Mycorrhizal fungi strain efficiently growing with rhododendron seedling

[0013] Example 1 Isolation and identification of rhododendron mycorrhizal fungi:

[0014] 1. Isolate the root system material: Take the living roots of the azalea species'Purple Crane' in Shanghai Binjiang Forest Park, put them in an ice bucket together with soil moisture, and store them in a refrigerator at 4°C until use.

[0015] 2. Separation and identification of culture medium:

[0016] (1) Separation medium: use Martin-Bengal Red Medium (referred to as MA) to adjust and improve its pH. The formula is: 10 grams of glucose, 5 grams of tryptone, 1 gram of dipotassium hydrogen phosphate, 0.5 grams of magnesium sulfate, 0.033 grams of Bengal red, 20 grams of agar, distilled water to a constant volume of 1000 ml, adjust the pH to 5.0, and then high pressure at 121°C Sterilize for 20 minutes, add 0.03 g of streptomycin when cooled to below 60°C, mix well, and pour the plate for use.

[0017] (2) Strain identification medium: use malt extract medium (MEA), the formula is: malt extract 20g, tryptone 1g, glucose 20g, agar 20g, distilled water to 1000ml, then high pressure at 121℃ Sterilize for 20 minutes and add 0.03 g of streptomycin when cooled to below 60°C.

[0018] (3) Mycelium identification medium (PDA medium): The formula is: 1000 grams of potato juice with a weight content of 20%, 20 grams of glucose, 0.6 grams of magnesium sulfate, 0.6 grams of potassium dihydrogen phosphate, and vitamins. B1 is 0.2 g, 20 g agar, and the pH of the medium is 5.0.

[0019] 3. Separation and morphological identification methods:

[0020] Isolation of mycorrhizal fungal strains: Take out the living roots of the azalea species'Purple Crane' from the refrigerator, gently rinse off the soil with tap water, and pick healthy living roots. After cleaning, rinse with 72% alcohol, wash in 10% household 84 disinfectant for 15~20 minutes, rinse with sterile water 3~5 times, cut into 0.3~0.5 cm roots, and place In the petri dishes of MA medium, 5 root segments are placed in each petri dish, and then placed in a 25°C incubator in the dark for 2 to 4 weeks to cultivate a total of 100 root segments.

[0021] Observation and identification of colony morphology and microscopic characteristics of mycelium: When the colony grows from the root segment, pick the colony with a toothpick to MEA medium, and continue the colony morphology identification. Each colony has 6 plaques. After 2 weeks of culture, observe the uniformity and uniformity of the colony morphology, and record the basic characteristics of the colony. If there is a difference in colony morphology, perform a second separation and identification. Strains with stable colony morphology were picked with a toothpick and cultured in the dark in a 22°C incubator on a PDA plate for 2 weeks, and then incubated in the dark in a 4°C incubator for 2 weeks. The mycelial morphology and spore shape were observed under an optical microscope at 100~400 times. . The incubator used in this example is Saifu constant temperature and humidity incubator HWS-350, and the optical microscope is Leica DM2000.

[0022] 4. Molecular identification method:

[0023] Collection of hyphae: MEA culture medium activated strains, inoculated into MEA liquid medium and shaken for 10-15 days (150rpm, 25℃), filter paper to collect hyphae for genomic DNA extraction. The CTAB method was used for total DNA extraction.

[0024] PCR amplification of ITS segment: rDNA ITS segment amplification uses universal primers ITS1 (5'TCC GTA GGT GAA CCT GCG G 3'), ITS4 (5'TCC TCC GCT TAT TGA TAT GC 3'). The composition of the PCR reaction system is 10×Buffer 5μl 25μmol/LM g2+3μl, 10mmol/L dNTP 1μl, 10μmol/L primers 1μl, template DNA 1μl (final concentration around 50ng/l), Taq enzyme 1U (0.25μl), Make up to 50μl with sterile pure water. The PCR reaction was performed using a BIORAD DNA Engine PCR instrument, and the cycle parameters were: pre-denaturation 95°C, 2min, denaturation 94°C, 40s, annealing 60°C, 40s, extension 72°C, 45s; fill-up 72°C, 5min. A total of 35 cycles were performed.

[0025] Product purification and sequencing: After the PCR product is purified by the kit, the vector is PMD18~T conversion connection, the sequencing primer is M13R (~48), and the sequencing instrument ABI 3730Xl is used for sequencing (sequencing is done by Shanghai Yingjun Biotechnology Co., Ltd.) . The obtained sequences were compared online using PRIMER3 software, supplemented by manual proofreading, to determine the reliability of the sequence. The obtained sequence is BLAST on NCBI, and the database used is GenBank+EMBL+DDBJ+PDB.

[0026] 5. Appraisal results:

[0027] Morphological characteristics of the strain: the colony of the strain of the present invention is off-white to brown tapetum-like, round, slightly raised in the center, 2.0~2.5cm in diameter; hyphae in diameter 2.0~3.0μm, conidia elliptical, in size 2.5~3.5 μm*1.5~2.0μm, with conidiophore, length 80~120μm. (Such as figure 1 with 2 Shown)

[0028] 6. Molecular identification results:

[0029] ITS sequence: as shown in SEQ ID NO:3.

[0030] The sequence was compared by BLAST, and the similarity with the ITS sequence of Oidiodendron sp.shylm72 and Oidiodendron maius isolate TR088 was 99%. The closest species was the fungus of Oidiodendron after ITS sequence alignment, which was identified as a new rhododendron. Mycorrhizal fungus strain, the preservation number is CGMCC No: 6010.

[0031] Example 2

[0032] The strain of the present invention is isolated from the root system of the azalea species'Zihe' in Shanghai Binjiang Forest Park. It belongs to the genus Oidiodendron OBJF31. It can be cultured on a flat plate or in a liquid. The MEA medium is used. The formula is: Malt The extract is 20g, tryptone 1g, glucose 20g, the pH value of the culture medium is 5.1, and the culture temperature is 22~24℃.

[0033] The macroscopic characteristics of the strain culture are: the colony grows slowly, initially grayish white, after 10 days, the color of the center of the colony becomes darker and brown, the texture of the colony is tapetum-like, round, and the center is slightly raised. The colony diameter is 2.0~2.5 for 14 days of plate culture. cm. Microscopic characteristics: The hyphae are 2.0~3.0μm in diameter, with single-celled conidia, oval, smooth surface, size 2.5~3.5μm*1.5~2.0μm, with conidiophore, length 80~120μm. The closest species in the ITS sequence alignment of the strains is Oidiodendron fungus, which is consistent with the identification results of mycelial morphological characteristics. The strain is identified as Oidiodendron fungus, and the deposit number is CGMCC No: 6010.

[0034] Rhododendron fortunei seedlings cultured aseptically for 9 weeks, about 2~4cm high, were inoculated with strain OBJF31 and the same type of excellent strains selected in 4 literatures (Zhang Chunying et al., 2010; Yu Fang et al., 2008), and no inoculation control . Holes were punched on both sides of the root system of the seedlings, and one circular bacterial block with a diameter of about 0.5 mm (preservation number: CGMCC No.6010) was buried in each. 30 seedlings were inoculated for each strain, and 30 seedlings were not inoculated. Seedlings Placed in a culture room for cultivation at a temperature of 25°C. After 20 weeks of inoculation treatment, the seedling infection rate and biomass were tested. The infection rate, average seedling height, average biomass and survival rate of inoculated seedlings of strain OBJF31 were higher than those of other strains inoculated seedlings and control seedlings (Table 1).

[0035] Table 1 Seedling growth performance indicators after 20 weeks of inoculation with different strains

[0036]

[0037] Example 3

[0038] Rhododendron fortunei seedlings cultured aseptically for 9 weeks, about 2 to 4 cm in height, were transplanted in sterilized substrates with different pH values. The method of adjusting the pH value of the cultivation substrate: Based on the original pH value of the cultivation substrate, add a certain amount of CaCO according to the volume ratio of the substrate 3 (Adding ratio is according to 0, 3, 5kg/m 3 ), adjust the pH value of the cultivation substrate to 5.5, 6.8, 7.2 respectively. The seedlings were inoculated after transplanting, and the inoculated strains were OBJF31 (preservation number: CGMCC No. 6010) and HS04. The inoculation method and seedling culture conditions were the same as those in Example 1. Each treatment was inoculated with 30 plants, and a non-inoculated control was set. After 20 weeks of inoculation, the infection rate and biomass of the seedlings were tested (Table 2). The biomass and survival rate of the inoculated seedlings of strain OBJF31 in the three pH matrixes were higher than those of the inoculated seedlings of strain HS04 and the control non-inoculated Seedlings, and the leaves are dark green.

[0039] Table 2 Effects of different strains in different pH substrates on the growth of Rhododendron fortunei seedlings

[0040]