Stiltaria italica, microbial inoculum prepared from same and application of microbial inoculum

Abstract

The invention discloses a Pleurotus citrinopileatus, a microbial agent prepared from the Pleurotus citrinopileatus and application of the Pleurotus citrinopileatus, and the Pleurotus citrinopileatus strain is named as Pleurotus citrinopileatus HSL and preserved in the China General Microbiological Culture Collection Center; the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation time is November 18, 2024; the preservation number of the strain is CGMCC No.41599. The Pleurotus citrinopileatus strain HSL provided by the invention can significantly promote root development of poplar seedlings, has a significant promotion effect on nitrogen and phosphorus absorption of seedlings, and can be used for efficient seedling raising of poplar.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular, to a Clitopilus, a bacterial agent prepared therefrom, and its application. Background Art

[0002] In the long-term evolution process, in order to adapt to the harsh natural environment, the roots of forest trees have established an inseparable symbiotic relationship with soil fungi. Forest trees provide carbon sources for soil fungi through photosynthesis, and soil fungi can promote the nutrient absorption, growth and development of plants, and improve the growth adaptability of plants. As the importance of symbiotic fungi in plant growth adaptability is increasingly recognized, more and more scientists believe that plants mainly exist in nature in the form of a "plant-symbiotic fungi" community with a shared destiny. In forest ecosystems, there are various types of root symbiotic fungi, such as mycorrhizal fungi, endophytic fungi, etc. They affect plant growth through various ways, such as directly promoting plant nutrient absorption through the hyphal pathway and indirectly promoting plant growth by regulating the expression pattern of root genes. Due to their high species diversity and functional diversity, root symbiotic fungi have great application potential in agricultural and forestry production. At present, the development of root symbiotic fungi is only limited to arbuscular mycorrhizal fungi and ectomycorrhizal fungi, and the development of the application potential of root endophytes, especially the root endophytes of native tree species, is still very lagging.

[0003] As China transforms from a major forestry country to a strong forestry country, this puts forward higher requirements for scientific and technological support for the quality improvement and efficiency increase of the forestry industry. The efficient breeding of forest trees is the main battlefield for forestry scientific and technological research. At present, some studies have created new forest tree germplasm resources through molecular improvement techniques and other means, which have promoted the efficient breeding of forest trees to a certain extent. However, limited by the fact that the stress resistance traits of plants are controlled by multiple quantitative trait genes (QTLs), the progress of molecular breeding research is slow. Therefore, it has become an urgent task to find more efficient, more economical and more environmentally friendly key technologies to solve the problem of efficient breeding of forest trees. Soil symbiotic bacteria are the core link connecting soil fertility and productivity. Forest soils contain rich symbiotic fungi, which play an important regulatory role in improving the growth adaptability of seedlings. However, the functional development research on these symbiotic fungi has not been systematically carried out yet. Deeply exploring the application potential of new forest soil symbiotic fungi helps to provide new solutions for the efficient breeding of forest trees in the direction of efficient cultivation of forestry biological resources.

[0004] Previous studies by this research team have shown that Clitopilus hobsonii QYL-10 can promote the root development and the absorption and accumulation of potassium elements in seedlings of Liquidambar styraciflua, but this strain has no contribution to the nitrogen and phosphorus absorption of seedlings. Although Clitopilus hobsonii XST (deposit number CGMCC No. 41176) has a promoting effect on the seed germination of Cymbidium pendulum, a orchid plant, its growth-promoting effect is average. Summary of the Invention

[0005] The present invention provides a Clitopilus sp. named HSL. Its taxonomic name is Clitopilus sp. This strain is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; the address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation time is November 18, 2024; the preservation number is CGMCC No. 41599.

[0006] The present invention also provides a bacterial agent prepared from the aforementioned Clitopilus sp. strain HSL. Specifically, the bacterial agent is a fungal block of the Clitopilus sp. strain HSL.

[0007] The present invention also provides an application of the aforementioned Clitopilus sp. or bacterial agent in promoting plant growth.

[0008] Specifically, the application is to place the mycelium or bacterial agent of the strain HSL on the surface of the plant root cultivation substrate or apply it to the cultivation substrate;

[0009] The plant is preferably Populus; more preferably, the plant is hybrid clone NL895 Poplar (Populus deltoids × P. euramericana) and Populus alba.

[0010] The promotion of plant growth is manifested in promoting the root development of plants and the absorption of nitrogen and phosphorus.

[0011] The beneficial effects of the present invention include: The Clitopilus sp. strain HSL provided by the present invention can significantly promote the root development of Populus seedlings and has a significant promoting effect on the absorption of nitrogen and phosphorus by Populus, and can be used for the efficient seedling raising of Populus. Brief Description of the Drawings

[0012] Figure 1 It is a culture characteristic diagram of strain HSL. Among them, A is the front side after culturing on PDA medium for 7 days; B is the back side after culturing on PDA medium for 7 days;

[0013] Figure 2 It is a phylogenetic relationship diagram of the used Clitopilus fungi;

[0014] Figure 3 It is the growth and development of NL895 Poplar seedlings after inoculating different Clitopilus fungal strains;

[0015] Figure 4 It is the influence of inoculating different Clitopilus fungal strains on NL895 Poplar seedlings;

[0016] Figure 5 It is the growth and development of Populus alba seedlings after inoculating different Clitopilus fungal strains;

[0017] Figure 6 Effect of inoculating different Clitopilus fungal strains on the roots of Populus alba seedlings.

[0018] Biological preservation information

[0019] The Clitopilus sp strain HSL is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms; the address of the preservation unit is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is November 18, 2024; the preservation number is CGMCC No. 41599. Specific implementation manners

[0020] The present invention will be further described and illustrated below in conjunction with embodiments. However, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0022] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0023] Example 1: Isolation and culture of strain HSL

[0024] 1. Preparation of isolation and culture media

[0025] Hagem medium: The preparation method of Hagem medium is 0.5 g / L NH4Cl, 1 g / L KH2PO4, 0.5 g / L (NH4)2HPO4, 1 g / L MgSO4·7H2O, 5 g / L glucose, 100 mg / L vitamin B1 solution, 20 mg / L benomyl (antifungal agent), and 10 g / L agar. After autoclaving the Hagem medium, 50 mg / L streptomycin sulfate and 20 mg / L tetracycline hydrochloride are added to the warm medium to inhibit bacterial growth.

[0026] PDA medium: 200 g of potatoes, peeled and cut into small pieces, boiled with ultrapure water until mushy, filtered through four layers of gauze, added with 20 g of glucose and 15 g of agar, and made up to 1 L. Autoclaved at 121 °C under high pressure for 25 min. The medium is cooled to 45 °C at room temperature, and streptomycin sulfate and tetracycline hydrochloride with final concentrations of 0.05 g / L and 0.02 g / L are added to inhibit bacterial contamination.

[0027] 2. Isolation and culture of the strain involved in the present invention

[0028] Fresh root samples of Quercus stewardii Rehder from Anqing, Anhui (N 31°06', E 116°18') were washed under distilled water and surface disinfected by soaking in 5% (v / v) H2O2 for 4 minutes. Then they were rinsed at least three times with sterile distilled water in 50 mL sterile centrifuge tubes (BD Falcon; BD Biosciences, San Jose, California, USA). Tissue blocks 2 - 3 mm long were cut from the root tips with a sterile scalpel and transferred onto the surface of Hagem medium. They were cultured in the dark at 26°C for 7 days and observed in a timely manner. When mycelial growth was observed on the medium surface, they were transferred to PDA medium for enrichment and purification until pure colonies were obtained. Under normal culture conditions, the growth of the strain was as Figure 1 shown.

[0029] Example 2. Molecular biological identification of strain HSL

[0030] 1. Extraction of strain DNA and PCR amplification

[0031] The pure cultured fungal mycelia obtained in step 2 of Example 1 were picked into a 1.5 ml sterile centrifuge tube, and the fungal DNA was extracted using the DNeasy Plant Mini Kit (Qiagen). Using the DNA as a template, gene fragments such as the ribosomal 28S large subunit (LSU), internal transcribed spacer sequence (ITS), RNA polymerase second large subunit (RPB2), and β - tubulin (TUB2) were amplified. The total volume of the amplification system was 25 μL: 1 μL of template DNA, 0.5 μL each of forward and reverse primers, 0.2 μL of Taq enzyme, 2 μL of dNTP, 2.5 μL of Buffer, and 18.3 μL of sterile ultrapure water. The PCR amplification program included: pre - denaturation at 94°C for 4 min; 35 cycles, including pre - denaturation at 94°C for 40 s, annealing at 55°C for 50 s, and extension at 72°C for 1 min; and final extension for 10 min.

[0032] 2. Sequence determination and species identification

[0033] After amplification and sequencing, the obtained strain HSL was confirmed to be Clitopilus sp. by comparison with the existing sequences in the NCBI database. This strain was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the deposit number CGMCC No. 41599. Orthofinder was used for clustering, and a total of 4,593 single-copy gene families were found in 6 species. Genes with an amino acid length of less than 100 bp were filtered out, and 2,798 were left for tree construction. First, the amino acid sequences were aligned using muscle v3.8 software, and the amino acid alignment was converted into a nucleic acid multiple sequence alignment according to the codon correspondence. Then, the trimal software was used to set -automated1 to filter the multiple sequence alignment results. The multiple sequence alignments of all single-copy families were concatenated and used with Raxml software to construct a phylogenetic tree, and the results are as Figure 2 shown.

[0034] Example 3: Verification of the root growth promotion effect of strain HSL on the hybrid excellent clone NL895 poplar (Populus deltoides×P.euramericana) and Populus alba

[0035] 1. Cultivation of sterile seedlings of NL895 poplar or Populus alba

[0036] A large number of high-quality sterile poplar seedlings were obtained in a short period by tissue culture rapid propagation. The explant materials selected for the tissue culture experiment were the young stems of tissue culture seedlings of poplar tree species from the same family and the same genetic background. After ultraviolet disinfection in the laminar flow hood, the young stems (with leaves) of poplar tissue culture seedlings were cut and transferred to the improved MS medium (the formula is shown in Table 1), making the tissue culture seedlings stand upright and trying to avoid the leaves touching the medium. 3 - 4 young stems of poplar were cut and inserted into each tissue culture bottle, the lid was covered, and then transferred to the tissue culture room. The temperature was 25±3°C, the humidity was 60 - 70%, and the photoperiod was 12 h. After 7 - 10 days of seedling cultivation, after rooting, sterile poplar seedlings with a root system and a plant height of 5 - 6 cm were selected, and seedlings with complete and relatively consistent root systems were picked in the laminar flow hood for co-culture experiments.

[0037] Table 1 Formula of improved MS rooting medium

[0038]

[0039]

[0040] 2. Preparation of Clitopilus fungi and strain HSL

[0041] The fungi of the genus Clitopilus, namely Clitopilus hobsonii QYL-10 (isolated from the roots of Quercus lyrata and preserved in our laboratory, hereinafter abbreviated as QYL-10), Clitopilus hobsonli XST-100 (isolated from the roots of Cymbidium Golden and preserved in our laboratory, hereinafter abbreviated as XST), Clitopilus hobsonli NL-19 (isolated from Quercus michauxii and preserved in our laboratory, hereinafter abbreviated as NL-19), Clitopilus scyphoides 50334 (isolated from the air in the Chinese Academy of Forestry and preserved in the China Forestry Microbial Culture Collection Center, strain number CFCC50334, hereinafter abbreviated as 50334), and the strain HSL of the present invention were cultured on PDA solid medium for 7 days. Then, plugs with a diameter of 5 mm were punched from the edges of the pure culture fungal plates using a borer and reserved for use.

[0042] 3. Co-culture of the fungal strains of the genus Clitopilus with the sterile seedlings of Populus × euramericana 'NL895' or Populus alba

[0043] Select relatively uniform sterile seedlings of Populus × euramericana 'NL895' or Populus alba and transfer them to a large petri dish (diameter 150 mm) containing 100 ml of solid medium, with 3 seedlings per dish, and 3 replicates for each treatment. The formula of the solid medium is as follows: add 100 ml of potato juice, 1 g of sucrose, 1 g of glucose, 0.33 g of NH4N O 3, 0.38 g of KNO3, 0.272 g of KH2PO4, 0.088 g of CaCl2·2H2O, 0.074 g of MgSO4·7H2O, 400 μl of 1× trace elements, 400 μl of 1× iron salt solution, add vitamin B1 with a final concentration of 100 μg / ml, and 10 g of agar; adjust the pH to 5.8 and make up the volume to 1 L. Autoclave at 121 °C for 25 min.

[0044] After culturing for 7 days under the same conditions, different Clitocybe fungal blocks obtained by punching were placed on the surface of the solid co-culture medium. Wait for the mycelium to grow radially and cover the roots of poplar seedlings (the fungi grow very fast and the culture containers are very small, so they can cover quickly) to complete inoculation. Use inoculating sterile PDA as the control treatment (Control), 5 fungal blocks per dish. After inoculation, in the light incubator, culture at 25 °C, 12 h light / 12 h dark for 14 days, observe the growth and development of poplar seedlings. At the same time, use a ruler to measure the plant height of poplar seedlings, use a vernier caliper to measure the stem diameter, use a balance to measure the root biomass and fresh weight of the stem and leaf parts, detect the N and P contents, and use the root scanning software (WinRHIZO Pro, Regent Instruments, Canada) to determine the total root surface area and root volume of each seedling. The results are as Figures 3 - 6 shown.

[0045] From Figure 3 and Figure 5 it can be seen that compared with the control group and the other Clitocybe fungi, the strain HSL of the present invention significantly promoted the growth and development of NL895 poplar and silver poplar seedlings. From Figure 4 it can be seen that compared with not inoculating Clitocybe fungi, inoculating the strain HSL of the present invention can significantly increase the plant height, fresh weight of the stem and leaf parts, stem diameter, root biomass and root volume of NL895 poplar seedlings; compared with inoculating other Clitocybe fungi, inoculating the strain HSL of the present invention can significantly increase the plant height, fresh weight of the stem and leaf parts, stem diameter, root biomass, root volume, total root surface area and N and P contents of NL895 poplar seedlings. In addition, from Figure 6 it can be seen that compared with not inoculating or inoculating other Clitocybe fungi, inoculating the strain HSL of the present invention can significantly increase the plant height, root biomass, stem diameter, fresh weight of the stem and leaf parts, total root surface area, root volume and N and P contents of silver poplar seedlings. Thus, it can be seen that the Clitocybe strain HSL provided by the present invention has a significant growth-promoting effect on poplar seedlings, especially better on silver poplar.

Claims

1. A Clitopilus sp., characterized in that: The Agaricus serrata strain is named Agaricus serrata HSL, and the strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration; the address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit time is November 18, 2024; the deposit number is CGMCC No.41599.

2. A bacterial agent prepared from the HSL strain of Agaricus oleifera according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is the HSL fungal block of the Agaricus serrata strain.

4. Use of the Agaricus bisporus described in claim 1 or the bacterial agent described in any one of claims 2 to 3 in promoting plant growth.

5. The use according to claim 4, characterized in that: The application is to place the mycelium of the strain HSL or the bacterial agent on the surface of the cultivation substrate of the plant roots or apply it into the cultivation substrate.

6. The use according to claim 4, characterized in that: The plant is poplar.

7. The use according to claim 6, characterized in that: The plant is a hybrid clone NL895 poplar or Populus alba.

8. The use according to claim 4, characterized in that: The promotion of plant growth is manifested in promoting the development of plant root systems and the absorption of nitrogen and phosphorus.

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