CN105794495B - Armillaria mellea strain symbiotic with gastrodia elata and application thereof - Google Patents
Armillaria mellea strain symbiotic with gastrodia elata and application thereof Download PDFInfo
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Abstract
The invention provides a Armillaria mellea strain related to high and stable yield of Gastrodia elata and a preparation method thereof. The taxonomic name of the strain is A.nabsnona T.J.Volk & Burds, which is a new recording species of the Armillaria fungus in China, and the preservation registration number is CGMCC No. 11901. The method comprises the steps of strain selection, separation and purification, identification and culture. By using the method, firstly, the risk of the phenomenon of 'empty pond' of low yield or no yield of the gastrodia elata caused by improper strain breeding is reduced; secondly, the strain and the gastrodia elata have better affinity, and the original quality of the gastrodia elata under the condition of large-scale artificial cultivation in the area where the gastrodia elata is mainly produced can be guaranteed.
Description
Technical Field
The invention relates to armillaria mellea symbiotic with gastrodia elata and a preparation method thereof, belonging to the technical field of biology.
Background
Armillaria mellea (Armillaria) fungi, commonly known as Corylus heterophyllus and Cucumaria bracteata, belong to the kingdom fungi, Basidiomycotina, Agaricales, and Sarcophythoraceae. The fungus is about 40 kinds in the world, is widely distributed in China, and is distributed in China, southwest areas and northeast areas. Armillaria mellea is an important symbiotic bacterium of a rare traditional Chinese medicine rhizoma gastrodiae (Gastrodia elata Bl.), and the armillaria mellea is required to provide nutrient substances in the form of rhizomorphous rhizomorph during 95% of the growth period of the rhizoma gastrodiae. Studies show that the yield and the quality of the gastrodia elata are different when the gastrodia elata is subjected to concomitant cultivation by using different sources of armillaria mellea strains. Therefore, the selection of Armillaria mellea strain with high affinity with Gastrodia elata for cultivating Gastrodia elata is one of the key factors for ensuring the production of Gastrodia elata.
So far, no gastrodia elata symbiotic armillaria mellea strain exists in the prior art, the classification and the designation of the strain are Armillaria nabsnona T.J.Volk & Burds, and the preservation registration number is as follows: CGMCC No.11901, and reports of its preparation method and application.
Disclosure of Invention
The invention aims to provide an Armillaria nabsona T.J.Volk & Burds, which is an Armillaria nabsona nana strain necessary for the growth of gastrodia elata, and the preservation registration number of the strain is as follows: CGMCC No.11901, and its preparation method and application, the strain can ensure artificial planting production of rhizoma Gastrodiae, and reduce risk of rhizoma Gastrodiae planting.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:
a symbiotic Armillaria gastrodiae strain is classified and named Armillaria nabsnona T.J. Volk & Burds, is a new record species of Armillaria fungus in China, and has the preservation registration number: CGMCC No. 11901.
The symbiotic armillaria mellea strain of gastrodia elata is characterized by being cultured by the following steps: on the general PDA culture medium, the hypha flourishing is white, the mycorrhiza in the medium is white to light yellow, and the aerial mycorrhiza is light reddish brown. The rhizomorph is a single-axis branch, the growth is fast, the water discharge is late, and the color of the culture medium is little or unchanged.
The preparation method of the Armillaria mellea strain comprises the steps of collecting Armillaria mellea strain in the field, screening, separating and purifying to obtain mother seeds, firstly collecting source materials of Armillaria mellea in the field as guiding bacteria, uniformly mixing the guiding bacteria with broad-leaved tree branches with the diameter of 50-80mm, burying the branches into a pond under forest to serve as bacterial materials, uniformly scattering seeds of Gastrodia elata mixed with germinating bacteria on the bacterial materials in the pond in 7-8 months of the next year, covering fresh leaves of the broad-leaved trees and sandy soil for cultivation, and selecting the bacterial materials with large quantity and good shape for growing Gastrodia elata in the pond to cultivate bacterial cables after 16-24 months; and then selecting and separating the stropharia rugoso which is proper in thickness, strong in growth and light in color on the cultured stropharia rugoso, intercepting a plurality of sections of stropharia rugoso by using a sterile scalpel, culturing the stropharia rugoso on a PDA culture medium for 7-10 days, then selecting and purifying the young stropharia rugoso growing into the culture medium until pure strains with consistent growth forms and heights of the stropharia rugoso are obtained by screening, and finally inoculating the pure strains on the slant culture medium of the PDA test tube according to the aseptic operation rules, wherein the constant temperature is 25 ℃, the relative humidity is 60-70%, and all the culture conditions are constant temperature.
The preparation method of the Armillaria mellea strain comprises the steps of further identifying and storing the purified strain, selecting more than 3 test tube strains for genetic identification, determining the strains to be the same species of Armillaria mellea, and storing the identified pure strain at the low temperature of 4 ℃ for subsequent production application in order to keep the activity of the pure strain.
The preparation method of the armillaria mellea strain comprises the following steps of firstly preparing test-tube strains, and then carrying out strain propagation, wherein the step of preparing the test-tube strains comprises the following steps:
1) selecting Armillaria mellea sources as introduced bacteria in the field, mixing with broad-leaved branches with the diameter of 50-80mm, implanting into a pond under the forest to serve as a bacterial material, uniformly scattering seeds of the Gastrodia elata mixed with germinating bacteria on the bacterial material in the pond in 7-8 months of the next year, covering fresh leaves of the broad-leaved trees and sandy soil for cultivation, and selecting the bacterial material with large quantity and good shape for growing the Gastrodia elata in the pond to cultivate bacterial cables after 16-24 months;
2) and (3) separating and purifying the rhizomorph: selecting a plurality of fasciculus with proper thickness, strong growth and white top on cultured mushroom materials, placing the fasciculus into a culture dish, inoculating the fasciculus to a PDA test tube slant culture medium according to an aseptic operation procedure, labeling the inoculated test tube for recording, placing the test tube into a constant temperature box with the temperature of 25 ℃, culturing for 7 to 10 days under the condition of relative humidity of 60 to 80 percent, and then selecting tender fasciculus growing into the culture medium for purification until pure strains with consistent fasciculus growth form height, namely test tube seed mother strains, are obtained by screening;
the strain propagation comprises the following steps: adding water according to the proportion of 20 percent of wheat bran or rice bran, 2 percent of glucose and 2 percent of agar to prepare a bottled solid culture medium of 500ml, and naturally adjusting the pH value. Transferring the test tube strain to a sterilized solid culture medium following an aseptic operation process, and culturing in a dark room at room temperature of 25 ℃ and humidity of 70-80% for 25 days to obtain a second-level strain; soaking fresh branches of broad-leaved trees with the length of 2-3cm and the diameter of 1-2cm for 24-48 hours, filling the fresh branches into a strain bottle with the volume of 750ml to reach the shoulder part of the bottle, injecting clean water into the bottle until the branches are half submerged, covering the bottle for sterilization, inoculating a second-level strain in an aseptic operation, putting the inoculated bottle into a dark room with the room temperature of 22-25 ℃ and the relative humidity of 70-80% for culturing for 50-60 days, and culturing a third-level strain; the produced third-level strain can be directly used for the companion planting of the gastrodia elata.
The preparation method of the armillaria mellea strain comprises the following steps of firstly preparing test-tube strains, and then carrying out strain propagation, wherein the step of preparing the test-tube strains comprises the following steps:
1) selecting Armillaria mellea sources in the field: collecting Armillaria mellea source materials in different Gastrodia elata producing areas of Zhaotong as introducing bacteria, mixing with broad-leaved tree branches with diameter of 50-80mm, planting in pond holes under forest as bacteria material, spreading seeds of Gastrodia elata mixed with germinating bacteria on the bacteria material in the pond holes 7-8 months next year, covering with fresh leaves of broad-leaved trees and sandy soil for cultivation, and selecting bacteria material with large amount of grown Gastrodia elata and good shape to culture bacteria in the pond holes after 16-24 months.
2) And (3) separating and purifying the rhizomorph: selecting a plurality of sections of light and strong strongylocentrotus obliquus on cultured strains, placing the sections in a culture dish, inoculating the sections to a PDA test tube slant culture medium in an aseptic operation, labeling the inoculated test tube, recording, placing the test tube in a constant temperature box at 25 ℃, culturing for 7-10 days under the condition that the relative humidity is 60% -80%, and then selecting tender strongylocentrotus obliquus growing in the culture medium for purification until pure strains with consistent strongylocentrotus growth form height are obtained by screening, namely the test tube seed mother strain.
3) The strain propagation comprises the following steps: adding water according to the proportion of 20 percent of wheat bran or rice bran, 2 percent of glucose and 2 percent of agar to prepare a bottled solid culture medium of 500ml, and naturally adjusting the pH value. Transferring the test tube seeds to a sterilized solid culture medium according to the sterile operation requirement, and placing the culture medium in a dark room with the room temperature of 25 ℃ and the humidity of 70-80% for culturing for 25 days to grow second-level strains; the third-class seed is prepared as follows: the base material 1 is composed of broad-leaved tree sawdust 78%, rice bran or wheat bran 20%, gypsum 10% and sucrose 1%. The base material 2 is a broad-leaved tree fresh branch knot which is soaked for 24-48 hours, has the length of 2-3cm and the diameter of 1-2cm, the base material 1: the base material 2 is 1: 2, the proportion of the total base material to water is 1: 1.5, the prepared base material is filled into a strain bottle of 750ml to the bottle shoulder, after capping and sterilization, a secondary strain is inoculated in an aseptic operation, and the inoculated bottle is placed into a dark room with the room temperature of 22-25 ℃ and the relative humidity of 70-80 percent for culturing for 50-60 days to obtain a third-level branch strain; the third branch strain can be directly used for the companion planting of gastrodia elata.
The application of the Armillaria mellea strain in the cultivation of the Gastrodia elata Blume comprises expanding the stock strain of the stock strain test tube strain into a second-stage solid strain, expanding and culturing the second-stage solid strain into a third-stage branch strain, and culturing the strain material by using the third-stage branch strain as a primer for the cultivation of the Gastrodia elata Blume and providing nutrient substances required by the growth of the Gastrodia elata Blume.
The application of the Armillaria mellea strain in the cultivation of the Gastrodia elata comprises the steps of firstly preparing Armillaria mellea test-tube strains, then carrying out strain propagation, and then carrying out accompanying cultivation on the Gastrodia elata by using three-level branch strains, wherein the preparation of the test-tube strains comprises the following steps:
1) selecting an Armillaria mellea source as a starter in the field, mixing the Armillaria mellea source with broadleaf branches with the diameter of 50-80mm, putting the Armillaria mellea source into a pond hole under the forest to serve as a fungus material, uniformly scattering the Gastrodia elata seeds mixed with germination bacteria on the fungus material in the pond hole in 7-8 months after the Gastrodia elata seeds naturally mature in the next year, covering fresh leaves of broadleaf trees and sandy soil for cultivation, and selecting the fungus material with large growth quantity and good shape in the pond hole to culture a fungus rope after 16-24 months;
2) and (3) separating and purifying the rhizomorph: selecting a plurality of strains which are clustered, have proper thickness, strong growth and white top ends on cultured strains, placing the strains in a culture dish, inoculating the strains to a PDA test tube slant culture medium according to a sterile operation rule, labeling the inoculated test tube for recording, placing the tubes in a constant temperature box at 25 ℃, culturing for 7-10 days at a relative humidity of 60-80%, and then selecting young strains growing in the culture medium for purification until pure strains with consistent growth forms of the strains, namely test tube seed mother strains, are obtained by screening;
3) the strain propagation comprises the following steps: adding water according to the proportion of 20 percent of wheat bran or rice bran, 2 percent of glucose and 2 percent of agar to prepare a bottled solid culture medium of 500ml, and naturally adjusting the pH value. Transferring the test tube strain to a sterilized solid culture medium according to an aseptic operation process, and placing the culture medium in a dark room with the room temperature of 25 ℃ and the humidity of 70-80% for culturing for 25 days to obtain a second-level strain; soaking fresh branches of broad-leaved trees with the length of 2-3cm and the diameter of 1-2cm for 24-48 hours, filling the fresh branches into a strain bottle with the volume of 750ml to reach the shoulder part of the bottle, injecting clean water into the bottle until the branches on the semi-submerged surface are covered and sterilized, inoculating a second-level strain in an aseptic operation, putting the inoculated bottle into a dark room with the room temperature of 22-25 ℃ and the relative humidity of 70-80% for culturing for 50-60 days, and culturing a third-level branch strain; the produced three-level branch strain is directly used for the companion planting of the gastrodia elata.
The application of the Armillaria mellea strain in the cultivation of the Gastrodia elata comprises the steps of firstly preparing Armillaria mellea test-tube strains, then carrying out strain propagation, and then carrying out accompanying cultivation on the Gastrodia elata with three-level branch strains, wherein the preparation of the test-tube strains comprises the following steps:
1) selecting Armillaria mellea sources in the field: collecting Armillaria mellea source materials in different areas of Gastrodia elata producing area of Zhaotong as introducing bacteria, mixing with broad-leaved tree branches with the diameter of 50-80mm, implanting into a pond hole under the forest as bacteria material, uniformly scattering the seeds of Gastrodia elata mixed with germination bacteria on the bacteria material in the pond hole in 7-8 months after the seeds of Gastrodia elata naturally mature in the next year, covering fresh leaves of broad-leaved trees and sandy soil for cultivation, and selecting the bacteria source materials with large growth amount and good shape of Gastrodia elata in the pond hole to culture a microbial inoculum after 16-24 months;
2) and (3) separating and purifying the rhizomorph: selecting a plurality of sections of light and strong strongylocentrotus obliquus on cultured strains, placing the sections in a culture dish, inoculating the sections to a PDA test tube slant culture medium in a sterile operation mode, labeling the inoculated test tube for recording, placing the test tube in a constant temperature box at 25 ℃, culturing for 7-10 days at a relative humidity of 60% -80%, and then selecting young strongylocentrotus obliquus growing in the culture medium for purification until pure strains with consistent strongylocentrotus growth form height are obtained through screening, namely the test tube seed mother strain.
3) The strain propagation comprises the following steps: the solid culture medium is prepared by adding water into 20% of wheat bran or rice bran, 2% of glucose and 2% of agar to prepare 500ml bottled solid culture medium, and the pH value is natural. Transferring the test tube seeds to a sterilized solid culture medium according to the sterile operation requirement, and placing the culture medium in a dark room with the room temperature of 25 ℃ and the humidity of 70-80% for culturing for 25 days to obtain a second-level strain; the third-class seed is prepared as follows: the base material 1 is composed of broad-leaved tree sawdust 78%, rice bran or wheat bran 20%, gypsum 10% and sucrose 1%. The base material 2 is a broad-leaved tree fresh branch knot which is soaked for 24-48 hours, has the length of 2-3cm and the diameter of 1-2cm, the base material 1: the base material 2 is 1: 2, the proportion of the total base material to water is 1: 1.5, the prepared base material is filled into a strain bottle of 750ml to the bottle shoulder, after capping and sterilization, a secondary strain is inoculated in an aseptic operation, and the inoculated bottle is placed into a dark room with the room temperature of 22-25 ℃ and the relative humidity of 70-80% for culturing for 50-60 days to obtain a tertiary strain; the third-level strain can be directly used for the companion planting of the gastrodia elata.
The invention is realized by screening, separating and purifying the Armillaria strain capable of keeping the excellent characteristics of Gastrodia elata Zhaotong by using dominant Armillaria strain collected in the field, the strain is classified and named as Armillaria nabsnona T.J.Volk & Burds, and is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 01, 18 days, and the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on North Chen, with the accession number: CGMCC No. 11901. The biomaterial (strain) identified as reference was HKAS 85500.
The technical scheme of the invention is as follows:
1. collecting wild Armillaria mellea source material as guiding bacteria, uniformly mixing with broadleaf branches with the diameter of 50-80mm, burying the mixture in a pond hole under forest as bacteria, uniformly scattering the seeds of Gastrodia elata mixed with germination bacteria on the bacteria in the pond hole in 7-8 months after the seeds of Gastrodia elata naturally mature in the next year, covering fresh leaves of broadleaf trees and sandy soil for cultivation, and selecting the bacteria with large quantity and good shape for growing Gastrodia elata in the pond hole after 16-24 months to culture the fungi.
2. Selecting a bacterial cord section with proper thickness, strong growth and light color on the cultured bacterial material, separating, cutting the bacterial cord section by using a sterile scalpel, culturing on a PDA culture medium, selecting a tender bacterial cord growing in the culture medium, and purifying until a pure bacterial strain with consistent bacterial cord form height is obtained by screening. Then inoculating the pure strain to a slant culture medium of a PDA test tube according to a sterile operation protocol, and labeling the inoculated test tube for recording. All the culture processes are constant temperature 25 deg.C, relative humidity 60-70%, and culture for 7-10 days.
3. Selecting more than 3 test tube strains for systematic classification and identification, and determining the test tube strains to be the same armillaria mellea.
4. The strain test tube strain is expanded into a second-stage solid strain, the second-stage solid strain is expanded and transformed to culture a third-stage bottled branch strain, and the branch strain is used as a 'guide bacterium' to culture a branch material or a 'bacterium material' for cultivating the gastrodia elata.
The characteristics of the strain are as follows: the strain has the advantages that hyphae flourish white on a general PDA culture medium, the rhizomorph is light yellow to light reddish brown, the number of branches is large, the growth is fast, the water spouting of the rhizomorph is late, and the color of the culture medium is little or unchanged.
Compared with the prior art, the invention has the following progress: firstly, the strain and the gastrodia elata have better affinity, and the occurrence of the phenomenon of 'empty pond' can be reduced by using the strain; secondly, the strain has natural distribution in the gastrodia elata growing area, and sufficient strain source can ensure the continuous and steady development of the local gastrodia elata industry. The Armillaria nobsona strain T.J.Volk & Burds, the preservation registration number is as follows: CGMCC No.11901, is used for accompanying cultivation of gastrodia elata, and the yield of the gastrodia elata can be improved by 3 to 5 times compared with that of the gastrodia elata by using a traditional method.
The specific implementation mode is as follows:
the following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the present invention.
Example 1:
1. preparing test tube strains:
1) selecting Armillaria mellea sources (collecting Armillaria mellea source materials in different sections of Gastrodia elata producing area) in the field as guiding bacteria, mixing with broadleaf branches with the diameter of 50-80mm, implanting into a pond under the forest as a bacterial material, uniformly scattering Gastrodia elata seeds mixed with germination bacteria on the bacterial material in the pond in 7-8 months when the Gastrodia elata seeds naturally mature in the next year, covering fresh leaves of the broadleaf trees and sandy soil for cultivation, and selecting the bacterial material with large growth quantity and good shape in the pond to culture the bacterial cables after 16-24 months.
2) And (3) separating and purifying the rhizomorph: selecting a plurality of fasciculus with proper thickness, strong growth and white top on the cultured mushroom material, placing the fasciculus into a culture dish, inoculating the fasciculus to a PDA test tube slant culture medium according to an aseptic operation rule, labeling the inoculated test tube for recording, placing the test tube into a constant temperature box with the temperature of 25 ℃, culturing for 3 to 10 days at the relative humidity of 60 to 80 percent, and then selecting the tender fasciculus growing in the culture medium for purification until a pure strain with consistent fasciculus growth form height is obtained by screening, namely a test tube seed (mother seed).
2. And (3) strain propagation:
the solid culture medium is prepared by adding water into 20% of wheat bran or rice bran, 2% of glucose and 2% of agar to prepare 500ml bottled solid culture medium, and the pH value is natural. Following the aseptic operation process, the purified test tube species (using dominant Armillaria nabsnona T.J.Volk & Burds. collected in the field to screen, separate and purify Armillaria nabsnona T.J.Volk & Burds. the strain is preserved in China general microbiological culture Collection center (CGMCC) 18 th 2016 (year 01, month 18), the preservation address is No.1 Hospital No. 3 of Beijing south Chen of the south China area, the preservation registration number is CGMCC No. 11901. the biological material (strain) for identifying the reference data is HKAS 85500. the strain is transferred to a sterilized solid culture medium and placed in a dark room with the room temperature of 25 ℃ and the humidity of 70-80% for 25 days to be cultured into a secondary strain. Soaking fresh branches of broad-leaved trees with the length of 2-3cm and the diameter of 1-2cm for 24-48 hours, filling the fresh branches into a strain bottle with the volume of 750ml to the position of the shoulder of the bottle, injecting clean water into the bottle until the branches are half submerged, covering the bottle for sterilization, inoculating a second-level strain in an aseptic operation, and culturing the inoculated bottle in a dark room with the room temperature of 22-25 ℃ and the relative humidity of 70-80% for 50-60 days to cultivate a third-level strain.
3. The produced third-level strain can be directly used for the companion planting of the gastrodia elata in the Yunbaicuan and other areas.
Example 2:
1. preparing test tube strains:
1) selecting Armillaria mellea sources in the field: the method comprises the steps of widely collecting Armillaria mellea source materials in different Gastrodia elata producing areas of Zhaotong as introducing bacteria, mixing with broadleaf branches with the diameter of 50-80mm, implanting into a pond hole under the forest as bacteria materials, uniformly scattering the Gastrodia elata seeds mixed with germination bacteria onto the bacteria materials in the pond hole after 7-8 months of natural maturity of the Gastrodia elata seeds in the next year, covering fresh leaves of the broadleaf trees and sandy soil for cultivation, and selecting the bacteria source materials with large growth quantity and good shape of Gastrodia elata in the pond hole to culture the rhizomorph 16-24 months later.
2) And (3) separating and purifying the rhizomorph: selecting a plurality of sections of light and strong strongylocentrotus obliquus on cultured strains, placing the sections in a culture dish, inoculating the sections on a PDA test tube slant culture medium according to an aseptic operation procedure, labeling the inoculated test tube for recording, placing the test tube in a constant temperature box at 25 ℃, culturing for 3-10 days at a relative humidity of 60% -80%, and then selecting young strongylocentrotus obliquus growing in the culture medium for purification until pure strains with consistent strongylocentrotus growth form height are obtained by screening, namely test tube strains (mother strains).
2. And (3) strain propagation:
the solid culture medium is prepared by adding water into 20% of wheat bran or rice bran, 2% of glucose and 2% of agar to prepare 500ml bottled solid culture medium, and the pH value is natural. Following the aseptic operation process, transferring the test tube species (preservation registration number is CGMCC No.11901) to a sterilized solid culture medium, and culturing in a dark room at room temperature of 25 ℃ and humidity of 70-80% for 25 days to obtain the second-level strain. The third-class seed is prepared as follows: the base material 1 is composed of broad-leaved tree sawdust 78%, rice bran or wheat bran 20%, gypsum 10% and sucrose 1%. The base material 2 is a broad-leaved tree fresh branch section with the length of 2-3cm and the diameter of 1-2cm, which is soaked for 24-48 hours. The ratio of the base material 1 to the base material 2 is 1: 2, and the ratio of the total base material to the water is 1: 1.5. Filling the prepared base material into a strain bottle of 750ml to the shoulder of the bottle, covering and sterilizing, inoculating a second-level strain in an aseptic operation, and culturing the inoculated bottle in a dark room at the room temperature of 22-25 ℃ and the relative humidity of 70-80% for 50-60 days to obtain a third-level strain.
3. The produced third-level strain can be directly used for the companion planting of the gastrodia elata in the Yunbaicuan and other areas.
Claims (1)
1. A preparation method of symbiotic armillaria mellea strains of gastrodia elata is characterized by comprising the following steps:
(1) preparing test tube strains:
selecting Armillaria mellea sources in the field: collecting Armillaria mellea source materials in different Gastrodia elata producing areas of Zhaotong as introducing bacteria, mixing with broad-leaved tree branches with the diameter of 50-80mm, implanting into a pond hole under forest as bacteria material, uniformly scattering the mixed seeds of Gastrodia elata with germination bacteria on the bacteria material in the pond hole in 7-8 months after the seeds of Gastrodia elata naturally mature in the next year, covering fresh leaves of broad-leaved trees and sandy soil for cultivation, and selecting the bacteria source materials with large growth amount and good shape of Gastrodia elata in the pond hole to culture bacteria cables after 16-24 months;
and (3) separating and purifying the rhizomorph: selecting a plurality of sections of light and strong strongylocentrotus obliquus on cultured strains, placing the sections in a culture dish, inoculating the sections to a PDA test tube slant culture medium according to a sterile operation procedure, labeling the inoculated test tube for recording, placing the test tube in a constant temperature box at 25 ℃, culturing for 3-10 days at a relative humidity of 60% -80%, and then selecting young strongylocentrotus obliquus growing in the culture medium for purification until pure strains with consistent strongylocentrotus growth form height are obtained by screening, namely, a test tube strain mother strain is obtained;
(2) and (3) strain propagation:
adding water according to the proportion of 20 percent of wheat bran or rice bran, 2 percent of glucose and 2 percent of agar to prepare a bottled solid culture medium of 500ml, and naturally adjusting the pH value; transferring the test tube with the preservation registration number of CGMCC No.11901 to a sterilized solid culture medium, and culturing in a dark room with the room temperature of 25 ℃ and the humidity of 70-80% for 25 days to obtain a secondary strain; the third-class seed is prepared as follows: the base material 1 is broad-leaved tree sawdust 78%, rice bran or wheat bran 20%, gypsum 10% and sucrose 1%; the base material 2 is a broad-leaved tree fresh branch knot which is soaked for 24-48 hours, has the length of 2-3cm and the diameter of 1-2 cm; the ratio of the base material 1 to the base material 2 is 1: 2, and the ratio of the total base material to water is 1: 1.5; filling the prepared base material into a strain bottle of 750ml to the shoulder of the bottle, covering and sterilizing, inoculating a second-level strain in an aseptic operation, and culturing the inoculated bottle in a dark room at the room temperature of 22-25 ℃ and the relative humidity of 70-80% for 50-60 days to obtain a third-level strain;
(3) the produced third-level strain can be directly used for the companion planting of the gastrodia elata in the Yunchuan region;
the gastrodia elata symbiotic armillaria mellea strains are classified and namedArmillaria nabsnonaT.J .Volk&Burds.And the preservation registration number is: CGMCC No. 11901; the culture characteristics of the strain are as follows: on a general PDA culture medium, hyphae flourish white, rhizomorph in the medium is white to light yellow, and aerial rhizomorph is light reddish brown; the rhizomorph is a single-axis branch, the growth is fast, the water discharge is late, and the color of the culture medium is little or unchanged.
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| CN106508359A (en) * | 2016-10-25 | 2017-03-22 | 成都中医药大学 | Method for cultivating gastrodia elata |
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| CN118120741A (en) * | 2024-04-28 | 2024-06-04 | 中国科学院昆明植物研究所 | Method for long-term preservation of seeds of Gastrodia plants of Orchidaceae |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1038743A (en) * | 1988-06-24 | 1990-01-17 | 中国医学科学院药用植物资源开发研究所 | Mycen osmundicola and gastrodia seed germination culture technique |
| CN101940116A (en) * | 2010-08-11 | 2011-01-12 | 北京中医药大学 | Technical method for culturing gastrodia elata at one time under forest |
| CN101999295A (en) * | 2010-10-26 | 2011-04-06 | 昭通神农乌天麻良种繁育有限公司 | Halimasch (XL-A0704) as well as preparation method and application thereof |
| CN102037853A (en) * | 2009-10-21 | 2011-05-04 | 辽宁省林业科学研究院 | Artificially cultured Grifulaumbelleta strain, Amillariella mellea and sclerotium production method for Grifulaumbelleta strain |
| CN102487716A (en) * | 2011-12-22 | 2012-06-13 | 大方县九龙天麻开发有限公司 | Gastrodiae elata planting method in primitive environment |
| CN104805023A (en) * | 2015-05-25 | 2015-07-29 | 北京农学院 | Halimasch fungicide preparing method and halimasch fungicide prepared by preparing method |
| CN104894006A (en) * | 2015-05-12 | 2015-09-09 | 贵阳医学院 | New brevibacillus brevis strain, cultivation method and application of new brevibacillus brevis strain |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR960003572B1 (en) * | 1993-09-03 | 1996-03-20 | 유연광 | Method for cultivating gastrodia elata blume |
-
2016
- 2016-03-18 CN CN201610155954.9A patent/CN105794495B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1038743A (en) * | 1988-06-24 | 1990-01-17 | 中国医学科学院药用植物资源开发研究所 | Mycen osmundicola and gastrodia seed germination culture technique |
| CN102037853A (en) * | 2009-10-21 | 2011-05-04 | 辽宁省林业科学研究院 | Artificially cultured Grifulaumbelleta strain, Amillariella mellea and sclerotium production method for Grifulaumbelleta strain |
| CN101940116A (en) * | 2010-08-11 | 2011-01-12 | 北京中医药大学 | Technical method for culturing gastrodia elata at one time under forest |
| CN101999295A (en) * | 2010-10-26 | 2011-04-06 | 昭通神农乌天麻良种繁育有限公司 | Halimasch (XL-A0704) as well as preparation method and application thereof |
| CN102487716A (en) * | 2011-12-22 | 2012-06-13 | 大方县九龙天麻开发有限公司 | Gastrodiae elata planting method in primitive environment |
| CN104894006A (en) * | 2015-05-12 | 2015-09-09 | 贵阳医学院 | New brevibacillus brevis strain, cultivation method and application of new brevibacillus brevis strain |
| CN104805023A (en) * | 2015-05-25 | 2015-07-29 | 北京农学院 | Halimasch fungicide preparing method and halimasch fungicide prepared by preparing method |
Non-Patent Citations (2)
| Title |
|---|
| Armillarian absnona,a new species from western North America;T.J.Volk等;《Mycologia》;19960331;第88卷(第3期);第484-491页 * |
| T.J.Volk等.Armillarian absnona,a new species from western North America.《Mycologia》.1996,第88卷(第3期),第484-491页. * |
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