CN117965324A - Russula YAFMF006, separation method thereof and mycorrhizal seedling infection method - Google Patents
Russula YAFMF006, separation method thereof and mycorrhizal seedling infection method Download PDFInfo
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a russula YAFMF006, a separation method thereof and a mycorrhizal seedling infection method, and relates to the technical field of agricultural planting. The preservation name of the russula vinosa YAFMF006 is Russula VIRESCENS YAFMF006; deposited in China center for type culture Collection, accession number: CCTCC M20232693 and russula vinosa YAFMF006 realize the infection of russula vinosa to the root bacteria of the pinus pinnatifida by inoculating the bacterial blocks to the root of the pinus pinnatifida seedlings for co-cultivation. The invention obviously improves the single infection rate of the russula vinosa, promotes the synthesis of the pinus massoniana seedlings and the mycorrhizal of the russula vinosa, and lays a foundation for the formation of fruiting bodies of the russula vinosa in the next step.
Description
Technical Field
The invention relates to the technical field of agricultural planting, in particular to russula YAFMF006, a separation method thereof and a mycorrhizal seedling infection method.
Background
Pinus koraiensis (Pinus kesiya var. Langbianensis) is a Pinaceae (PINEACEAE) Pinus (Pinus) plant, is a geographical variant of Pinus koraiensis (Pinus kesiya), and is naturally distributed in the humid and semi-humid areas of the southern and quasi-tropical regions of Yunnan in China. The tree species has the characteristics of excellent materials, fast growth, long fibers, high rosin yield and the like, is one of excellent rural tree species in the Yunnan hot zone, plays an important role in regional forestry, and has obvious economic value, forest ecological service function and carbon sink benefit. However, the breeding mode of pinus massoniana has some problems including low survival rate of seedlings, poor transplanting adaptability, slow growth and the like, and limits the development of artificial forests.
Pinus koraiensis belongs to a typical symbiotic tree species of ectomycorrhizal fungi (Ectomycorrhizal fungi, ECF) with a high dependency, which may play a synergistic role in their nutritional metabolism. ECF has ecological effects of maintaining stability and diversity, promoting plant rhizosphere nutrient circulation, restoring ecological vegetation, improving soil and the like in a ecological system, and has potential application value in breeding and cultivation management. The mycorrhiza technology is widely applied to agriculture and forestry production and environmental protection, and is mainly used for introducing, raising seedlings, forestation, preventing and controlling seedling root diseases, producing edible fungi and the like, and forming a seedling raising forestation-mycorrhiza edible fungi mode. The mode selects proper tree species and precious edible mycorrhizal fungi symbiotic with the tree species before forestation, and artificially synthesizes mycorrhizal fungi, thereby realizing the transformation from single forestation to seedling forestation and precious mycorrhizal fungi as main materials, not only increasing the economic income, but also fully playing the advantages of mycorrhizal symbiotic relationship. At present, mycorrhizal fungi become an important factor for forestation, and not only are the mycorrhizal fungi unique in edible and medicinal values, but also the growth of trees can be promoted, the yield can be increased, and the stress resistance of the trees can be enhanced.
Russula vinosa (Russula virescens) belongs to russulaceae, russula genus, and is distributed in the provinces of black longjiang, sichuan, yunnan, guizhou and the like in China. The strain forms exogenous mycorrhiza with broad-leaved tree species and conifer species such as Yunnan pine. The russula in russula has symbiotic relationship with trees such as Betula (Betula sp.), beech (Fagus sylvatica), aspen (Populus tremula) and chestnut (Quercus sp.) to form ectomycorrhizas. The ectomycorrhizal fungi can resist pathogenic bacteria by producing chemical inhibitors, forming mycorrhizal rhizosphere microbial communities with protective effect, enhancing nutrient absorption and the like, so that the disease resistance of plants is improved, the survival rate of seedlings is enhanced, the quality of artificial forests is improved, the stability of forests is enhanced, the wood reserves are improved, ecological benefits are brought into play, and a foundation is laid for efficient directional cultivation of pinus massoniana and industrialized cultivation of mycorrhizal edible fungi.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the russula YAFMF006, the separation method and the mycorrhizal seedling infection method thereof, so as to improve the survival rate of the pinus massoniana seedlings and the yield of mycorrhizal edible fungi.
In order to achieve the above object, the technical scheme of the present invention is realized by the following technical scheme:
The russula vinosa YAFMF006, russula vinosa YAFMF06 belongs to russula vinosa Russula virescens, and the preservation name is russula vinosa YAFMF006 Russula VIRESCENS YAFMF006; the Chinese culture medium is preserved in China center for type culture Collection, and the preservation address is university of Wuhan; preservation date: 2023, 12, 27, deposit number: cctccc M20232693.
Preferably, the russula-emitting diode YAFMF006 gene sequence comprises a nucleotide sequence shown as SEQ ID No. 1.
The separation method of the russula-emitting YAFMF006 comprises the following steps:
s1-1, collecting fruiting body samples of the russula vinosa, and carrying out disinfection and crushing treatment to obtain tissue crushing liquid;
S1-2, diluting the tissue disruption solution, coating the tissue disruption solution on a PDMYKAS flat plate, culturing in the dark for 15 days, separating bacterial colonies, and identifying to obtain russula vinosa;
S1-3, transferring the identified mycelium of the russula vinosa to PDMY solid culture medium for continuous culture for 20d, extracting mycelium DNA, and carrying out molecular identification on the strain to obtain the russula vinosa schrader YAFMF006.
Preferably, the PDMYKAS plate has the formula: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose, 100 mu g/ml of calicheamicin, 100 mu g/ml of ampicillin, 100 mu g/ml of streptomycin and 10g/L of agar.
Preferably, the PDMY solid medium has the formula: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose and 16g/L of agar.
The mycorrhizal seedling infection method of the russula vinosa YAFMF006 is that the russula vinosa YAFMF006 carries out mycorrhizal seedling infection on the pinus massoniana, and the specific method comprises the following steps:
S2-1, strain preparation: transferring the russula mycelium to PDMY solid culture medium, culturing for 20d, identifying to obtain russula YAFMF006 bacterial colony, and obtaining russula YAFMF006 standby bacterial block;
S2-2, cultivation of mycorrhizal seedlings: obtaining pinus massoniana seeds with sprouted young roots, placing the pinus massoniana seeds in a tissue culture bottle containing a culture medium, simultaneously inoculating standby fungus blocks around the root parts of the sprouted seeds, enabling the inoculated fungus blocks to be consistent with the sprouted seeds in number, and sealing the culture room for culture to obtain fungus root seedlings;
S2-3, transplanting seedlings: filling the seedling substrate subjected to autoclaving into a standby seedling tray, and transplanting mycorrhizal seedlings into the seedling tray for planting.
Preferably, the formula of the PDMY solid medium in the step S2-1 is as follows: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose and 16g/L of agar.
Preferably, the root length of the pinus massoniana seed growing out of the root obtained in the step S2-2 is 5-10mm.
Preferably, the composition of the culture medium in each tissue culture flask in the step S2-2 is as follows: 10.5g of rice, 9g of raw soil, 1.5g of turf, 0.3g of monopotassium phosphate, 0.3g of magnesium sulfate, 0.3g of calcium sulfate, 0.6g of glucose and 55mL of tap water.
Preferably, the seedling substrate in the step S2-3 is a mixture of vermiculite, humus soil, perlite and organic fertilizer.
The invention provides a russula YAFMF006, a separation method thereof and a mycorrhizal seedling infection method, which have the advantages that compared with the prior art:
The invention has the advantages that a new strain russula viridis YAFMF is preserved, the strain preparation and mycorrhizal seedling cultivation under the aseptic condition are comprehensively utilized, and the successful inoculation and aseptic matrix transplanting are combined, so that the single infection rate of russula viridis is remarkably improved, the mycorrhizal synthesis of pinus koraiensis seedlings and russula viridis is promoted, and a foundation is laid for the formation of russula viridis fruiting bodies in the next step. Meanwhile, after the pinus koraiensis seedlings and the russula vinosa are formed into mycorrhizas, the growth index of the pinus koraiensis seedlings can be remarkably improved, so that excellent pinus koraiensis seedlings are cultivated. In addition, the invention adopts new strains for cultivation, has high infection rate and high transplanting survival rate, and has obvious economic benefit.
Drawings
FIG. 1 is a diagram showing the growth of russula mycelium in example 1 of the present invention;
FIG. 2 is a phylogenetic tree display diagram of Pleurotus nebrodensis constructed based on ITS in example 1 of the present invention;
FIG. 3 is a schematic diagram showing the inoculation and cultivation of pinus massoniana bacterins and russula vinosa in example 2 of the present invention;
FIG. 4 is a schematic diagram showing the growth of seedlings inoculated with 10d of the pinus massoniana seeds and the russula vinosa fungus pieces in example 2 of the present invention;
FIG. 5 is a graph showing the root infection of the seedlings of example 2 inoculated with 10d of the seed of Pinus massoniana and the fungus block of Pleurotus citrinopileatus (right side of the figure) compared with the root of the seedlings of Pinus massoniana of the control group (left side of the figure);
FIG. 6 is a schematic diagram showing the growth of seedlings inoculated with the seed of Pinus massoniana and the russula vinosa in example 2 of the present invention for 20 d;
FIG. 7 is a schematic diagram showing the infection of the root of the seedling (3 rd and 4 th root seedlings from the left of the figure) inoculated with the seed of Pinus massoniana and the russula vinosa in example 2 of the present invention, and the root of the seedling of Pinus massoniana planted in the control group (1 st and 2 nd root seedlings from the left of the figure);
FIG. 8 is a schematic diagram showing the growth of seedlings inoculated with the seed of Pinus massoniana and the russula vinosa in example 2 of the present invention for 30 d;
FIG. 9 is a schematic diagram showing the infection of the root of the seedling inoculated with 30d of the seed of Pinus massoniana and the russula vinosa in example 2 of the present invention;
FIG. 10 is a schematic view of the root of the seedling of Pinus massoniana wrapped with the mycelium of Pinus viridis of Pinus massoniana seed and Pinus viridis of the inoculation 30d in example 2 of the present invention;
FIG. 11 is a graph showing the comparison of the infection of the root of seedlings (mycelium wrapped by the root of the cleaned seedling on the left side of the figure) inoculated with 30d by the seed of Pinus massoniana and the fungus block of Pleurotus ostreatus in example 2 of the present invention with the root of seedlings of Pinus massoniana in the control group (right side of the figure).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
Separation of russula YAFMF 006:
1. Collecting fresh fruiting bodies of russula vinosa with the maturity of 70% -80% and fresh fruiting bodies of russula vinosa without diseases and insect pests as samples, cleaning the collected fruiting body samples of russula vinosa, washing with flowing water 12 h, cutting a stipe into small pieces of 2cm 2, washing with sterilized water (500 ml), soaking in 75% ethanol for 1min, rinsing with sterilized water for 4 times, rinsing for 1min each time, sucking excessive water on the surface with sterilized filter paper, and airing for later use;
2. Selecting a small tissue at the junction of a sprout cover and a stipe or at a fungus pleat of a fresh fruiting body of russula vinosa, cutting the tissue into tissue blocks with the length of about 0.5cm 2 to be used as culture tissue blocks, placing the tissue blocks into a centrifuge tube, adding small steel balls and 500 mu l of sterilized water, and crushing for 1min (180 rpm) to obtain russula vinosa schradson broken tissue liquid;
3. The russula vinosa crushed tissue fluid is diluted by 100 times and coated on 10 PDMYKAS plates (PDMYKAS: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose, 100. Mu.g/ml of caliamycin, 100. Mu.g/ml of ampicillin, 100. Mu.g/ml of streptomycin and 10g/L of agar), cultured for 15 days, colonies are observed and selected, and single colonies are identified, wherein one is identified as russula vinosa;
5. Inoculating mycelium of Pleurotus Eryngii colony into PDMY (fresh potato juice 500 ml/L+yeast extract 1 g/L+malt extract 2.1 g/L+glucose 5 g/L+agar 16 g/L) solid culture medium, and culturing for 20d to obtain colony;
6. Identification of morphology: fungi grow well on PDMY solid culture medium, and the colony is milky white, round, compact in hypha, undeveloped in aerial hypha, thicker in fungus layer and irregularly shaped (as shown in figure 1).
7. DNA extraction:
① CTAB (cetyltrimethylammonium bromide) was heated in a 65℃water bath for 30min prior to the experiment;
② Taking 50mg of dried russula mycelium, adding 3 small steel balls into a 2mL centrifuge tube, soaking the centrifuge tube in liquid nitrogen for 6min, immediately crushing the centrifuge tube by a crusher for 1.5min, adding 1mL of preheated CTAB solution, blowing and mixing the mixture uniformly by a pipetting gun, transferring the mixture into a centrifuge tube filled with 200 mu L of PVP (polyvinylpyrrolidone), suspending the mixture in a fume hood, adding 20 mu L of beta-mercaptoethanol, vibrating the mixture for 15s for full grinding, then placing the mixture into a 65 ℃ water bath kettle for water bath for 1.5h, turning the mixture up and down for 5-6 times every 10min, and centrifuging the mixture at a temperature of 12000r/min and a temperature of 4 ℃ for 10min;
③ Taking 1mL of supernatant into a new centrifuge tube, adding 500 mu L of DNA phenol reagent and chloroform-isoamyl alcohol mixed solution, turning over up and down for 10min, centrifuging (4 ℃ C. 12000 r/min) for 10min (③ steps are repeated twice);
④ Taking 900 mu L of supernatant, placing into a new centrifuge tube, adding 50 mu L of 3mol sodium acetate solution and 900 mu L of 95% absolute glacial ethanol (-20 ℃), shaking, and placing into a refrigerator at-20 ℃ for precipitation for 3 hours;
⑤ Centrifuging (4 ℃ C. 12000 r/min) for 10min after precipitation, discarding supernatant, adding 500 μL 75% alcohol, turning over up and down for 2-3 times, standing for 3min, and discarding supernatant;
⑥ Adding 500 μL of 95% alcohol, turning over for 2-3 times, standing for 3min, centrifuging at room temperature (13000 rpm) for 3min, discarding ethanol, standing and drying;
⑦ Adding 40 mu L of elution buffer EB, and centrifuging at normal temperature (13000 rpm) for 1.5min to obtain the genome DNA of the fungus russula vinosa.
8. ITS analysis and identification:
The fungal rDNA spacer sequence (containing ITS1 region, 5.8S region, ITS4 region) was amplified using fungal universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and the resulting ITS sequencing sequence was as shown in SEQ ID No.1:
CTTCCGTAGGTGAACCTGCGGAAGGATCATTATCGTATAACAGAGATGCCTAGGGCTGTCGCTGACCCTTTATGGGTCGTGCACGCCCAGAGGTGTTCTCTTATGTCCGTCTCACCCCTTTGTGCATCACCGCGTGTGCCTCCCCCCTATTGACTTTGTTGTGAAAGGGCGGGTTCACGTTTTAACACAAACACTATTTTTAATGAGCATGTGTAGAATGTCTTACTTTTTGCGATTATACGCAATCAATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACATAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCACACCCGTTGAGTGTCGTGAACACCCTCAACCTTTTTGGTTTTTTAACCGGGAAGGCTTGGACTTTGGAGGTTTCCTTGCTGGCCTTCCTTTGAAGCCAGCTCCTCCTAAATGAATTAGTGGGGTTTGCCTTGCTGATCCTTGATGTGATAAAGTATGCTTCTTACGTCTTGGGTTTTGAAGGAACCTGCTTCAAGTGGTGTTTGAACAAAGACAATGTTCGAGTTGCATTGCGACTCAAACTCAGCGATCTTGACCTCAAATCGGGTGAGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAATT;
9. construction of developmental trees
Based on ITS phylogenetic tree (fig. 2) using MEGA software to make a russula, the morphological identification and molecular biological identification result of the strain were combined, and the fungus was closest to Russula virescens, so YAFMF006 strain was identified as russula (Russula virescens).
Example 2:
Culturing mycorrhizal seedlings:
1. selecting the culture medium for culturing the bacterial colony in the step 5 in the embodiment 1, shearing, and cutting off bacterial blocks containing the bacterial colony of the russula YAFMF for later use;
2. Selecting uniform and full pinus massoniana seeds stored for 6d at the temperature of minus 28 ℃, washing off ash layers on the surfaces of the seeds by tap water, sterilizing 30 percent of H 2O2 for min times, washing 2 times by sterile water, soaking the seeds in warm water with the initial temperature of 60 ℃ for 24 hours in a refrigerator with the temperature of 4 ℃, sterilizing the seeds for 10 minutes by using 0.1 percent of HgCl 2 under the sterile condition, and washing 5 times by sterile water. Subsequently, soaking for 3min with 75% ethanol, and washing with sterile water for 5 times again;
After autoclaving the filter paper-laid flasks, the treated seeds were placed in the flasks for germination testing. Each culture bottle contains 30 seeds, sterile water is added into the culture bottle to keep moist, meanwhile, water films are avoided from appearing around the seeds, and germinated seeds with young root seedlings of 5-10mm in length are collected for later use;
3. the culture medium (10.5 g of rice, 9g of raw soil, 1.5g of turf, 0.3g of monopotassium phosphate, 0.3g of magnesium sulfate, 0.3g of calcium sulfate, 0.6g of glucose and 55mL of tap water) is filled into a 250mL tissue culture bottle, sterilized at high temperature and high pressure for 2 hours, and then moved into an ultra-clean workbench for standby. Moving 5-7 germinated seeds into each tissue culture bottle under aseptic conditions in an ultra-clean workbench, and simultaneously inoculating bacterial blocks (shown in figure 3) with about 0.5cm 2 around the root of each germinated seed; sealing, and culturing in an illumination culture room;
4. Sterilizing the seedling tray with 10% H 2O2 solution for 10min, washing with tap water for three times, washing with sterile water, and air drying. The seedling substrate is a mixture of vermiculite, humus soil, perlite and organic fertilizer, and is put into a standby seedling tray after high-pressure sterilization. Transplanting the mycorrhizal seedlings infected by the russula YAFMF006, then performing watering, weeding and fertilizing, and prohibiting herbicide use during weeding, so that ventilation and ventilation of a nursery are ensured, diseases are prevented, and no fungicidal agent is applied during seedling raising.
Seedling detection:
The growth and infection of mycorrhizal seedlings under different conditions in step 3 of the above example 2 were detected, and a conventional pinus massoniana root seedling growth control group was set, i.e., the operation according to the above example 2 was performed, except that no fungus block was inserted into the tissue culture flask, and the other steps were the same as those in example 2.
Specific results of root fungus infection are shown in fig. 4, fig. 5, fig. 6, fig. 7, fig. 8, fig. 9, fig. 10 and fig. 11, and it can be seen from the figure that the russula viridis YAFMF006 can effectively carry out root fungus infection on root seedlings of pinus massoniana by adopting the method of example 2.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. The russula vinosa YAFMF006 is characterized in that russula vinosa YAFMF06 belongs to russula vinosa Russula virescens and has the preservation name of russula vinosa YAFMF006 Russula VIRESCENS YAFMF006; the Chinese culture medium is preserved in China center for type culture Collection, and the preservation address is university of Wuhan; preservation number: cctccc M20232693.
2. The russula-greening-emitting mushroom YAFMF006 as defined in claim 1, wherein: the russula viridis YAFMF gene sequence comprises a nucleotide sequence shown as SEQ ID No. 1.
3. The separation method of the russula YAFMF006 is characterized by comprising the following steps of: the separation method comprises the following steps:
s1-1, collecting fruiting body samples of the russula vinosa, and carrying out disinfection and crushing treatment to obtain tissue crushing liquid;
S1-2, diluting the tissue disruption solution, coating the tissue disruption solution on a PDMYKAS plate, culturing in the dark for 15d, separating bacterial colonies, and identifying to obtain russula vinosa, wherein the formula of the PDMYKAS plate is as follows: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose, 100 mu g/ml of calicheamicin, 100 mu g/ml of ampicillin, 100 mu g/ml of streptomycin and 10g/L of agar;
S1-3, transferring the identified russula vinosa mycelia to PDMY solid culture medium for continuous culture for 20d, extracting mycelium DNA, and carrying out molecular identification on the strain to obtain russula vinosa schrader YAFMF006, wherein the formula of the PDMY solid culture medium is as follows: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose and 16g/L of agar.
4. The mycorrhizal seedling infection method of russula vinosa YAFMF006 is characterized in that russula vinosa YAFMF006 carries out mycorrhizal seedling infection on pinus massoniana, and the specific method comprises the following steps:
S2-1, strain preparation: transferring the russula mycelium to PDMY solid culture medium, culturing for 20d, identifying to obtain russula YAFMF bacterial colony, and obtaining russula YAFMF006 standby bacterial mass, wherein the formula of the PDMY solid culture medium is as follows: 500ml/L of fresh potato juice, 1g/L of yeast extract, 2.1g/L of malt extract, 5g/L of glucose and 16g/L of agar;
S2-2, cultivation of mycorrhizal seedlings: obtaining pinus massoniana seeds with the sprouting young root length of 5-10mm, placing the pinus massoniana seeds in a tissue culture bottle containing a culture medium, simultaneously inoculating standby fungus blocks around the root of the sprouting seeds, enabling the quantity of the inoculated fungus blocks to be consistent with that of the sprouting seeds, and sealing the inoculated fungus blocks to be placed in an illumination culture chamber for culture to obtain fungus root seedlings;
S2-3, transplanting seedlings: filling the seedling culture matrix subjected to autoclaving into a standby seedling culture tray, and transplanting mycorrhizal seedlings into the seedling culture tray for planting.
5. The method for mycorrhizal seedling infection of russula bicolor YAFMF006 according to claim 4, wherein the method comprises the steps of: the composition of the culture medium in each tissue culture bottle in the step S2-2 is as follows: 10.5g of rice, 9g of raw soil, 1.5g of turf, 0.3g of monopotassium phosphate, 0.3g of magnesium sulfate, 0.3g of calcium sulfate, 0.6g of glucose and 55mL of tap water.
6. The method for mycorrhizal seedling infection of russula YAFMF006 in step S2-3, wherein the seedling substrate is a mixture of vermiculite, humus soil, perlite and organic fertilizer.
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