CN115824757B - Dyeing method for endophytic fungi of camellia oleifera root system - Google Patents
Dyeing method for endophytic fungi of camellia oleifera root system Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Coloring (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a dyeing method of endophytic fungi of a camellia oleifera root system, which belongs to the technical field of plant tissue observation, and comprises the steps of firstly placing healthy camellia oleifera root segments in a KOH solution for water bath at 90 ℃ for 1H, and then transferring the healthy camellia oleifera root segments into H 2 O 2 And (3) the solution is transparent for 30min, and is soaked in HCl solution for 5min after the transparency is finished, and then is put into a coloring agent for dyeing, and decolorization is carried out after the dyeing is finished. The sasanqua root section treated by the sasanqua root endophytic fungus dyeing method has the advantages of transparency, good dyeing and decoloring effects, obvious contrast between the background and the endophytic fungus structure, and clear and visible endophytic fungus structure; the method has the advantages of simple operation, strong practicability, low cost, low expensive equipment dependence, small workload and high success rate, and can effectively solve the technical problem that the existing dyeing method of plant root fungi and leaf fungi is not suitable for dyeing endophytic fungi of the tea-oil tree root system.
Description
Technical Field
The invention relates to the technical field of plant tissue observation, in particular to a dyeing method of endophytic fungi of a tea-oil tree root system.
Background
The Camellia oleifera (Camellia oleifera Abel) refers generally to a species with higher seed oil content in Camellia genus (Camellia), and is a woody oil plant specific to subtropical regions in China. The tea oil is rich in unsaturated fatty acids mainly comprising oleic acid and linoleic acid, has high nutritive value and good health care effect, and is more and more favored by people. The key of healthy development of the oil tea industry is to ensure high yield and high efficiency, but the problems of aging of oil tea varieties and low single yield of oil tea still exist in the current development process of the oil tea industry, especially, the soil in an oil tea planting area is acidic, the physical and chemical properties of the soil are changed, the quality is reduced, and the oil tea is seriously degraded, so that the method becomes one of important factors for restricting the development of the oil tea industry. Endophytic fungi are fungi which are parasitic in healthy plants, complete all or nearly all life cycles in the tissues, organs or cell gaps of host plants, and cause no obvious disease symptoms to plant tissues. The plant endophytic fungi not only can help the host to absorb nutrient elements, synthesize plant hormones and enhance photosynthesis of plants, but also can improve the resistance of the host to interference and stress of biological factors and abiotic factors, and play an important role in different ecological systems. Therefore, research on endophytic fungi of the tea-oil tree root system is developed, and the method has important significance for developing biological bacterial fertilizer suitable for the growth and development requirements of the tea-oil tree, improving the soil ecology of the tea-oil tree forest land and promoting the high yield of the tea-oil tree.
The observation of the infection condition and the structural characteristics of the plant endophytic fungi is the basis for researching the plant endophytic fungi, and the observation is mainly carried out by adopting a color-dyeing inspection method at present. The dyeing effect of 9 plant root systems endophytic fungi such as juniper, masson pine, cyclobalanopsis glauca, loropetalum chinense, blueberry, dayflower, castanopsis sclerophylla, moringa oleifera, nutgrass galingale rhizome and the like is analyzed in the early stage of the subject group, and the obvious difference of the dyeing effect of the same dyeing method on different plant root systems endophytic fungi is found. The early stage of the subject group also adopts a dyeing method for observing poplar root system fungi disclosed in CN104472331B and a dyeing method for observing plant leaf fungal disease histopathological process disclosed in CN106383047 to dye camellia oleifera root system fungi, and no good dyeing effect is realized. Therefore, the invention provides a dyeing method of endophytic fungi of a tea-oil tree root system.
Disclosure of Invention
The invention provides a dyeing method of endophytic fungi of a camellia oleifera root system, which effectively solves the technical problem that the existing dyeing method of the fungi of the plant root system and the fungi of the leaf parts is not suitable for dyeing the endophytic fungi of the camellia oleifera root system, and simultaneously provides a dyeing method of the endophytic fungi of the camellia oleifera root system with excellent dyeing effect.
The invention provides a dyeing method of endophytic fungi of a tea-oil tree root system, which is characterized by comprising the following steps of:
s1, soaking the oil tea root system in KOH solution after ultrasonic treatment, and washing in a water bath at 90 ℃ for 1h to obtain a pretreated oil tea root system;
s2, putting the pretreated camellia oleifera root system described in S1 in H 2 O 2 Soaking the tea-oil tree root in the solution for 30min, cleaning, soaking the tea-oil tree root in the HCl solution for 5min, and cleaning to obtain the transparent tea-oil tree root system;
s3, dyeing: and (3) placing the transparent-treated tea-oil camellia root system in a staining solution, staining, and decoloring with ethanol to obtain a stained tea-oil camellia root system.
Preferably, in S3, the staining solution includes trypan blue staining solution and water-soluble aniline blue staining solution.
Preferably, in S3, the dyeing time is 3 to 5 hours.
Preferably, in S3, the decolorization is performed by adopting an ethanol solution with the mass concentration of 70% for 50-70 min.
Preferably, in S1, the mass concentration of the KOH solution is 8% -12%.
Preferably, in S1, the feed liquid ratio of the tea-oil camellia root system to the KOH solution is 1 g:6-12 ml.
Preferably, in S2, the H 2 O 2 The mass concentration of the solution is 9-11%, and the pH value is 6.8-7.5.
Preferably, the pretreatment of the root system and H of the oil tea 2 O 2 The feed liquid ratio of the solution is 1 g:6-12 ml.
Preferably, in S2, the mass concentration of the HCl solution is 1%, and the feed liquid ratio of the pretreated camellia oleifera root system to the HCl solution is 1 g:6-12 ml.
Preferably, the preparation method of the trypan blue dye liquor comprises the following steps: uniformly mixing glycerol, lactic acid and distilled water according to the volume ratio of 1:3:1, adding trypan blue according to the feed liquid ratio of 1g to 400ml of trypan blue to distilled water, and fully dissolving to obtain the preparation; the preparation method of the water-soluble aniline blue dye solution comprises the following steps: mixing water-soluble aniline blue and distilled water according to a feed liquid ratio of 1g to 1000 mL.
Compared with the prior art, the invention has the beneficial effects that:
(1) Firstly, putting healthy camellia oleifera root segments into KOH solution for water bath at 90 ℃ for 1H, and then transferring into H 2 O 2 And (3) the solution is transparent for 30min, and is soaked in HCl solution for 5min after the transparency is finished, and then is put into a coloring agent for dyeing, and decolorization is carried out after the dyeing is finished. The sasanqua root section treated by the sasanqua root endophytic fungus dyeing method has the advantages of transparency, good dyeing and decoloring effects, obvious contrast between the background and the endophytic fungus structure, and clear and visible endophytic fungus structure.
(2) The dyeing method of the oil tea root system endophytic fungi provided by the invention has the advantages of simple operation method, strong practicability, low cost, low expensive equipment dependence, small workload and high success rate, and can effectively solve the technical problem that the existing dyeing method of plant root system fungi and leaf fungi is not suitable for dyeing the oil tea root system endophytic fungi.
Drawings
FIG. 1 is an optical microscope image of the tea-oil camellia root system of example 1 after passing through aniline blue dye liquor; wherein a is hypha; b is an intracellular hypha; c-e are hyphae and microsclerotia; f is that hypha is connected with microsclerotium; g is intracellular hooked hypha; h is an intracellular annular hypha; i is intracellular hypha and microsclerotia; j-l are microsclerotia;
FIG. 2 is an optical microscope image of the tea-oil camellia root system of example 2 after being dyed by trypan blue dye liquor; wherein a is hypha; b is hypha and intracellular annular hypha; c is an intracellular mycelium pellet; d-e is vesicle; f is intracellular annular hypha and microsclerotium; g is spore; h-j is microsclerotia; k is hypha enlargement and microsclerotium; l is hypha and microsclerotium;
FIG. 3 is an optical microscope image of a tea-oil camellia root system of comparative example 1 after dyeing with a cotton blue dye liquor; wherein a-b are hyphae; c is that hypha is connected with microsclerotium; d is intracellular hooked hypha; e-f are intracellular annular hyphae; g-l is microsclerotia;
FIG. 4 is an optical microscope image of a tea-oil camellia root system of comparative example 2 after being dyed by an acid fuchsin dye liquor; wherein a-b are hyphae; c is hypha and microsclerotium; d-e is intracellular hypha; f is an intracellular mycelium pellet; g is intercellular hypha; h is vesicle; i-j is microsclerotia; k-l is hypha enlargement and microsclerotia;
FIG. 5 is an optical microscope image of a tea-oil camellia root system of comparative example 3 after being dyed by a black ink dye liquor; wherein a is hypha; b is an intracellular mycelium pellet; c-e are vesicles; f is that microsclerotium is connected with hypha; g is intracellular annular hypha and microsclerotium; h is microsclerotium and intracellular hooked hypha; i-l is microsclerotia;
FIG. 6 is an optical microscope image of the tea-oil camellia root system of comparative example 4 after being dyed by a methylene blue dye solution; wherein a is an intracellular annular hypha; b is hypha; c is a vesicle; d is microsclerotium;
FIG. 7 is an optical microscope image of a tea-oil camellia root system of comparative example 5 after being dyed with Sudan red IV dye liquor; wherein a is hypha and spore; b is hypha; c is an intracellular annular hypha; d is a vesicle; e-h is microsclerotia; i is a spore;
FIG. 8 is an optical microscope image of a tea-oil camellia root system of comparative example 6 after being dyed with a red ink dye liquor; wherein a-b are hyphae; c-d is that hypha is connected with microsclerotium; e is intracellular hooked hypha; f is hypha; g-l is microsclerotia;
FIG. 9 is an optical microscope image of a tea-oil camellia root system of comparative example 7 after dyeing with a blue ink dye liquor; wherein a is background control; b is hypha; c is intracellular hooked hypha; d is microsclerotium;
FIG. 10 is an optical microscope image of a tea-oil camellia root system of comparative example 8 after Congo red dye liquor staining; wherein a is background control; b is microsclerotium; c is an intracellular hypha; d is an intracellular bacterial screen;
FIG. 11 is an optical microscope image of a tea-oil camellia root system of comparative example 9 after staining with a staining method of poplar root systems; wherein a-e are hyphae; f is microsclerotia; g-i is hypha;
FIG. 12 is an optical microscope image of a tea-oil camellia root system of comparative example 10 after staining with a plant leaf staining method; wherein a-e are hyphae; f is hyphae and microsclerotia.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention. The following test methods and detection methods, if not specified, are conventional methods; the reagents and starting materials, unless otherwise specified, are commercially available.
The invention uses the tea-oil camellia root system as raw material to carry out dyeing experiment.
An ultrasonic cleaning instrument (model: shumei KQ 3200E) is adopted, the working frequency is 40kHz, the ultrasonic electric power is 150W, and the ultrasonic time is 15-30 min.
The preparation method comprises the following steps: transferring the camellia oleifera root segments onto a glass slide, and carrying out tabletting observation by using distilled water.
The preparation method of the trypan blue dye liquor comprises the following steps: mixing 20mL of glycerol, 60mL of lactic acid and 20mL of distilled water, adding 0.05g of trypan blue, and fully dissolving.
The preparation method of the water-soluble aniline blue dye solution comprises the following steps: dissolving 0.1g of water-soluble aniline blue in 100mL of distilled water, and uniformly mixing to obtain the aqueous aniline blue.
Example 1
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 6mL of feed liquid ratio in KOH solution with the mass concentration of 8%, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:6mL of feed liquid is compared with H with the mass concentration of 9% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 6mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated camellia oleifera root system in a water-soluble aniline blue staining solution, staining for 4 hours, and decolorizing for 50 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain the stained camellia oleifera root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Example 2
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 12mL of feed liquid in KOH solution with the mass concentration of 12%, and washing in a water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:12mL of feed liquid is compared with H with the mass concentration of 11% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 12mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated camellia oleifera root system in a water-soluble aniline blue staining solution, staining for 5h, and decolorizing for 70min by adopting an ethanol solution with the mass concentration of 70% to obtain the stained camellia oleifera root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Example 3
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated camellia oleifera root system in a water-soluble aniline blue staining solution, staining for 3h, and decolorizing for 60min by adopting an ethanol solution with the mass concentration of 70% to obtain the stained camellia oleifera root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Example 4
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 6mL of feed liquid ratio in KOH solution with the mass concentration of 8%, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:6mL of feed liquid is compared with H with the mass concentration of 9% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 6mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated tea-oil camellia root system in trypan blue staining solution, staining for 4 hours, and decolorizing for 50 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain the stained tea-oil camellia root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Example 5
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 12mL of feed liquid in KOH solution with the mass concentration of 12%, and washing in a water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:12mL of feed liquid is compared with H with the mass concentration of 11% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 12mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated tea-oil camellia root system in trypan blue staining solution, staining for 5h, and decolorizing for 70min by adopting an ethanol solution with the mass concentration of 70% to obtain the stained tea-oil camellia root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Example 6
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: and (3) placing the transparent-treated tea-oil camellia root system in trypan blue staining solution, staining for 3h, and decolorizing for 60min by adopting an ethanol solution with the mass concentration of 70% to obtain the stained tea-oil camellia root system.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
In order to further illustrate the technical effects of the present invention, the present invention is further provided with comparative examples, specifically as follows:
comparative example 1
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in the cotton-blue staining solution, staining for 3 hours, and decolorizing for 60 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain a stained tea-oil camellia root system;
the preparation method of the cotton-blue staining solution comprises the following steps: adding 0.2g of cymbidium into 120g of lactic acid, and uniformly mixing.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 2
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated camellia oleifera root system in the S2 in an acidic fuchsin staining solution, staining for 3h, and decolorizing with 70% ethanol solution for 60min to obtain a stained camellia oleifera root system;
the preparation method of the acid fuchsin staining solution comprises the following steps: mixing 20mL of glycerol, 60mL of lactic acid and 20mL of distilled water, adding 0.05g of acid fuchsin, and dissolving completely.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 3
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in the black ink dyeing liquid for dyeing for 3 hours, and decoloring for 60 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain a dyed tea-oil camellia root system;
the preparation method of the black ink dyeing liquid comprises the following steps: and adding 5mL hero black ink into 95mL glacial acetic acid with mass concentration of 5%, and fully and uniformly mixing to obtain the hero black ink.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 4
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated camellia oleifera root system in the methine blue staining solution, staining for 3 hours, and decolorizing for 60 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain a stained camellia oleifera root system;
the preparation method of the methylene blue staining solution comprises the following steps: and fully dissolving 0.5g of methylene blue in 100mL of distilled water.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 5
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in Sudan red IV staining solution, staining for 3h, and decolorizing with 70% ethanol solution for 60min to obtain a stained tea-oil camellia root system;
the preparation method of the Sudan IV staining solution comprises the following steps: mixing 10mL of 95% ethanol with 10mL of glycerol, adding 0.1g of Sudan IV, and mixing.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 6
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in the red ink dyeing liquid for dyeing for 3 hours, and decoloring for 60 minutes by adopting an ethanol solution with the mass concentration of 70% to obtain a dyed tea-oil camellia root system;
the preparation method of the red ink dyeing liquid comprises the following steps: and adding 5mL of Boss card red ink into 95mL of 5% glacial acetic acid, and fully and uniformly mixing to obtain the ink.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 7
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated camellia oleifera root system in the blue ink dyeing liquid, dyeing for 3h, and decoloring for 60min by adopting an ethanol solution with the mass concentration of 70% to obtain a dyed camellia oleifera root system;
the preparation method of the blue ink dyeing liquid comprises the following steps: and adding 5mL of 'Boss' blue ink into 95mL of glacial acetic acid with the mass concentration of 5%, and fully and uniformly mixing to obtain the ink.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 8
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, after ultrasonic treatment of the root system of the oil tea, 1g of the oil tea is as follows: soaking 8mL of feed liquid ratio in 10% KOH solution, and washing in water bath at 90 ℃ for 1h to obtain pretreated camellia oleifera root systems;
s2, the pretreated camellia oleifera root system in the step S1 is prepared according to the following weight of 1g:8mL of feed liquid is compared with H with the mass concentration of 10% and the pH value of 6.8-7.5 2 O 2 Soaking the solution for 30min, cleaning, and then mixing according to 1g: feed to liquid ratio of 8mL will be H 2 O 2 Soaking the camellia oleifera root system soaked in the solution in an HCl solution with the mass concentration of 1% for 5min, and cleaning to obtain a transparent camellia oleifera root system;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in Congo red staining solution for staining for 3 hours, and decolorizing with 70% ethanol solution for 60min to obtain a stained tea-oil camellia root system;
the preparation method of the Congo red staining solution comprises the following steps: 1g Congo red is weighed and fully dissolved in 100mL of distilled water to obtain the compound.
S4, tabletting and observing: transferring the root system of the dyed oil tea to a glass slide, tabletting with distilled water, observing under an optical fiber, photographing, and calculating the colonisation rate.
Comparative example 9 (dyeing method of poplar root system)
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, preparing a mixed solution according to the following mass components: naCl8g, KCl0.2g, na 2 HPO 4 ·12H 2 O3.6g、KH 2 PO 4 0.24g, regulating the pH to 7.4 by using NaOH and HCl, adding water to a volume of 1L, taking 95mL of mixed solution, uniformly mixing with 5mL of 8% NaClO solution to form a transparent solution, and preserving at 4 ℃;
s2, collecting fresh camellia oleifera root systems, flushing with running water, and cutting into root sections with the length of 5 cm;
s3, immersing the root segments of the camellia oleifera root system into the transparent solution completely, and immersing at 20 ℃ for 10min.
S4, washing clean with clear water until no transparent solution residue exists, and finishing the transparent root system of the oil tea;
s5, carrying out fungus colonization microscopic observation on the transparent camellia oleifera root system: the transparent tea-oil camellia root system uses 10% H 2 O 2 Softening for 2 minutes, acidifying for 3 minutes by 2% HCl, then dyeing by trypan blue dye liquor, and mixing lactic acid and glycerin according to a volume ratio of 1:1, after decolorization, making a tablet, observing and photographing, and calculating the colonisation rate.
Comparative example 10 (dyeing method of plant leaves)
The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, preparing a reagent according to the following steps: (1) preparing a fixed decoloring liquid: 100% trichloroacetic acid is prepared by adding 227ml of water into 500g of trichloroacetic acid, mixing 100% trichloroacetic acid solution, ethanol and chloroform according to the ratio of 3/150/50, and storing in brown bottle; (2) preparing a transparent coloring agent: 20g of chloral hydrate is added with 4-5 ml of glycerin and 5-6 ml of water, and is stored in a glass jar after being fully dissolved. Uniformly mixing the chloral hydrate solution and the staining solution in a ratio of 6/1-4/1, and preheating at 60 ℃ for 1min.
S2, collecting fresh camellia oleifera root systems, flushing with running water, and cutting into root sections with the length of 5 cm;
s3, fixed decolorization: immersing the sample block in a fixed decolorizing solution, and after the fixed decolorizing is carried out for 4 to 6 hours, replacing the fixed decolorizing solution, and carrying out fixed decolorizing for 4 to 6 hours;
s4, transparent dyeing: immersing the fixed decolored sample block into a transparent coloring agent, and heating at 40-60 ℃ for 4-6 h;
s5, tabletting and observing.
In the invention, the dyeing effect of the water-soluble aniline blue adopted in the embodiments 1-3 is similar, the trypan blue adopted in the embodiments 4-6 is similar, the dyeing effect of the water-soluble aniline blue dye liquor and the trypan blue dye liquor on the endophytic fungi of the camellia root system is illustrated by taking the embodiment 3 and the embodiment 6 as examples, and as can be seen from the figures 1-2, the dyeing effect of the water-soluble aniline blue is good, the faded background is light green, and the blue-dyed intracellular hypha, brown endophyte and microsclerotium can be observed to be distributed in root tissues, but the structures such as vesicles and the radicle are not observed; the camellia root cortex dyed by trypan blue is dyed into blue green, brown endophytic hypha, microsclerotium and endophytic fungi structures dyed into deep blue intracellular hypha, vesicle, microsclerotium and the like can be clearly observed under a microscope.
As can be seen from fig. 3 to 10, the camellia root cortex cells dyed with cymbidium are blue, the endophytic hyphae and the microsclerotia are brown, and the intracellular hooked hyphae are blue. The background fading effect is poor, so that the observation effect is slightly deficient; after the tea-oil tree root system is dyed by acid fuchsin, structures such as intracellular hyphae, vesicles and partial microsclerotia in root cortex cells are dyed into red, the whole root system is transparent enough, brown endophytic hyphae and microsclerotia can be seen, and although the observation effect is good after the acid fuchsin is dyed, the dyeing effect is unstable and can be continuously faded; after the tea-oil camellia root system is dyed by black ink, structures such as light black intracellular hooked hyphae, vesicles, brown endophytic hyphae, microsclerotium and the like can be observed. However, the background cannot fade completely, impurities in the root system are dyed into dark colors, and the observation effect is slightly lacking; effect of methine blue on dyeing of sasanqua root endophytic fungi: the dyeing is too deep, the fading is not obvious, the structures such as endophytic hypha, microsclerotium, vesicle and the like and root tissues are dyed into deep blue, the background is not obvious in contrast with the structure of endophytic fungi, and the structures are not easy to observe and distinguish; after being dyed, sudan IV can clearly see the structures of lipid-rich vesicles, rhizogenes, microsclerotia and the like dyed in red, but partial intracellular hyphae and microsclerotia are not colored or have poor coloring effect, more impurities and poor observation effect; the coloring effect of the endophytic fungus structure of the tea-oil camellia root system dyed by the red ink is poor, but brown endophytic hypha, microsclerotium and other structures can be seen due to the good tissue transparency effect. The impurities in the root system are more, and the structures such as the background and hypha are not easy to distinguish, so that experimental observation is not facilitated; blue ink has poor dyeing effect on endophytic fungi of the root system of the camellia oleifera, is too deep in dyeing and has poor background fading effect, endophytic hypha and microsclerotium are dyed into the same deep blue, the contrast is not obvious, and root cortex impurities are dyed into deep colors, so that the observation is not facilitated; the congo red has extremely poor dyeing effect on the endophytic fungi of the tea-oil tree root system, the root system is difficult to fade after dyeing, the root tissue and the endophytic fungi are dyed red, and the endophytic fungi structure is difficult to observe.
As can be seen from fig. 11 to 12, the solutions of comparative example 9 (poplar root system dyeing method) and comparative example 10 (plant leaf dyeing method) are adopted, the observation effect of the endophytic fungi structure of the camellia root treated by the poplar root system dyeing method is extremely poor, the transparency effect of the root system is poor, the background is dark, the impurities are more, and only the bluish endophytic fungi structure dyed in dark blue can be observed; the dyeing effect of the plant leaf dyeing method on endophytic fungi of the tea-oil tree root system is also poor, the treated root system is not transparent enough, the contrast with the endophytic fungi structure is not obvious, and the structures such as endophytic fungi hypha and microsclerotium are invisible.
Under an optical microscope, the oil tea root systems treated by 10 different staining solutions in examples 1-2 and comparative examples 1-8 were observed, and the structures of hyphae, microsclerotium, vesicles, spores and the like of endophytes were observed in each treatment root system, but the results of the calculation of the staining effect, the fading effect, the observation effect and the colonization rate of endophytes in the oil tea root systems by different staining agents were greatly different, and the results are shown in table 1.
Table 110 comparison of staining effect and colonisation ratio of staining solutions
As is clear from Table 1, the camellia oleifera roots dyed with methylene blue are difficult to fade, the observation effect is poor, and the calculated colonisation rate is only 50%. The camellia oleifera roots dyed by acid fuchsin have good observation effect, and endophytic fungus structures can be observed in 20 root segments, but dyeing is unstable and continuous fading can be realized. The blue ink has poor dyeing effect, is difficult to fade after dyeing, has unobvious contrast between the background and the endophytic fungus structure, and has the colonization rate of 60 percent. The black ink is generally dyed in a dark color with incomplete fading and impurities, and the calculated colonisation rate is 90%. The red ink is not colored on the endophytic fungi of the camellia oleifera root, but the endophytic fungi structure can be observed in 20 root segments due to the enough transparency of the root system. Sudan IV can only dye part of endophytic fungi structure, and after dyeing, the background impurities are more, and the calculated colonisation rate is only 55%. The background was not completely discolored after dyeing of cotton blue, but the structure of the endophyte was basically observed, and the calculated colonisation rate was 95%. The dyeing effect of the water-soluble aniline blue is good, the endophytic fungus structures can be observed in root segments, but the endophytic fungus structures such as vesicles and the like can not be observed. Congo red is too deeply dyed, has extremely poor observation effect, is difficult to observe endophytic fungus structures, and has a colonization rate as low as 20%. The dyeing effect of trypan blue is optimal, and the endophytic fungi structures of 20 root segments are clearly visible.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (1)
1. The dyeing method of the endophytic fungi of the tea-oil tree root system is characterized by comprising the following steps of:
s1, soaking the oil tea root system in KOH solution after ultrasonic treatment, and washing in a water bath at 90 ℃ for 1h to obtain a pretreated oil tea root system; the mass concentration of the KOH solution is 8% -12%; the feed liquid ratio of the tea-oil camellia root system to the KOH solution is 1 g:6-12 ml;
s2, putting the pretreated camellia oleifera root system described in S1 in H 2 O 2 Soaking the tea-oil tree root in the solution for 30min, cleaning, soaking the tea-oil tree root in the HCl solution for 5min, and cleaning to obtain the transparent tea-oil tree root system; the H is 2 O 2 The mass concentration of the solution is 9% -11%, and the pH value is 6.8-7.5; the pretreatment of the root system and H of the oil tea 2 O 2 The feed liquid ratio of the solution is 1 g:6-12 ml; the mass concentration of the HCl solution is 1%, and the feed liquid ratio of the pretreated camellia oleifera root system to the HCl solution is 1 g:6-12 ml;
s3, dyeing: placing the transparent-treated tea-oil camellia root system in the S2 in a dyeing liquid, dyeing for 3-5 h, and decoloring for 50-70 min by adopting an ethanol solution with the mass concentration of 70% to obtain a dyed tea-oil camellia root system; the dyeing liquid comprises trypan blue dyeing liquid and water-soluble aniline blue dyeing liquid;
the preparation method of the trypan blue dye liquor comprises the following steps: uniformly mixing glycerol, lactic acid and distilled water according to the volume ratio of 1:3:1, adding trypan blue according to the feed liquid ratio of 1g to 400ml of trypan blue to distilled water, and fully dissolving to obtain the preparation; the preparation method of the water-soluble aniline blue dye solution comprises the following steps: mixing water-soluble aniline blue and distilled water according to a feed liquid ratio of 1g to 1000 mL.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181511A (en) * | 2011-02-22 | 2011-09-14 | 贵州省果树科学研究所 | Method for simultaneously dyeing mycelial masses and starch in mycorhiza of arethusa |
| CN103411813A (en) * | 2013-05-28 | 2013-11-27 | 江苏大学 | Method for rapidly and efficiently dyeing arbuscular mycorrhizal fungi |
| CN104764646A (en) * | 2015-03-06 | 2015-07-08 | 广西壮族自治区农业科学院花卉研究所 | A paphiopedilum concolor mycorrhiza staining method |
| CN106383047A (en) * | 2016-11-09 | 2017-02-08 | 北京林业大学 | Staining method for observing histopathologic process of fungus disease in leaf segment of plant |
| CN107446985A (en) * | 2017-08-30 | 2017-12-08 | 李保华 | A set of Apple top layer colonizes and infected the rapid examination method of fungi |
| CN108760446A (en) * | 2018-05-18 | 2018-11-06 | 北京林业大学 | A kind of one body swimming bath for arbuscular mycorrhiza trypan blue staining |
| CN114459860A (en) * | 2021-12-23 | 2022-05-10 | 华南农业大学 | Efficient dyeing method for arbuscular mycorrhizal fungi in root of woody plant and application of efficient dyeing method |
-
2022
- 2022-12-28 CN CN202211697076.5A patent/CN115824757B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181511A (en) * | 2011-02-22 | 2011-09-14 | 贵州省果树科学研究所 | Method for simultaneously dyeing mycelial masses and starch in mycorhiza of arethusa |
| CN103411813A (en) * | 2013-05-28 | 2013-11-27 | 江苏大学 | Method for rapidly and efficiently dyeing arbuscular mycorrhizal fungi |
| CN104764646A (en) * | 2015-03-06 | 2015-07-08 | 广西壮族自治区农业科学院花卉研究所 | A paphiopedilum concolor mycorrhiza staining method |
| CN106383047A (en) * | 2016-11-09 | 2017-02-08 | 北京林业大学 | Staining method for observing histopathologic process of fungus disease in leaf segment of plant |
| CN107446985A (en) * | 2017-08-30 | 2017-12-08 | 李保华 | A set of Apple top layer colonizes and infected the rapid examination method of fungi |
| CN108760446A (en) * | 2018-05-18 | 2018-11-06 | 北京林业大学 | A kind of one body swimming bath for arbuscular mycorrhiza trypan blue staining |
| CN114459860A (en) * | 2021-12-23 | 2022-05-10 | 华南农业大学 | Efficient dyeing method for arbuscular mycorrhizal fungi in root of woody plant and application of efficient dyeing method |
Non-Patent Citations (2)
| Title |
|---|
| D. R. Bryla et al..Effects of mycorrhizal infection on drought tolerance and recovery in safflower and wheat.《Plant and Soil》.1997,第197卷95-103. * |
| 郑世学等.以新鲜根段进行AM真菌的分子检测及竞争性侵染研究.《菌物学报》.2004,第23卷(第01期),126-132. * |
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