and each bottle was filled with about 100 mL. The bottles were sterilized at 121℃ with high-pressure steam for 60 minutes. The Rhododendron dahurica seedlings with good growth and the same growth were transplanted into the soil matrix on a sterile clean bench and then sterilized at 25℃. The sterile punching method was used, and a sterile hole was used to pick a fungal piece with a diameter of 1 cm. It was inoculated in the surrounding soil matrix about 1 cm away from the root of the Rhododendron dahurica seedlings and about 0.5 cm deep. It was placed in a 25℃ environment for culture. The uninoculated Rhododendron dahurica seedlings were used as the control. 50 bottles of Rhododendron dahurica seedlings were treated with each inoculation, totaling 250 bottles. The plants were 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 trypan blue staining method was used to observe the mycorrhizal root type and mycelial structure under a microscope. The staining and microscopic examination steps 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 phosphorus 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-solubilizing 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, first inoculate the strain on PDA medium and culture it until the colonies cover the surface of the medium. Then take bacterial pieces with a diameter of about 2 mm and inoculate them into King culture medium without tryptophan. Each triangular flask is filled with 40 mL of culture medium, and repeat it 3 times in total. Then, culture it in a shaker at 28°C and 120r / min for 8 days. After the culture is completed, take the clarified bacterial solution and blank control, add 100uL of colorimetric solution to each, and observe the color change after standing in the dark for 0.5 hours. Check whether the solution is colored (pink or pink) by comparing the color with the standard solution. 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 of secretion.

[0152] Quantitative determination: The preparation method is to prepare IAA standard solutions with concentrations of 0, 1, 2, 3, 4, 6, 8, and 10 ug / mL by using auxin standards, then color development is performed according to the method, the OD=530 value at each concentration is measured, and finally a standard curve is drawn. Then the Salkowski colorimetric solution is mixed with the fermentation broth supernatant after centrifugation, and the mixture is allowed to stand in the dark for 30 minutes, and the OD=530 value is measured. Subsequently, the concentration is used as the horizontal axis and the OD=530 value is used as the vertical axis, and finally the IAA content in the mycorrhizal fungi is calculated according to the standard curve equation.

[0153] 1.2.9 Determination of the effect of mycorrhizal fungi inoculation

[0154] 1.2.9.1 Growth index determination

[0155] The height of Rhododendron dahurica was determined by measuring the distance from the plant root to the stem tip using an electronic digital display vernier caliper (accuracy 0.01 mm). The leaf samples to be tested were placed on a grid paper with a spacing of 0.5 cm to measure the length and width of the leaves. When measuring the root length, the mud and sand on the surface of the root tissue should be washed off with tap water in advance, and the surface moisture should be dried with absorbent paper to avoid moisture interference. The uninoculated control group was used, and 20 Rhododendron dahurica seedlings were taken for each inoculation treatment, with 3 biological replicates.

[0156] 1.2.9.2 Biomass determination

[0157] Fresh weight determination method: Take out 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 drying, 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 and fix it at 100°C for 30 minutes, then cool to 70°C and dry to constant weight. Record the dry weight value. Use the uninoculated sample as the control group. Take 20 Rhododendron dasyphylla seedlings for each inoculation treatment and perform three biological replicates.

[0159] 1.2.9.3 Determination of photosynthetic physiological indicators

[0160] (1) Determination of chlorophyll content

[0161] Soak the Rhododendron dahurica leaves with different inoculation treatments in a dark environment for 24 hours. The extraction is completed when the leaf tissue turns white. Centrifuge at 4000r / min for 6 minutes, take the supernatant, and use a UV spectrophotometer to measure the absorbance of A665 and A649. Calculate according to 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 dasyphylla 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, 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, of the third fully expanded leaf of Rhododendron dasyphylla from the top under different inoculation treatments. The uninoculated leaves were used as the control. Five leaves were collected for each inoculation treatment, with three biological replicates.

[0173] (3) Determination of chlorophyll fluorescence parameters

[0174] The third round of fully expanded leaves of Rhododendron dasyphylla seedlings with the same growth were taken. After 15 minutes of dark treatment, the chlorophyll fluorescence parameters of the leaves were measured using a chlorophyll fluorescence meter, mainly including the 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 Determination of root activity (TTC method)

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

[0177] 2 Results and analysis

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

[0179] 2.1.1 Morphological structure of mycorrhiza

[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 dasyphylla taken from the sampling site were also observed and measured microscopically. Figure 3 As shown:

[0181] Depend on Figure 3It can be seen that the root system of wild Rhododendron dasyphylla is a fibrous root system, which is composed of many thin hair roots. The diameter of the hair root 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 b), young hairy roots are the main objects of observation and analysis in this experiment.

[0182] 2.1.1.1 Mycorrhizal anatomy

[0183] Using the sterile seedling root system as a control, 20 young Rhododendron dahurica root segments were taken by paraffin section method, a total of 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 a: The cross-section of the mycorrhiza of wild Rhododendron dasyphylla 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 has basically not changed the anatomical structure of the hair roots, except that there are hyphae or hyphae ring structures in the cortical cells of the mycorrhiza.

[0185] like Figure 4 As shown in b: The cross-section anatomical structure of the sterile cultured hairy root segment is basically the same as the cross-section of the wild Rhododendron dasyphylla, and the shape is also circular. The epidermal cells and cortical cells of the mycorrhiza are clearly visible, and no hyphae or hyphae ring structure is seen, indicating that it is not infected by mycorrhizal fungi.

[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 circle structures were observed in the epidermal and cortical cells.

[0188] 1.1.1.2 Mycorrhizal submicroscopic structure

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

[0190] like Figure 5As shown: Different from the anatomical structure of mycorrhiza, by scanning and observing 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 is either dense or loose, and the thickness of the hyphae is uneven. Since the hyphae are entangled on the surface of the roots, it is difficult to observe other structures on the root surface. From the electron microscope scans 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 Morphological structure of mycelium in mycorrhiza

[0192] After the fungus infects the root of the host plant, the external morphology is very similar to that of the normal root. Therefore, it is necessary to use trypan blue staining and slide processing 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 sterile seedling root system was used as a control. 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 Rhododendron dahurica, and no mycelium 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: The morphological structure of mycelium in the mycorrhiza of Rhododendron dasyphylla 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 attached to the outer wall of the root (such as Figure 7 In addition, some hyphae form a mycelial network on the surface of the root segment (such as Figure 7 2. The hyphae are distributed in the root cells, and their morphological changes are more than those on the surface of the root cells. Some thick hyphae form dense hyphal nodes, showing typical structural characteristics of azalea mycorrhizal fungi (such as Figure 7 i), often filling the entire cell, in addition to forming intracellular hyphae and interpenetrating hyphae structures (e.g. Figure 7 b), in a few root samples, vesicles may appear, and the vesicles are round or oblong (such as d and e in question 7). 3. Mycelia are distributed between root cells. Most of the intercellular hyphae grow longitudinally along the root cells (such as 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 knot-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, which is often distributed in the epidermis, cortical cells or intercellular spaces, and rarely 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 enlarged image of the "microsclerotia", it can be seen that its structure is composed of closely arranged granular cells with thickened and swollen cell walls, with different shapes and sizes, mostly dark brown in color, and the color depth of the same microsclerotium group is uneven.

[0198] Table 4 Morphological characteristics of mycelium in the mycorrhiza of Rhododendron dasyphylla in the sampling area

[0199]

[0200]

[0201] As shown in Table 4, the mycelium in the mycorrhiza of Rhododendron dasyphylla in the sampling area has rich morphological structures, including mycelial 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, which 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, and 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 dasyphylla in the sampling area

[0206]

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

[0208] It can be seen from Table 5 that the roots of Rhododendron dahurica 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 of 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 Dahurian rhododendron at the sampling site. The results are shown in the following table.

[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 effective phosphorus content is 27.28mg / kg.

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

[0215]

[0216] IBM SPSS Statistics26 software was used to perform one-way ANOVA (Duncan multiple comparison (P)) and Spearman correlation analysis on the data. It was found that the chemical properties of the soil had an effect 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, and negatively correlated with total phosphorus, available phosphorus, and pH, with correlation coefficients of -0.933, -0.983, and -0.608, respectively, and the correlation was 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 taken from Huilong Mountain, Wuchang City. After culturing for 2-4 weeks, the growth rates were slightly different. Most of them were 3-4 cm in diameter. The colony colors were mainly black, gray, and white. Most of the colonies were velvety, and some were tapered. Pigments could be observed in some hyphae. The morphological characteristic indicators are shown in Table 8. The fungal colony morphology is shown in Fig. 9 .

[0220] Table 8 Morphological characteristics of mycorrhizal fungi in the sampling area of ​​Rhododendron dasyphylla

[0221]

[0222] 2.3.2 Molecular biological identification of mycorrhizal fungi of Rhododendron xing'anensis

[0223] Since fungi have complex morphological characteristics and are unstable due to the influence of the external environment during growth, some isolated fungi are not easy to produce reproductive structures when they are cultured in pure culture and cannot be classified only based on the mycelial characteristics, molecular biological identification methods are introduced into the identification of fungi. The present invention uses rDNA-ITS sequence method to carry out molecular biological identification of mycorrhizal fungi.

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

[0225] Four different forms of mycorrhizal fungi were isolated from the root tissue of Rhododendron dasyphylla in the sampling area. 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: Fig.10 .

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

[0227] The sequencing results of 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 No. GenBank Number Coverage ratio (%) Sequence similarity (%) Fungi with the highest 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] As shown in Table 9, the four mycorrhizal fungi isolated from the root segments of Rhododendron dasyphyllum in the sampling area are Phialocephala fortinii, Uncultured phialocephala, Rhizodermea veluwensis, and Pezicula ericae, with the highest similarity of 98%-100%. All four mycorrhizal fungi belong to the subphylum Ascomycota. At the genus level, there are three genera, including Phialocephala, Pezicula, and Rhizodermea.

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

[0232] 2.4.1 Microstructure 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: Fig.11 shown.

[0234] like Fig.11As shown: The front of the XA1 strain is gray in the middle, black at the edge, black on the back, with a fluffy texture, mycelium branches with septa, and round spores. The front and back of the XA2 strain are brown to gray, with a tapety texture, thick mycelium with septa, and scattered spores, mostly round in shape. The front and back of the XA3 strain are gray-black, fluffy, and mycelium with septa, but no conidia are observed. The front and back of the XA4 strain are white, with a tapety texture, and it can be observed that the mycelium is thick and has a septate structure, but no spores are found.

[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. Fig.12 As shown:

[0237] like Fig.12 As shown, 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 was 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

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

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

[0244] Phosphorus is one of the three essential nutrients for plant growth and plays a very important 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 circle method was used to conduct a preliminary test on the phosphorus solubilization ability of four mycorrhizal fungi. The results are as follows: Fig.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. Fig.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-dissolving circles, indicating that the four mycorrhizal fungi had no phosphate-dissolving 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, plant hormones (auxin, cytokinin, gibberellin), etc., during the symbiosis with the host plant, thereby achieving the purpose of promoting plant growth and development. Therefore, this experiment conducted a preliminary determination of the ability of four mycorrhizal fungi to secrete IAA.

[0248] (1) IAA qualitative determination:

[0249] Depend on Fig.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 were colored (pink or pink). It was found that the four solutions XA1, XA2, XA3, and XA4 were all colored, indicating that the four mycorrhizal fungi can secrete indole substances. The darker the color, the more secretion, among which XA1 and XA4 are darker in color.

[0250] (2) Quantitative determination of IAA:

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

[0252] Fig.16 is the IAA standard curve, such as Fig.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 inoculation

[0254] The inoculation effect is an important part of the study of mycorrhizal fungi, and is the ultimate goal of strain isolation and identification. At the same time, it is also the basis for the screening of excellent strains and the application of mycorrhizal technology in gardening and horticultural production practices. The mycorrhizal fungi isolated from the root system of Dahurian Rhododendron in the present invention, but whether these mycorrhizal fungi have a promoting effect on the growth of Dahurian Rhododendron still needs to be verified by inoculating the strains into Dahurian Rhododendron tissue culture seedlings. Through the above experiments, the present invention found that XA1 and XA4 have strong ability to secrete IAA, and it is speculated that they will promote root growth, but XA1 grows slowly, is easily inactivated, and is not easy to preserve, but XA4 grows fast, is not easy to inactivate, is easy to preserve, and can be used in practical production processes, so the present invention selected XA4 for inoculation to observe the growth status of Dahurian Rhododendron, the sequence of XA4 in the ITS segment is as follows Fig.18 shown.

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

[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 to measure the plant height of Rhododendron dahurica at 1 month, 2 months, 3 months and 4 months. The results are as follows: Fig.19 As shown:

[0258] like Fig.19 As shown: 4 months after inoculation, compared with the control group, the height of the Dahurian Rhododendron inoculated with XA4 increased significantly, with a plant height of 7.9-8.2 cm, an increase of 41.87% over CK. This shows that artificial inoculation of mycorrhizal fungi XA4 can promote the growth of Dahurian Rhododendron 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 width of the Rhododendron dasyphylla plants under different treatments. The results are as follows: Fig. 20 As shown:

[0261] like Fig. 20 As shown: 4 months after inoculation, the leaf length of the plants inoculated with XA4 mycorrhizal fungi 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 Dahurian Rhododendron plants.

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

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

[0264] like Fig.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 biomass of Rhododendron dasyphylla

[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. In this study, the biomass of Rhododendron dasyphylla seedlings was detected 4 months after artificial inoculation with XA4 mycorrhizal fungi. Five seedlings were selected for each treatment, and three biological replicates were performed. The results are shown in Fig. 22 .

[0267] like Fig. 22 As shown in the figure, compared with the control group, the biomass of the aboveground and underground parts of the Dahurian Rhododendron inoculated with XA4 mycorrhizal fungi increased. In terms of biomass distribution, the biomass of the underground part of the Dahurian Rhododendron plants treated with the fungus was greater than the biomass of the aboveground part. This shows that the inoculation of XA4 mycorrhizal fungi has different degrees of promoting effect on the nutritional growth of Dahurian Rhododendron in different positions, and the effect on the underground part is more significant.

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

[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 its content can reflect the photosynthesis potential of plants to a certain extent. 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: Fig.23 As shown:

[0271] like Fig.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 means of studying photosynthesis, and the chlorophyll fluorescence parameter Fv / Fm is an important indicator of the degree of light inhibition. 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 photosynthesis efficiency and light energy utilization rate of the plant have been improved. This experiment measured the FV / FM of the leaves of Rhododendron dasyphylla after 4 months of different treatments. The results are as follows: Fig.24 As shown:

[0274] Depend on Fig.24 It can be seen that 4 months after artificial inoculation with XA4 mycorrhizal fungi, 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. It can be seen that inoculation with mycorrhizal fungi 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 indexes can not only reflect the growth characteristics and physiological characteristics of plants, but also understand the needs of plants for light, water, temperature and CO2 concentration by analyzing the photosynthetic parameters of plants, and clarify their ecological adaptability to the environment. Therefore, this experiment measured four photosynthetic characteristic indexes of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration rate under different treatments. The measurement results are as follows: Fig.25 As shown:

[0277] Depend on Fig.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 Fig.25 As shown in Figure b, inoculation with XA4 mycorrhizal fungi had a significant effect on the intercellular CO2 concentration of the leaves of Rhododendron dahurica seedlings. Compared with the uninoculated leaves, the intercellular CO2 concentration of the leaves increased after inoculation.

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

[0280] Depend on Fig.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 of mycorrhizal fungus XA4 significantly improved the photosynthetic indexes of Rhododendron dasyphylla, 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 root activity standard curve

[0284] Based on 0 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, and 150 μg of standard colorimetric solutions of triphenyltetrazolium, a standard curve was drawn according to the absorbance at OD=485 nm. Fig.26 is the standard curve of root activity,

[0285] The formula for calculating the root activity standard curve 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 dasyphylla 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: Fig. 27 As shown:

[0288] like Fig. 27 As shown in the figure, the root activity of Rhododendron dahurica inoculated with XA4 was significantly improved to 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 Characteristics of mycorrhizal infection of Rhododendron dasyphylla

[0291] Studies have shown that most Ericaceae plants live in soils with low mineral nutrition and high organic matter content. And at this time, the infection rate of mycorrhizal fungi of rhododendron is relatively high. The present invention has also obtained the same conclusion. This finding shows that the nutrient content and pH value in the soil have an important influence on the mycorrhizal infection rate of Dahurian rhododendron. Therefore, when planting Dahurian rhododendron, it is necessary to pay attention to the nutrient content and pH value in the soil to improve the mycorrhizal infection rate of Dahurian rhododendron, thereby promoting plant growth and development. In addition, the present invention found that the microhabitats of different sampling sites were different during sampling, and the mycorrhizal infection rate may also have a greater relationship with relevant ecological factors such as plant density, soil thickness, slope, illumination, associated tree species, and community type. Studies have shown that tree age has an influence on mycorrhizal infection rate. In the study of the mycorrhizal infection rate of rabbit eye blueberry, it was found that the mycorrhizal infection rate of trees aged between 8-18 years was the highest, while the mycorrhizal infection rate of 2-4 year old seedlings was relatively low. Therefore, in the process of introduction, cultivation, protection and development and utilization of Dahurian 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 play its full role in a good environment.

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

[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 vitis-idaea, and 23 fungi were obtained after identification. However, the separation and purification culture medium in the present invention only uses potato agar medium (PDA), which is widely used in China. Finally, the number of fungi isolated and purified from the hairy roots of Dahurian Rhododendron was small, and only 20 mycorrhizal fungi with different morphologies were obtained. It may be due to the single culture conditions and the small number of culture medium types that some strains are difficult to culture in pure form. 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 Dahurian 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 grayish black. Although belonging to the same type of fungi, there are large differences in their colony morphology. Some Phialocephala fungi have a darker overall color, black or grayish black, while other colonies are black in the middle and have obvious grayish white edges on the periphery, but the molecular identification results show that the two different morphological fungi 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] Studies have shown that the fungi that can form mycorrhizal fungi of azalea are mainly members of the Ascomycotina and Basidiomycetes, and there are also a small amount of Zygomycotina. The fungi separated from the Xing'an Rhododendron by the present invention are all Ascomycota after morphological and molecular identification, which is consistent with the results of Jia Rui's separation and identification of Xing'an Rhododendron in Greater Khingan Mountains. However, a very typical azalea mycorrhizal fungi, Oidiodendron fungi, was found in the study, which was not separated. It is speculated that it is related to the location and time of sample collection, which may be caused by different habitats or host plants. This difference is manifested in the types of symbiotic fungi. Some fungi may be more inclined to grow in specific locations or times, while others may be more adapted to different environmental conditions. Therefore, for the phenomenon of differences in the collected samples, this difference also provides valuable information for studying and understanding the distribution and ecological functions of fungi.

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

[0298] In the present invention, since the inoculation experiment is carried out with tissue culture seedlings, it is found that compared with the uninoculated bacteria, the inoculation of Pezicula ericae XA4 has a growth-promoting effect on the tissue culture seedlings of Dahurian rhododendron, but further verification is still needed on the seedlings. After the strains are inoculated, strains that can promote the growth and development of Dahurian rhododendron are screened out, but the stress resistance, seed germination rate, and cutting survival rate of Dahurian rhododendron are not tested, and further research is needed. When the growth-promoting ability of four mycorrhizal fungi is measured, it is found that the four fungi have no phosphorus solubilizing ability, but can all produce IAA, which may be that the fungi promote the growth and development of Dahurian rhododendron plants by producing plant hormones.

[0299] 4 Conclusion

[0300] The present invention selects the concentrated distribution sites of wild Dahurian rhododendron, randomly samples and detects the symbiotic mycorrhiza, uses trypan blue staining to study the morphological structure and infection characteristics of the mycorrhiza; separates and purifies the mycorrhizal fungi and uses a combination of morphological identification and molecular biological identification to determine the type of mycorrhizal fungi; screens and re-grafts Dahurian rhododendron tissue culture seedlings again to detect the growth-promoting effect of mycorrhizal fungi. The main research conclusions are as follows:

[0301] (1) Trypan blue staining results show that the mycorrhizal morphology of Rhododendron dahurica is relatively rich, but there are 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 thick hyphae form dense hyphal nodes, showing the typical structural characteristics of Rhododendron mycorrhizal fungi, and vesicles appear 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. Through paraffin section observation, it was found that the mycorrhizal structure is very simple, consisting of a layer of epidermal cells and a narrow central column surrounded by one or two layers of cortical cells. Mycorrhizal fungal infection generally does not change the anatomical structure of the hairy roots. Scanning electron microscopy observation found that the surface of the hairy roots of Rhododendron dahurica sampled in the field was covered with a layer of loose or dense hyphae.

[0302] (2) The roots of Rhododendron xing'anensis 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 third-level 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 ability and IAA secretion ability of four mycorrhizal fungi were measured, and the dominant fungus Pezicula ericae XA4 was selected and analyzed for back-grafting on sterile cultured Dahurian Rhododendron seedlings. Trypan blue staining was used to detect that all mycorrhizal fungi could be back-grafted successfully. Compared with the uninoculated ones, 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 ones.

[0305] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

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

2. Use of XA4 described in claim 1 in infecting the roots of Rhododendron dahurica.

3. Use of XA4 according to claim 1 in phosphate dissolution.

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

5. Use of the XA4 described in claim 1 in promoting the growth of Rhododendron dahurica.

6. Use of XA4 described in claim 1 in improving the growth rate of Rhododendron dahurica.

7. Use of the XA4 described in claim 1 in promoting the growth of Rhododendron dahurica leaves.

8. Use of the XA4 described in claim 1 in promoting the root growth of Rhododendron dahurica.

9. Use of the XA4 described in claim 1 in enhancing the photosynthesis of Rhododendron dahurica.

10. Use of the XA4 described in claim 1 in improving the root activity of Rhododendron dahurica.

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