CN105794495A - Armillaria mellea strain symbiotic with rhizoma gastrodiae and application of stain - Google Patents
Armillaria mellea strain symbiotic with rhizoma gastrodiae and application of stain Download PDFInfo
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Abstract
The invention provides an armillaria mellea strain related to the high yield and stable yield of rhizoma gastrodiae and a preparation method of the strain. The taxonomy name of the strain is A.nabsnona T.J.Volk&Burds., the strain is a newly recorded species of armillaria fungi in China, and the preservation registration number is CGMCC No.11901. The method comprises the steps of strain selection, separation and purification, identification and cultivation. By means of the armillaria mellea strain related to the high yield and the stable yield of rhizoma gastrodiae and the preparation method of the strain, a risk of low yield of rhizoma gastrodiae or 'empty pond' phenomenon of no yield caused by the fact that selected stains are improper is reduced; the strain and gastrodia elata f.glauca have a better dependency, and the original quality of gastrodia elata f.glauca under the large-scale artificial cultivation condition in the region mainly producing gastrodia elata f.glauca can be guaranteed.
Description
Technical field
The present invention relates to a kind of Armillaria mellea with Rhizoma Gastrodiae symbiosis and preparation method thereof, belong to biological technical field.
Background technology
Armillaria fungus (Armillaria) popular name Armillariella mellea, maize bacterium, be under the jurisdiction of mycota, Basidiomycotina (Basdiomyeotina), gill fungi guiding principle (Hymenomyeetes), Agaricales (Agaricales), swollen coral Cordycepps (Physalacriaceae).This alive boundary treaty of genus fungus has 40 kinds, and also relatively broad at distribution in China, all there is distribution Central China, southwest and the Northeast.Armillaria mellea is the fungal component that rare Chinese medicine Rhizoma Gastrodiae (GastrodiaelataBl.) is important, is both needed to want Armillaria mellea to provide nutrient substance with rhizomorphic form during the growth of Rhizoma Gastrodiae 95%.There are some researches show and plant Rhizoma Gastrodiae with the Armillaria mellea bacterial strain companion of separate sources, the yield of Rhizoma Gastrodiae is different with quality.Therefore, selecting the Armillaria mellea cultivation Rhizoma Gastrodiae high with Rhizoma Gastrodiae affinity is one of key factor ensureing Rhizoma Gastrodiae production.
So far, in prior art, the Classification And Nomenclature there are no Rhizoma Gastrodiae symbiosis this bacterial strain of Armillaria mellea is ArmillarianabsnonaT.J.Volk&Burds., and preservation registration number is: CGMCCNo.11901, and its preparation method and application report.
Summary of the invention
It is an object of the invention to provide a kind of necessary Armillaria mellea ArmillarianabsnonaT.J.Volk&Burds. of Rhizoma Gastrodiae growth, bacterial strain preservation registration number is: CGMCCNo.11901, and its preparation method and application, this strain can ensure that the artificial growth of Rhizoma Gastrodiae produces, and decreases the risk of Rhizoma Gastrodiae plantation.
In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of Rhizoma Gastrodiae symbiosis Armillaria mellea bacterial strain, the Classification And Nomenclature of this bacterial strain is ArmillarianabsnonaT.J.Volk&Burds., and for Armillaria fungus at Chinese New Records, preservation registration number is: CGMCCNo.11901.
A kind of Rhizoma Gastrodiae symbiosis Armillaria mellea bacterial strain as mentioned, the cultural characteristic of this bacterial strain is: in general PDA culture medium, mycelia is vigorous for white, and in base, shoestring is that white is extremely light yellow, and air rhizomorph is light reddish brown color.Shoestring is monopodial branching, and growth is fast, and guttation is later, and culture medium color becomes or constant less.
The preparation method of described Armillaria mellea bacterial strain, the method includes field acquisition Armillaria mellea bacterial strain, screen, separate, be purified into female kind, first field Armillaria mellea source material is gathered as drawing bacterium, mix thoroughly with diameter 50-80mm broad-leaved branch, embedment cave, the sylvan life pool is as bacterium material, in month next year 7-8, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16-24 month, the bacterium material many, shapeliness of growth Rhizoma Gastrodiae quantity in cave, the pool is selected to cultivate shoestring；Then on cultured bacterium material, select the shoestring that thickness is suitable, robust growth, color are shallow be easily separated, several sections of shoestrings are intercepted with aseptic operation cutter, it is placed in PDA culture medium to cultivate and selects after 7-10 days the tender shoestring of children growing in culture medium to be purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, finally by sterile working's code by pure inoculation to PDA test tube slant culture medium, all of condition of culture is constant temperature 25 DEG C, relative humidity 60～70%.
The preparation method of Armillaria mellea bacterial strain as mentioned, the method includes further purification obtained strains being identified and preserving, the test tube bacterial strain choosing more than 3 carries out Genetic identification, determining that it is Armillaria mellea of the same race, the pure bacterial strain after identifying is keep its activity to place 4 DEG C of cryopreservation for follow-up production application.
The preparation method of described Armillaria mellea bacterial strain, first prepares test tube strains, then carries out strain expanding propagation, and described preparation test tube strains includes:
1) field selects Armillaria mellea source as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in August next year 7, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium material many, shapeliness of growth Rhizoma Gastrodiae quantity in cave, the pool is selected to cultivate shoestring；
2) Rhizomorph isolation method, purification: select on cultured bacterium material grow thickly, thickness is suitable, the shoestring number section of robust growth, top white is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, cultivate the tender shoestring of children selecting to grow into culture medium after 7 10 days to be purified when relative humidity 60%～80%, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, i.e. test tube kind mother kind；
Described strain expanding propagation is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.Following sterile working's process, test tube kind be transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to second class inoculum；The fresh minor matters of broad leaf tree of length 2 3cm, diameter 1 2cm are soaked 24 48 hours, load in the seed bottle of 750 milliliters to shoulder position, cleaning water is injected to partly flooding surface minor matters in bottle, after adding a cover sterilizing, sterile working accesses second class inoculum, postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% is cultivated 50 60 days, cultivates three-class strain；The three-class strain produced can be directly used for G. Elata B1f. G1auca S Chow companion and plants.
The preparation method of described Armillaria mellea bacterial strain, first prepares test tube strains, then carries out strain expanding propagation, and described preparation test tube strains includes:
1) field selects Armillaria mellea source: extensively gather the Armillaria mellea source material in difference G. Elata B1f. G1auca S Chow producing region, Zhaotong as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in August next year 7, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium material many, shapeliness of growth Rhizoma Gastrodiae quantity in cave, the pool is selected to cultivate shoestring.
2) Rhizomorph isolation method, purification: the shoestring number section selecting the shallow stalwartness of color on cultured bacterium material is placed in culture dish, sterile working is inoculated in PDA test tube slant culture medium, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, cultivate the tender shoestring of children selecting to grow into culture medium after 7 10 days to be purified when relative humidity 60%～80%, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube kind mother's kind.
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.According to sterile working's requirement, test tube kind being transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, grows up to second class inoculum；Three grades of kinds are formulated as: base material 1 is hardwood sawdust 78%, Testa oryzae or Testa Tritici 20%, Gypsum Fibrosum 10%, sucrose 1%.Base material 2 is the fresh minor matters of broad leaf tree of immersion length 2 3cm of 24 48 hours, thick 1 2cm in footpath, base material 1: base material 2 is 1: 2, the ratio of total base material and water is 1: 1.5, the base material prepared is loaded in the seed bottle of 750ml to shoulder place, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, the darkroom of relative humidity 70% 80% is cultivated 50 60 days, obtains three grades of branch strains；Three grades of branch strains can be directly used for G. Elata B1f. G1auca S Chow companion and plant.
The application in gastrodin cultivation of the described Armillaria mellea bacterial strain, this strain test tube kind mother is planted expansion and changes into two grades of solid spawns, expanding with two grades of solid spawns and turning cultivation is three grades of branch strains, use three grades of branch strains to make to draw bacterium and cultivate bacterium material, for gastrodin cultivation, it is provided that to the nutrient substance that Rhizoma Gastrodiae growth is required.
The application in gastrodin cultivation of the described Armillaria mellea bacterial strain, first prepares Armillaria mellea test tube strains, then carries out strain expanding propagation, then plants Rhizoma Gastrodiae with three grades of branch strain companions, and described preparation test tube strains includes:
1) field selects Armillaria mellea source as drawing bacterium, it is placed into cave, the sylvan life pool as bacterium material with diameter 50 80mm broad-leaved branch mix, in 7 Augusts of next year seeds of Gastrodia elata natural maturity, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, select the bacterium material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool to cultivate shoestring；
2) Rhizomorph isolation method, purification: be chosen as on cultured bacterium material clump raw, thickness suitable, the shoestring number section of robust growth, top white is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, relative humidity 60% 80%, cultivate 7 10 days, the reselection tender shoestring of the children in culture medium of growing into is purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, i.e. test tube kind mother kind；
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to the bottled solid medium of 500ml, natural ph.Following sterile working's process test tube kind to be transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to second class inoculum；The fresh minor matters of broad leaf tree of length 2 3cm, diameter 1 2cm are soaked 24 48 hours, load in the seed bottle of 750 milliliters to shoulder position, cleaning water is injected to partly flooding surface minor matters in bottle, after adding a cover sterilizing, sterile working accesses second class inoculum, postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% is cultivated 50 60 days, cultivates three grades of branch strains；The three grades of branch strains produced are directly used in G. Elata B1f. G1auca S Chow companion and plant.
The application in gastrodin cultivation of the described Armillaria mellea bacterial strain, first prepares Armillaria mellea test tube strains, then carries out strain expanding propagation, then three grades of branch strain companions is planted Rhizoma Gastrodiae, and described preparation test tube strains includes:
1) field selects Armillaria mellea source: extensively gather the Armillaria mellea source material of different regions, G. Elata B1f. G1auca S Chow producing region, Zhaotong as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, 7 Augusts in next year seeds of Gastrodia elata natural maturity, again by bacterium material in cave, the pool of the seeds of Gastrodia elata uniform broadcasting mixed with Germination Strain, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium source material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool is selected to cultivate shoestring；
2) Rhizomorph isolation method, purification: the shoestring number section selecting the shallow stalwartness of color on cultured bacterium material is placed in culture dish, sterile working is inoculated in PDA test tube slant culture medium, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, relative humidity 60% 80%, cultivates 7 10 days, and the grow into tender shoestring of children of culture medium of reselection is purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube kind mother's kind.
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, and glucose is 2%, and the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.According to sterile working's requirement, test tube kind being transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, obtains second class inoculum；Three grades of kinds are formulated as: base material 1 is hardwood sawdust 78%, Testa oryzae or Testa Tritici 20%, Gypsum Fibrosum 10%, sucrose 1%.Base material 2 is the fresh minor matters of broad leaf tree of immersion length 2 3cm of 24 48 hours, thick 1 2cm in footpath, base material 1: base material 2 is 1: 2, the ratio of total base material and water is 1: 1.5, the base material prepared is loaded in the seed bottle of 750ml to shoulder place, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, the darkroom of relative humidity 70% 80% is cultivated 50 60 days, obtains three-class strain；Three-class strain can be directly used for G. Elata B1f. G1auca S Chow companion and plants.
The present invention is realized in, utilize the advantage Armillaria mellea bacterial strain of field acquisition, screen, separate, be purified into the Armillaria mellea bacterial strain that can keep Zhaotong Rhizoma Gastrodiae good characteristic, the Classification And Nomenclature of this bacterial strain is ArmillarianabsnonaT.J.Volk&Burds., it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 01 18th, 2016, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation registration number is: CGMCCNo.11901.Identify that the biomaterial (strain) joining evidence is HKAS85500.
The technical scheme is that
1, field Armillaria mellea source material is gathered as drawing bacterium, embedment cave, the sylvan life pool is mixed thoroughly as bacterium material with diameter 50 80mm broad-leaved branch, in 7 Augusts of next year seeds of Gastrodia elata natural maturity, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, select the bacterium material many, shapeliness of growth Rhizoma Gastrodiae quantity in cave, the pool to cultivate shoestring.
2, on cultured bacterium material, select the shoestring number section that thickness is suitable, robust growth, color are shallow be easily separated, intercept several sections with aseptic operation cutter to be placed in PDA culture medium and cultivate, the reselection tender shoestring of the children in culture medium of growing into is purified, until screening obtains the pure bacterial strain that shoestring form is highly consistent.Then by sterile working's code by pure inoculation to PDA test tube slant culture medium, record is performed labelled for the test tube inoculated.All of incubation is constant temperature 25 DEG C, relative humidity 60 70%, cultivates 7 10 days.
3, the test tube bacterial strain choosing more than 3 carries out system classification qualification, it is determined that it is same Armillaria mellea.
4, the expansion of strain tube kind changes into two grades of solid spawns, expands to turn with two grades of solid spawns and cultivates three grades of bottled branch strains, uses branch strain work " drawing bacterium " to cultivate branch material or " bacterium material " for gastrodin cultivation.
This strain characteristic: this strain mycelia in general PDA culture medium is vigorous is white, and shoestring is that light yellow branch is many to light reddish brown color, and growth is fast, and shoestring guttation is later, and culture medium color becomes or constant less.
Compared with prior art, the present invention possesses following progress: one is this strain and G. Elata B1f. G1auca S Chow has good affinity, uses this strain can reduce the appearance of " the empty pool " phenomenon；Two is that this strain has natural distribution in G. Elata B1f. G1auca S Chow growth area, and sufficient bacterium source can guarantee that the lasting sane development of local Tianma industry.Armillaria mellea strains A rmillarianabsnonaT.J.Volk&Burds. of the present invention, preservation registration number is: CGMCCNo.11901, is used for accompanying cultivation Rhizoma Gastrodiae, and the yield of Rhizoma Gastrodiae can improve 3 to 5 times than traditional method.
Detailed description of the invention:
Further illustrate the essentiality content of the present invention below with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
1, preparation test tube strains:
1) field selects Armillaria mellea source (gathering the Armillaria mellea source material in difference location, Rhizoma Gastrodiae producing region, Zhaotong) as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in 7 Augusts of next year seeds of Gastrodia elata natural maturity, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, select the bacterium material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool to cultivate shoestring.
2) Rhizomorph isolation method, purification: select on cultured bacterium material grow thickly, thickness is suitable, the shoestring number section of robust growth, top white is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, relative humidity 60% 80%, cultivate 3 10 days, the reselection tender shoestring of the children in culture medium of growing into is purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, i.e. test tube kind (female kind).
2, strain expanding propagation:
According to Testa Tritici or Testa oryzae 20%, glucose is 2%, and the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.Follow sterile working's process, the test tube kind obtained by purification (utilizes the advantage Armillaria mellea bacterial strain of field acquisition, screen, separate, be purified into the Armillaria mellea bacterial strain that can keep Zhaotong Rhizoma Gastrodiae good characteristic, the Classification And Nomenclature of this bacterial strain is ArmillarianabsnonaT.J.Volk&Burds., it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 01 18th, 2016, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation registration number is: CGMCCNo.11901.The biomaterial (strain) identifying ginseng evidence is HKAS85500) it is transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to second class inoculum.The fresh minor matters of broad leaf tree of length 2 3cm, diameter 1 2cm are soaked 24 48 hours, load in the seed bottle of 750ml to shoulder position, cleaning water is injected to partly flooding surface minor matters in bottle, after adding a cover sterilizing, sterile working accesses second class inoculum, postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% is cultivated 50 60 days, can cultivate three-class strain.
3, namely the three-class strain produced can be directly used for the G. Elata B1f. G1auca S Chow companion in the areas such as river, Yunnan-Guizhou and plants use.
Embodiment 2:
1, preparation test tube strains:
1) field selects Armillaria mellea source: extensively gather the Armillaria mellea source material in difference G. Elata B1f. G1auca S Chow producing region, Zhaotong as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in 7 Augusts of next year seeds of Gastrodia elata natural maturity again by bacterium material in cave, the pool of the seeds of Gastrodia elata uniform broadcasting mixed with Germination Strain, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium source material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool is selected to cultivate shoestring.
2) Rhizomorph isolation method, purification: the shoestring number section selecting the shallow stalwartness of color on cultured bacterium material is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, relative humidity 60% 80%, cultivates 3 10 days, and the grow into tender shoestring of children of culture medium of reselection is purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube strains (female kind).
2, strain expanding propagation:
According to Testa Tritici or Testa oryzae 20%, glucose is 2%, and the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.Following sterile working's process, test tube kind (preservation registration number is CGMCCNo.11901) be transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to second class inoculum.Three grades of kinds are formulated as: base material 1 is hardwood sawdust 78%, Testa oryzae or Testa Tritici 20%, Gypsum Fibrosum 10%, sucrose 1%.Base material 2 is the fresh minor matters of broad leaf tree of immersion length 2 3cm of 24 48 hours, thick 1 2cm in footpath.Base material 1: base material 2 is 1: 2, the ratio of total base material and water is 1: 1.5.The base material for preparing being loaded in the seed bottle of 750ml to shoulder place, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% is cultivated 50 60 days, can cultivate three-class strain.
3, namely the three-class strain produced can be directly used for the G. Elata B1f. G1auca S Chow companion in the areas such as river, Yunnan-Guizhou and plants use.
Claims (9)
1. a Rhizoma Gastrodiae symbiosis Armillaria mellea bacterial strain, it is characterised in that the Classification And Nomenclature of this bacterial strain is ArmillarianabsnonaT.J.Volk&Burds., and preservation registration number is: CGMCCNo.11901.
2. a kind of Rhizoma Gastrodiae symbiosis Armillaria mellea bacterial strain as claimed in claim 1, it is characterised in that the cultural characteristic of this bacterial strain is that mycelia is vigorous for white in general PDA culture medium, and in base, shoestring is white to light yellow, and air rhizomorph is light reddish brown color.Shoestring is monopodial branching, and growth is fast, and guttation is later, and culture medium color becomes or constant less.
3. the preparation method of the Armillaria mellea bacterial strain described in claim 1, it is characterised in that the method includes field acquisition Armillaria mellea bacterial strain, screens, separates, is purified into female kind.First field Armillaria mellea source material (wild Armillaria mellea shoestring or sporophore) is gathered as drawing bacterium, mix thoroughly with diameter 50-80mm broad-leaved branch, embedment cave, the sylvan life pool is as bacterium material, in month next year 7-8, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon in cave on bacterium material, covers the fresh leaf of broad leaf tree and sand is cultivated.Select Rhizoma Gastrodiae growth population in cave, the pool many after 16-24 month, shoestring cultivated by the bacterium material of shapeliness, bacterium material is selected thickness be suitable for, robust growth, the shoestring that color is shallow carries out strain separating, namely several sections of shoestrings are intercepted with aseptic operation cutter, it is placed in PDA culture medium and cultivates, after cultivating 7-10 days, the grow into tender shoestring of children of culture medium of reselection is purified and obtains the pure bacterial strain that shoestring growthform is highly consistent, finally by sterile working's code by pure inoculation to PDA test tube slant culture medium, inclined-plane is unsuitable long, all of condition of culture is constant temperature 25 DEG C, relative humidity 60-70%.
4. the preparation method of Armillaria mellea bacterial strain as claimed in claim 3, it is characterized in that the method includes purification obtained strains being identified and preserving, the test tube bacterial strain choosing more than 3 carries out system classification qualification, determining that it is Armillaria mellea of the same race, the pure bacterial strain after identifying is keep its activity to place 4 DEG C of cryopreservation for follow-up production application.
5. the preparation method of the Armillaria mellea bacterial strain described in claim 1, it is characterised in that first preparing test tube strains, then carry out strain expanding propagation, the preparation of described test tube strains includes:
1) field selects Armillaria mellea source as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, again the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool in August next year 7, covers the fresh leaf of broad leaf tree and sand is cultivated.After 16 24 months, the bacterium source material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool is selected to cultivate shoestring；
2) Rhizomorph isolation method, purification: select on cultured bacterium material grow thickly, thickness is suitable, the shoestring number section of robust growth, top white is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, cultivate 7 10 days when relative humidity 60%～80%, the reselection tender shoestring of the children in culture medium of growing into is purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube stock；
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to the bottled solid medium of 500ml, natural ph, follow sterile working's process, test tube kind is transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to second class inoculum.The fresh minor matters of broad leaf tree of length 2 3cm, diameter 1 2cm are soaked 24 48 hours, load in the seed bottle of 750ml to shoulder position, cleaning water is injected to partly flooding surface minor matters in bottle, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% cultivates 50 60 days, cultivating three-class strain (cultigen), the three-class strain produced can be directly used for companion and plants G. Elata B1f. G1auca S Chow.
6. the preparation method of the Armillaria mellea bacterial strain described in claim 1, it is characterised in that first preparing test tube strains, then carry out strain expanding propagation, described preparation test tube strains includes:
1) field selects Armillaria mellea source: extensively gather the Armillaria mellea source material of different regions, G. Elata B1f. G1auca S Chow producing region, Zhaotong as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in August next year 7, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium source material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool is selected to cultivate shoestring.
2) Rhizomorph isolation method, purification: the shoestring number section selecting the shallow stalwartness of color on cultured bacterium material is placed in culture dish, it is inoculated in PDA test tube slant culture medium according to sterile working, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, the tender shoestring of children grown in culture medium is selected to be purified after cultivating 7 10 days when relative humidity 60%～80%, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube kind mother's kind.
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph.According to sterile working's requirement, test tube kind being transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, grows up to second class inoculum；Three grades of kinds are formulated as: base material 1 is hardwood sawdust 78%, Testa oryzae or Testa Tritici 20%, Gypsum Fibrosum 10%, sucrose 1%.Base material 2 is the fresh minor matters of broad leaf tree of immersion length 2 3cm of 24 48 hours, thick 1 2cm in footpath, base material 1: base material 2 is 1: 2, the ratio of total base material and water is 1: 1.5, the base material prepared is loaded in the seed bottle of 750ml to shoulder place, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, the darkroom of relative humidity 70% 80% is cultivated 50 60 days, obtains three-class strain；The three grades of branch strains produced can be directly used for G. Elata B1f. G1auca S Chow companion and plant.
7. the application in gastrodin cultivation of the Armillaria mellea bacterial strain described in claim 1, it is characterized in that this strain test tube kind mother is planted expansion changes into two grades of solid spawns, expand to turn with two grades of solid spawns and cultivate three grades of branch strains, use three grades of branch strains to make to draw bacterium and cultivate bacterium material, for gastrodin cultivation, it is provided that to Gastrodiac nutritive material.
8. then three grades of branch strain companions are planted Rhizoma Gastrodiae by the application in gastrodin cultivation of the Armillaria mellea bacterial strain described in claim 1, it is characterised in that described application is first to prepare Armillaria mellea test tube strains, then carries out strain expanding propagation, and described preparation test tube strains includes:
1) field selects Armillaria mellea source as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, in August next year 7, the seeds of Gastrodia elata mixed with Germination Strain is uniformly sprinkling upon on the bacterium material in cave, the pool, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium source material many, shapeliness of Rhizoma Gastrodiae growth population in cave, the pool is selected to cultivate shoestring；
2) Rhizomorph isolation method, purification: be chosen as on cultured bacterium material clump raw, thickness suitable, the shoestring number section of robust growth, top white is placed in culture dish, it is inoculated in PDA test tube slant culture medium by sterile working's code, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, the tender shoestring of children grown in culture medium is selected to be purified after cultivating 7 10 days when relative humidity 60%～80%, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, i.e. test tube kind mother kind；
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose is 2%, the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph, follow sterile working's process, test tube kind is transferred on sterilized solid medium, put into room temperature 25 DEG C, the darkroom of humidity 70% 80% is cultivated 25 days, can grow up to for second class inoculum；The fresh minor matters of broad leaf tree of length 2 3cm, diameter 1 2cm are soaked 24 48 hours, load in the seed bottle of 750 milliliters to shoulder position, cleaning water is injected to partly flooding surface minor matters in bottle, after adding a cover sterilizing, sterile working accesses second class inoculum, postvaccinal bottle is put into room temperature 22 DEG C 25 DEG C, and the darkroom of relative humidity 70% 80% is cultivated 50 60 days, cultivates three grades of branch strains；The three grades of branch strains produced can be directly used for G. Elata B1f. G1auca S Chow companion and plant.
9. then three grades of branch strain companions are planted Rhizoma Gastrodiae by the application in gastrodin cultivation of the Armillaria mellea bacterial strain described in claim 1, it is characterised in that described application is first to prepare Armillaria mellea test tube strains, then carries out strain expanding propagation, and described preparation test tube strains includes:
1) field selects Armillaria mellea source: extensively gather the Armillaria mellea source material of different regions, G. Elata B1f. G1auca S Chow producing region, Zhaotong as drawing bacterium, with diameter 50 80mm broad-leaved branch mix implant cave, the sylvan life pool as bacterium material, 7 Augusts in next year seeds of Gastrodia elata natural maturity, again by bacterium material in cave, the pool of the seeds of Gastrodia elata uniform broadcasting mixed with Germination Strain, cover the fresh leaf of broad leaf tree and sand is cultivated, after 16 24 months, the bacterium material many, shapeliness of growth Rhizoma Gastrodiae quantity in cave, the pool is selected to cultivate shoestring；
2) Rhizomorph isolation method, purification: the shoestring number section selecting the shallow stalwartness of color on cultured bacterium material is placed in culture dish, sterile working is inoculated in PDA test tube slant culture medium, record is performed labelled for the test tube inoculated, put into 25 DEG C of calorstats, under relative humidity 60%～80% condition, cultivate the tender shoestring of children selecting to grow into culture medium after 7 10 days be purified, until screening obtains the pure bacterial strain that shoestring growthform is highly consistent, it is test tube kind mother's kind；
3) the strain expanding propagation described in is: according to Testa Tritici or Testa oryzae 20%, glucose 2%, the ratio of agar 2% adds water and is configured to 500 milliliters of bottled solid mediums, natural ph, require to be transferred on sterilized solid medium by test tube kind according to sterile working, putting into room temperature 25 DEG C, the darkroom of humidity 70%-80% is cultivated 25 days, obtains second class inoculum；Three grades of kinds are formulated as: base material 1 is hardwood sawdust 78%, Testa oryzae or Testa Tritici 20%, Gypsum Fibrosum 10%, sucrose 1%.Base material 2 is the fresh minor matters of broad leaf tree of the immersion length 2-3cm of 24-48 hour, the thick 1-2cm in footpath, base material 1: base material 2 is 1: 2, the ratio of total base material and water is 1: 1.5, the base material prepared is loaded in the seed bottle of 750ml to shoulder place, after adding a cover sterilizing, sterile working accesses second class inoculum, and postvaccinal bottle is put into room temperature 22 DEG C-25 DEG C, the darkroom of relative humidity 70%-80% is cultivated 50-60 days, obtains three grades of branch strains；Three grades of branch strains can be directly used for G. Elata B1f. G1auca S Chow companion and plant.
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