CN114459860A - Efficient dyeing method for arbuscular mycorrhizal fungi in root of woody plant and application of efficient dyeing method - Google Patents
Efficient dyeing method for arbuscular mycorrhizal fungi in root of woody plant and application of efficient dyeing method Download PDFInfo
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention relates to the technical field of applied microbiology, in particular to a high-efficiency dyeing method for arbuscular mycorrhizal fungi in woody plant roots and application thereof. The efficient dyeing method of the arbuscular mycorrhizal fungi in the woody plant roots is applied to observing the growth state of the arbuscular mycorrhizal fungi in the woody plant roots or measuring the infection rate of the arbuscular mycorrhizal fungi in the woody plant roots.
Description
Technical Field
The invention relates to the technical field of applied microbiology, in particular to a high-efficiency dyeing method for arbuscular mycorrhizal fungi in woody plants and application thereof.
Background
Arbuscular Mycorrhizal Fungi (AMF) belong to Glomeomyceta fungi, are obligate living nutrition symbiotic fungi, and can form a reciprocal symbiont with more than about 80 percent of plant root systems (about 20 ten thousand) on land, namely Arbuscular Mycorrhiza (AM). The arbuscular mycorrhiza has a hypha network which is widely distributed in the soil, so that the absorption of mineral nutrients such as nitrogen and phosphorus by the root system of the plant is promoted, the soil structure is improved, the stress resistance of the plant is improved, and the improvement of the yield and the quality of the plant is facilitated. Therefore, the AM fungus has wide application potential in agricultural production.
The basic work of mycorrhizal research is to observe the growth state of mycorrhiza and determine the infection rate of mycorrhiza, and almost all mycorrhizal fungi inoculation tests relate to the work, so that people pay great attention to the research of the observation and determination methods for more than half a century and establish a series of important methods for researching mycorrhizal fungi. Mycorrhizal fungi is usually dyed and then observed for characteristics, but due to the fact that woody plants are high in lignification degree, thick and deep in pigment, microscopic observation and photographing record of AM fungal microstructures (such as arbuscular branches) are not facilitated, whether the plants are infected by the AM fungi can only be judged reluctantly, and data such as infection strength (M%) and arbuscular abundance (A%) cannot be calculated accurately. Therefore, the research on the dyeing method capable of clearly observing the AM fungus microstructure in the root system of the woody plant has important significance.
Disclosure of Invention
One of the purposes of the invention is to provide an efficient dyeing method of arbuscular mycorrhizal fungi in woody plant roots, which can efficiently dye the arbuscular mycorrhizal fungi in the woody plant roots, so that the arbuscular mycorrhizal fungi in the woody plant roots are uniformly dyed and have clear images, and the observation of the fine structure of the arbuscular mycorrhizal fungi in the woody plant roots is facilitated.
The invention also aims to provide an application of the efficient dyeing method of the arbuscular mycorrhizal fungi in the roots of the woody plants.
In order to achieve one of the above purposes, the invention provides the following technical scheme:
the method is characterized by carrying out tissue slicing on the arbuscular mycorrhiza in the woody plant root to be stained to ensure that the thickness of the slice is 60-100 mu m, and carrying out staining treatment on the obtained slice.
In some embodiments, the slice thickness is 80 μm
In some embodiments, the step of tissue sectioning comprises selecting a root segment of arbuscular mycorrhiza within woody plant roots to be stained having a length of 0.5cm to 2cm, embedding and fixing it on a slide with low melting point agarose, sectioning using a vibrating microtome Leica VT 1000S, the sectioning procedure being: the slicing speed is 0.165 mm/s-0.175 mm/s, and the slicing frequency is 40 Hz-60 Hz.
In some embodiments, the method of staining treatment is trypan blue staining comprising the steps of:
a) digestion: adding 6-10% (W/V) KOH into the slices, and removing cytoplasm in the cells by water bath for 50-60 min at 80-90 ℃;
b) acidifying: taking out the slices, washing, adding 0.15-0.2M HCl, and standing at room temperature for 4-5 min;
c) dyeing: adding 0.03-0.05% (W/V) trypan blue dye solution for dyeing, and dyeing for 6-10 min at room temperature;
d) and (3) decoloring: placing the mixture in a decoloring solution for decoloring for 1 d-2 d to decolor the structure of the non-arbuscular mycorrhizal fungi;
e) observation and recording of photographs.
In the trypan blue staining method, the arbuscular mycorrhiza in the woody plant root can be effectively stained without a bleaching step, and the effect of simplifying experimental steps is achieved.
In some embodiments, in step d), the decolorized solution is obtained by mixing lactic acid, glycerin and distilled water, wherein the volume ratio of the lactic acid to the glycerin to the distilled water is (1-2): (1-2): (1-2).
In some embodiments, the method of staining treatment is a dual fluorescent staining method comprising the steps of:
a) bleaching: putting the slices into 40-50% (W/V) ethanol for incubation for 1-3 h, and bleaching the root system;
b) and (3) transparency: adding 15-20% (W/V) KOH solution to incubate for 1-2 d to make the root system transparent;
c) dyeing: taking out the slices, washing the slices with PBS (phosphate buffer solution) with the pH value of 7-7.4, and adding 0.15 ug/ml-0.2 ug/ml Alexa
WGA488 fluorescent dye, incubating for 10-15 h at room temperature, and then adding 0.1-2 ug/ml Sigma-
Carrying out vacuum incubation for 20-30 min by using Nile Red fluorescent dye;
d) cleaning: washing the substrate for more than 3 times by using PBS (phosphate buffer solution) with the pH value of 7-7.4 to wash away the flooding;
e) observation and recording of photographs.
The efficient dyeing method of the arbuscular mycorrhizal fungi in the root of the woody plant has the beneficial effects that:
(1) the invention slices the arbuscular mycorrhiza in the woody plant root, and overcomes the problem that the arbuscular mycorrhiza in the woody plant root is difficult to stain. The sliced arbuscular mycorrhiza in the woody plant root has proper thickness, the dyeing effect is greatly enhanced, and the slice thickness is thinner, so that the arbuscular mycorrhiza in the woody plant root is uniformly dyed in layers and clear in image, the fine structure of the arbuscular mycorrhiza in the woody plant root can be clearly observed, the efficient dyeing of the arbuscular mycorrhiza in the woody plant root is realized, an effective dyeing method is provided for mycorrhiza researchers, and the method has strong practical application value and scientific research theoretical value.
(2) The efficient dyeing method of the arbuscular mycorrhizal fungi in the woody plant roots is convenient and fast, and is suitable for large-scale application.
In order to achieve the second purpose, the invention provides the following technical scheme:
the application of the efficient dyeing method of the arbuscular mycorrhizal fungi in the woody plant roots is provided, and the efficient dyeing method of the arbuscular mycorrhizal fungi in the woody plant roots is applied to observation of the growth state of the arbuscular mycorrhizal fungi in the woody plant roots or measurement of the infection rate of the arbuscular mycorrhizal fungi in the woody plant roots.
The application of the efficient dyeing method for the arbuscular mycorrhizal fungi in the roots of the woody plants can clearly observe the fine structure of the mycorrhiza and provide a foundation for mycorrhizal research.
Drawings
FIG. 1 shows the staining of the mycorrhiza of bitter orange by the conventional Trypan blue staining method, and the scales are 200 μm, 100 μm and 50 μm from left to right.
FIG. 2 shows the staining of the mycorrhiza of bitter orange by trypan blue staining method of example 1, and the scales are 200 μm, 100 μm and 50 μm from left to right.
FIG. 3 shows staining conditions of mycorrhiza of fructus Aurantii by conventional double fluorescent staining method, wherein the scales are 50 μm, 20 μm and 10 μm from top to bottom, and the views from left to right are WGA-488 staining pattern, Nile Red staining pattern and double staining pattern.
FIG. 4 shows how the mycorrhiza of Aurantii Immaturus is stained by the double fluorescent staining method of example 4, wherein the scale is 50 μm, 20 μm and 10 μm from top to bottom, and the WGA-488 staining pattern, Nile Red staining pattern and double staining pattern are shown from left to right.
Reference numerals
Ad-developing arbuscular; am — mature arbuscular; as-senescent arbuscular; a-multiple branches; h-hyphae; v-vesicles; NL-neutral lipid.
Detailed Description
The present invention will be further described with reference to the following specific examples and accompanying drawings, in order to illustrate the efficacy of the staining method of the present invention, the examples used a model strain of AM fungus, Rhizopus angus irregularis DAOM 197198, as a microbial inoculum, and a representative woody plant, Poncirus trifoliata (L.) Raf, as a test plant, and the seeds of Citrus aurantium were surface-sterilized with 1.5% NaClO solution, and then placed in an incubator at 28 ℃ for germination for one week. After the seeds germinate, the seeds are uniformly spread into a plastic pot filled with sterilized turf (121 ℃, 1h, 2 times), after 8 weeks of cultivation, the bitter orange seedlings are transplanted into a plastic flowerpot filled with 1kg of matrix (diatomite: river sand is 2: 1), and the plastic flowerpot is placed in a greenhouse for cultivation for 6 weeks and sampling is carried out.
The following examples are not intended to limit the invention in any way.
Example 1
The embodiment discloses a trypan blue staining method for staining the mycorrhiza of fructus aurantii, which comprises the following specific steps:
s1, patch: selecting more than 30 root segments with the length of 1cm, embedding the root segments by using low-melting-point agarose, and fixing the root segments on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.175mm/s, the slicing frequency is 50Hz, and the slicing thickness is 80 μm; wherein 80 μm is the optimum slice thickness;
s3, digestion: adding 10% (W/V) KOH, and water bathing at 90 deg.C for 60min to remove cytoplasm from cells for easy observation;
s4, acidification: after washing, adding 0.2M HCl, standing at room temperature for 5min, creating an acid environment and facilitating the coloring of trypan blue dye;
s5, dyeing: adding 0.05% (W/V) trypan blue dye solution for dyeing, and dyeing for 10min at room temperature;
s6, decoloring: placing in decolorizing solution (mixing lactic acid, glycerol and distilled water at volume ratio of 1: 1: 1) for decolorizing for 1d to decolorize non-AM fungus structure, increase contrast and improve observation effect;
s7, slicing, and placing under an optical microscope to observe the AM fungal structure in the root system, wherein the observation result is shown in figure 2.
Example 2
The embodiment discloses a trypan blue staining method for staining the mycorrhiza of fructus aurantii, which comprises the following specific steps:
s1, patch: selecting more than 30 root segments with the length of 0.5cm, embedding the root segments by using low-melting-point agarose, and fixing the root segments on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.165mm/s, the slicing frequency is 40Hz, and the slicing thickness is 60 mu m;
s3, digestion: adding 6% (W/V) KOH, and water bathing at 80 deg.C for 50min to remove cytoplasm from cells for easy observation;
s4, acidification: after washing, adding 0.15M HCl, standing for 4min at room temperature, creating an acid environment and facilitating the coloring of trypan blue dye;
s5, dyeing: adding 0.03% (W/V) trypan blue dye solution for dyeing, and dyeing for 6min at room temperature;
s6, decoloring: placing in decolorizing solution (mixing lactic acid, glycerol and distilled water at volume ratio of 1: 2: 2) for decolorizing for 2d to decolorize non-AM fungus structure, increase contrast and improve observation effect;
and S7, slicing, and placing under an optical microscope to observe the AM fungal structure in the root system.
Example 3
The embodiment discloses a trypan blue staining method for staining the mycorrhiza of fructus aurantii, which comprises the following specific steps:
s1, patch: selecting more than 30 root segments with the length of 2cm, embedding the root segments by using low-melting-point agarose, and fixing the root segments on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.170mm/s, the slicing frequency is 60Hz, and the slicing thickness is 100 μm;
s3, digestion: adding 7% (W/V) KOH, and water bathing at 82 deg.C for 55min to remove cytoplasm from cells for easy observation;
s4, acidification: after washing, adding 0.18M HCl, standing at room temperature for 4.5min, creating an acidic environment, and facilitating the coloring of trypan blue dye;
s5, dyeing: adding 0.05% (W/V) trypan blue dye solution for dyeing, and dyeing for 10min at room temperature;
s6, decoloring: placing in decolorizing solution (mixing lactic acid, glycerol and distilled water at volume ratio of 2: 1: 1) for decolorizing for 1.5d to decolorize non-AM fungus structure, increase contrast and improve observation effect;
and S7, slicing, and placing under an optical microscope to observe the AM fungal structure in the root system.
Example 4
The embodiment discloses a method for staining mycorrhiza of fructus aurantii by adopting a double fluorescence staining method, which comprises the following specific steps:
s1, patch: selecting more than 30 1cm sections, embedding the sections by using low-melting-point agarose, and fixing the sections on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.175mm/s, the slicing frequency is 50Hz, and the slicing thickness is 80 μm; wherein 80 μm is the optimum slice thickness;
s3, bleaching: putting the bitter orange slices into 50% (W/V) ethanol for incubation for 2h, and bleaching root systems;
s4, transparency: adding 20% (W/V) KOH solution to incubate for 2d to make the root system transparent;
s5, dyeing: after washing, 0.2ug/ml Alexa was added
WGA488 fluorescent dye, incubated for 15h at room temperature, followed by addition of 2ug/ml Sigma-
Carrying out vacuum incubation for 30min by using Nile Red fluorescent dye;
s6, cleaning: washed 3 times with PBS buffer (pH7.4) and washed to remove color bloom.
After flaking, the AM fungal structure in the root system was observed by confocal laser microscopy, and the observation results are shown in FIG. 4.
Example 5
The embodiment discloses a method for staining mycorrhiza of fructus aurantii by adopting a double fluorescence staining method, which comprises the following specific steps:
s1, patch: selecting more than 30 1cm sections, embedding the sections by using low-melting-point agarose, and fixing the sections on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.165mm/s, the slicing frequency is 40Hz, and the slicing thickness is 60 mu m;
s3, bleaching: putting the bitter orange slices into 40% (W/V) ethanol, incubating for 1h, and bleaching root systems;
s4, transparency: adding 20% (W/V) KOH solution to incubate for 2d to make the root system transparent;
s5, dyeing: after washing, 0.2ug/ml Alexa was added
WGA488 fluorescent dye, incubated for 15h at room temperature, followed by addition of 2ug/ml Sigma-
Nile Red fluorescent dye, extractVacuum incubation for 30 min;
s6, cleaning: washed 4 times with PBS buffer (pH7) and washed loose.
And (3) observing the AM fungal structure in the root system by using a laser confocal microscope after flaking.
Example 6
The embodiment discloses a method for staining mycorrhiza of fructus aurantii by adopting a double fluorescence staining method, which comprises the following specific steps:
s1, patch: selecting more than 30 1cm sections, embedding the sections by using low-melting-point agarose, and fixing the sections on a glass slide;
s2, slicing: sections were taken using a Leica VT 1000S vibrating microtome, with the slicing program set to: the slicing speed is 0.170mm/s, the slicing frequency is 60Hz, and the slicing thickness is 100 μm;
s3, bleaching: putting the bitter orange slices into 45% (W/V) ethanol, incubating for 1.5h, and bleaching root systems;
s4, transparency: adding 18% (W/V) KOH solution to incubate for 1.5d to make the root system transparent;
s5, dyeing: after rinsing, 0.18ug/ml Alexa was added
WGA488 fluorescent dye, incubated for 12h at room temperature, followed by addition of 1ug/ml Sigma-
Carrying out vacuum incubation for 25min by using Nile Red fluorescent dye;
s6, cleaning: washed 3 times with PBS buffer (pH7.4) and washed to remove color bloom.
And (3) observing the AM fungal structure in the root system by using a laser confocal microscope after flaking.
Comparative example 1
The method for staining the mycorrhiza of the fructus aurantii by adopting a trypan blue staining method comprises the following specific steps:
s1, digestion: soaking 1cm long root segment of fructus Aurantii fungi in 10% (W/V) KOH solution, digesting in 90 deg.C water bath for 60min to obtain transparent root;
s2, bleaching: immersing the roots in 3% (W/V) alkaline H2O2Rinsing in the solution for 15min to bleach the root system;
s3, acidification: washing, placing in 0.2M HC1, acidifying for 5min, and making acidic condition for coloring trypan blue dye;
s4, dyeing: immersing in 0.05% (W/V) Trypan blue (Trypan blue) staining solution, and incubating at 90 deg.C for 60 min;
s5, decoloring: soaking the root system in a decolorizing solution (lactic acid, glycerol and distilled water are uniformly mixed according to the volume ratio of 1: 1: 1) to decolorize for 1 d.
The AM fungal structure in the root system was observed under an optical microscope, and the observation results are shown in FIG. 1.
Comparative example 2
The comparative example adopts a double fluorescence staining method to stain the mycorrhiza of the bitter orange, and comprises the following specific steps:
s1, bleaching: putting 1 cm-long fructus Aurantii mycorrhizae section in 50% (W/V) ethanol, incubating for 2h, and bleaching root system;
s2, transparency: adding 20% (W/V) KOH solution to incubate for 2d to make the root system transparent;
s3, dyeing: after washing, 0.2ug/ml Alexa was added
WGA488 fluorescent dye, after incubation for 15h at room temperature, 2ug/ml Sigma-
Carrying out vacuum incubation for 30min by using Nile Red fluorescent dye;
s4, cleaning: washing with PBS buffer (pH7.4) for 3 times with shaking to remove the floating color, and the results are shown in FIG. 3.
Comparison of Experimental results
1. Comparison of staining effects between conventional Trypan blue staining method and Trypan blue staining method of the present invention (biological microscope observation)
Fig. 1 is a graph of the result of staining the mycorrhiza of bitter orange by the traditional trypan blue staining method obtained in comparative example 1, and it can be seen that the observation effect of the traditional staining method is often poor due to factors such as high lignification degree, stout and deep pigment of the root system of woody plant, so that by using the traditional trypan blue staining method, only whether the root system is infected by AM fungus can be judged, but the infection intensity (M%) and the abundance (a%) of the arbuscular branches cannot be accurately calculated. In FIG. 1A and V are the arbuscular and vesicular, respectively, and are relatively faint.
FIG. 2 is a graph showing the results of the trypan blue staining method for the mycorrhiza of bitter orange obtained in example 1, and it can be seen that the microscopic structure (arbuscular branches) of AM fungus in the mycorrhiza of bitter orange can be clearly observed after the section of the mycorrhiza of bitter orange is processed, so that the infection intensity (M%) and the abundance (A%) of arbuscular branches can be objectively calculated. A, V and H in FIG. 2 are the arbuscular, vesicular and endophytic hyphae, respectively, which are clearly evident in the color picture.
As can be seen from comparison of FIG. 1 and FIG. 2, the staining method of the present invention can effectively improve the staining effect of arbuscular mycorrhiza in woody plant roots, and is helpful for clearly observing AM fungal structures such as arbuscular and endophytic hyphae.
2. The dyeing effect of the traditional double-fluorescence dyeing method is compared with that of the double-fluorescence dyeing method of the invention (laser confocal visualization)
Micro-mirror observation)
FIG. 3 is a graph showing the results of staining of a mycorrhiza of bitter orange by the conventional double fluorescent staining method obtained in comparative example 2, in which it can be seen that the fine structure (arbuscular) of AM fungus in the root system and the accumulation state of neutral lipid cannot be observed. V, NL and As in FIG. 3 are vesicles, neutral lipids and aged twigs, respectively, and are more blurred.
FIG. 4 is a graph showing the results of staining of mycorrhiza of bitter orange by the double fluorescent staining method of the present invention obtained in example 4, in which the developmental state of arbuscular branches of AM fungus and the accumulation ratio of neutral lipids in degraded arbuscular branches can be observed in detail. In FIG. 4V, NL, Ad, Am and As are vesicles, neutral lipids, developing shoots, maturing shoots and senescent shoots, respectively, As is clear and more evident in the color picture.
As can be seen from comparison of FIG. 3 and FIG. 4, the staining method of the present invention can effectively improve the staining effect of arbuscular mycorrhiza in woody plant roots, and can observe finer arbuscular structures, which is helpful for determining the development status of arbuscular.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. An efficient dyeing method of arbuscular mycorrhizal fungi in woody plants is characterized by comprising the following steps: carrying out tissue slicing on the arbuscular mycorrhiza in the woody plant root to be dyed to ensure that the thickness of the slice is 60-100 mu m, and dyeing the obtained slice.
2. The method for high-efficiency dyeing of arbuscular mycorrhizal fungi of woody plant according to claim 1, which is characterized by comprising the following steps: the optimum slice thickness was 80 μm.
3. The method for efficiently staining arbuscular mycorrhizal fungi in woody plants according to claim 1, which is characterized in that: the tissue slicing step comprises the steps of selecting woody plant root segments which are about 0.5 cm-2 cm in length and infected by arbuscular mycorrhizal fungi, embedding and fixing the root segments on a glass slide by using low-melting-point agarose, and slicing by using a vibrating microtome Leica VT 1000S, wherein the slicing procedure is as follows: the slicing speed is 0.165 mm/s-0.175 mm/s, and the slicing frequency is 40 Hz-60 Hz.
4. The method for efficiently staining arbuscular mycorrhizal fungi in woody plants according to claim 1, which is characterized in that: the method for the dyeing treatment is a trypan blue dyeing method, and the trypan blue dyeing method comprises the following steps:
a) digestion: adding 6-10% (W/V) KOH into the slices, and carrying out water bath at 80-90 ℃ for 50-60 min to remove cytoplasm in cells;
b) acidifying: taking out the slices, washing, adding 0.15-0.2M HCl, and standing at room temperature for 4-5 min;
c) dyeing: adding 0.03-0.05% (W/V) trypan blue dye solution for dyeing, and dyeing for 6-10 min at room temperature;
d) and (3) decoloring: placing the mixture in a decoloring solution for decoloring for 1 d-2 d to decolor the structure of the non-arbuscular mycorrhizal fungi;
e) observation and recording of photographs.
5. The method for efficiently staining arbuscular mycorrhizal fungi in woody plants according to claim 4, characterized in that: in the step d), the destaining solution is obtained by mixing lactic acid, glycerol and distilled water, wherein the volume ratio of the lactic acid to the glycerol to the distilled water is (1-2): (1-2): (1-2).
6. The method for efficiently staining arbuscular mycorrhizal fungi in woody plants according to claim 1, which is characterized in that: the dyeing treatment method is double fluorescent dyeing, and the double fluorescent dyeing method comprises the following steps:
a) bleaching: putting the slices into 40-50% (W/V) ethanol for incubation for 1-3 h, and bleaching the root system;
b) and (3) transparency: adding 15-20% (W/V) KOH solution to incubate for 1-2 d to make the root system transparent;
c) dyeing: taking out the slices, washing the slices by using PBS (phosphate buffer solution) with the pH of 7-7.4, and adding 0.15 ug/ml-0.2 ug/ml Alexa
WGA488 fluorescent dye, incubating for 10-15 h at room temperature, and then adding 0.1-2 ug/ml Sigma-
Carrying out vacuum incubation on the Nile Red fluorescent dye for 20-30 min;
d) cleaning: washing the substrate for more than 3 times by using PBS (phosphate buffer solution) with the pH value of 7-7.4 to wash away the flooding;
e) observation and recording of photographs.
7. The application of the efficient dyeing method of the arbuscular mycorrhizal fungi in the roots of woody plants is characterized in that: the method for efficiently staining an arbuscular mycorrhizal fungus in woody plant roots according to any one of claims 1 to 6, which is applied to the observation of the growth state of the arbuscular mycorrhizal fungus in woody plant roots or the measurement of the rate of infestation of the arbuscular mycorrhizal fungus in woody plant roots.
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| CN115824757B (en) * | 2022-12-28 | 2024-03-26 | 九江学院 | Dyeing method for endophytic fungi of camellia oleifera root system |
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