Simple and rapid rubber tree powdery mildew vacuole observation method

Abstract

The invention discloses a simple and rapid rubber tree powdery mildew vacuole observation method, and belongs to the technical field of phytopathology and microbiological microscopic observation. The invention discloses a simple and rapid method for observing vacuoles of powdery mildew of a rubber tree. The vacuoles of powdery mildew are observed by using cytochemical staining and a microscopic technology. The form of the vacuoles treated by the method is not influenced, the form and the number of the vacuoles are easy to microscopically observe, and a foundation is laid for developing invasion of the powdery mildew of the rubber trees, responding to a drought stress mechanism and the functions of the vacuoles; technical support is provided for studying the growth and development state and metabolic activity of the bacterium, regulating the transport process of cell environment substances and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant pathology and micro-observation technology, and more particularly to a simple and rapid method for observing liquid vacuoles of Oidium heveae. BACKGROUND

[0002] Natural rubber is an important strategic material in national production and one of the four major industrial raw materials in the world. China is the largest consumer and importer of natural rubber in the world. As a constraint resource, the stable and safe supply of natural rubber is related to the healthy and sustainable development of the entire industry and even the national economy. In 1918, the first report of rubber tree powdery mildew was reported in Java, Indonesia. Currently, the disease has spread to all rubber-growing regions around the world. Powdery mildew is one of the most serious leaf diseases of rubber trees. It mainly damages young tissues such as new leaves, young shoots, and inflorescences, and has a great impact on growth and latex yield. Severe damage to powdery mildew leads to leaf fall, young shoot death, delayed plant growth, delayed tapping of rubber trees, reduced latex yield, and significant economic losses. In 2008, an outbreak of powdery mildew on rubber trees in Xishuangbanna caused an estimated loss of 400-500 million yuan.

[0003] Vacuoles are membrane-bound vesicular structures commonly found in fungal cells. The main functions of vacuoles are to regulate cell osmotic pressure, maintain water balance in cells, accumulate and store nutrients and various metabolic products. The vacuole membrane has special selective permeability, making the vacuole have high osmotic properties, which causes water to move into the vacuole, greatly affecting the regulation of cell osmotic pressure and maintaining turgor pressure, and enabling the storage and accumulation of various substances in the vacuole, which is crucial for fungal invasion and resistance to drought stress. Through cell chemical staining and microscopic techniques, the morphology, number, size, and distribution of vacuoles can be observed, which is beneficial for studying the growth and development state, metabolic activity, invasion behavior, response to drought stress, and regulation of cell environment substance transport processes of the fungus.

[0004] Vacuole is an important organelle in cells, which exists widely in the cells of organisms. Vacuole is closely related to the invasion of powdery mildew and the resistance to drought and other adverse environmental factors. The observation of the number and morphology of vacuoles is needed for the study of the mechanism of pathogen invasion, response to drought stress and the function of vacuoles. The traditional method for observing vacuoles is neutral red staining method. Neutral red is a weak alkaline pH indicator. In neutral or weak alkaline environment, living cells have the ability to absorb a large amount of neutral red and transport it to vacuoles. Because the liquid in vacuoles is generally acidic, when neutral red enters the vacuole, it will release a large number of cations, so that the vacuole is dyed cherry red. After the vacuoles of rubber tree powdery mildew spores are dyed with neutral red, the vacuoles of the active powdery mildew spores are dyed light red. However, after the rubber tree powdery mildew vacuoles are dyed with neutral red, the morphology of the rubber tree powdery mildew vacuoles is obviously affected, irregular deformation occurs, shrinkage occurs, and the boundary between the vacuoles is not clear. The difficulty of counting the number of vacuoles and observing the morphology of vacuoles is increased.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a simple and rapid method for observing the vacuoles of rubber tree powdery mildew. SUMMARY

[0006] Therefore, the present application provides a simple and rapid method for observing the vacuoles of rubber tree powdery mildew, which uses cell chemical staining and microscopic observation to observe the vacuoles of powdery mildew. The morphology of the vacuoles is not affected after treatment, and the morphology and number of the vacuoles are easy to observe under a microscope. The present application lays a foundation for the study of the mechanism of rubber tree powdery mildew invasion, response to drought stress and the function of vacuoles, and provides technical support for the study of the growth and development state, metabolic activity and regulation of the transport process of cell environment substances of the fungus. The problem of the prior art that the morphology of the rubber tree powdery mildew vacuoles is obviously affected after being dyed with neutral red, irregular deformation occurs, shrinkage occurs, and the boundary between the vacuoles becomes unclear, which increases the difficulty of counting the number of vacuoles and observing the morphology of vacuoles, is solved.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A simple and rapid method for observing the vacuoles of rubber tree powdery mildew, comprising the following steps:

[0009] (1) Paste transparent tape on the leaf part of newly picked rubber tree powdery mildew, and gently press the transparent tape with hands to make the transparent tape tightly adhere to the leaf, so as to avoid the transparent tape from being creased or hollow;

[0010] (2) Then hold one end of the transparent tape with hands, gently tear the transparent tape with rubber tree powdery mildew from the rubber leaf, and cut the transparent tape with rubber tree powdery mildew into a size of 1.5 cm x 2.0 cm with scissors;

[0011] (3) Place a 1.5cm×2.0cm transparent tape with rubber tree powdery mildew attached into a beaker, pour in 0.02-0.04% trypan blue solution and soak for 20-40 minutes, then rinse with distilled water 1-2 times, and then soak in 0.025-0.05% Congo red solution for 15-30 minutes.

[0012] (4) After the above treatment, place the transparent tape with rubber tree powdery mildew in a new container and rinse it with distilled water 1 to 2 times.

[0013] (5) Use a pipette to draw 100 μL of distilled water onto a glass slide. Place the transparent tape with rubber tree powdery mildew attached flat in the distilled water, cover with a slide, and flatten it. Select rubber tree powdery mildew spores with a clear field of view. First, find the target to be observed under low magnification, and then adjust the microscope to high magnification to observe the vacuoles and take pictures.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a simple and rapid method for observing vacuoles of powdery mildew on rubber trees, which has the following beneficial effects:

[0015] (1) This invention uses transparent tape to adhere powdery mildew spores to rubber tree leaves. After treatment with trypan blue and Congo red staining solutions, the vacuolar morphology of the powdery mildew spores remains unaffected, showing no irregular deformation or shrinkage. Furthermore, the boundaries between vacuoles within the powdery mildew spores are clear, facilitating microscopic observation of their morphology and quantity. This invention has advantages such as readily available reagents and equipment, low cost, simple operation, and high efficiency. It provides a technical guarantee for the study of the vacuolar function of powdery mildew spores on rubber trees.

[0016] (2) Congo red solution stains powdery mildew mycelium orange-yellow, while powdery mildew spores remain unstained, creating a significant contrast that facilitates target observation. Trypan blue, as a live biological stain, can be used to detect the activity of powdery mildew spores: healthy and active powdery mildew spores have selective permeability to trypan blue due to their cell membranes, and are not stained blue under microscopic observation after staining; while inactive rubber tree powdery mildew spores, with damaged cell membranes and increased permeability, are stained blue by trypan blue. Using trypan blue staining can standardize the observed state of powdery mildew spores, facilitating subsequent statistical analysis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1Observation results of the spores of E. leucotricha for Example 1;

[0019] In the figure, the active spores of E. leucotricha, the inactive spores of E. leucotricha and the vacuoles are respectively indicated by black arrows, red arrows and green arrows;

[0020] Figure 2 Observation results of the spores of E. leucotricha for Example 2;

[0021] In the figure, the active spores of E. leucotricha, the inactive spores of E. leucotricha and the vacuoles are respectively indicated by black arrows, red arrows and green arrows;

[0022] Figure 3 Observation results of the spores of E. leucotricha for Example 3;

[0023] In the figure, the active spores of E. leucotricha, the inactive spores of E. leucotricha and the vacuoles are respectively indicated by black arrows, red arrows and green arrows;

[0024] Figure 4 Observation results of the vacuole staining for Comparative Example 1;

[0025] Figure 5 Observation results of the vacuole staining for Comparative Example 2;

[0026] Figure 6 Observation results of the vacuole staining for Comparative Example 3;

[0027] Figure 7 Observation results of the vacuole staining for Comparative Example 4;

[0028] Figure 8 Observation results of the vacuole staining for Comparative Example 5;

[0029] Figure 9 Observation results of the vacuole staining for Comparative Example 6. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0031] The microscopic observation methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0032] The reagents, apparatuses and the like used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0033] Example 1

[0034] A simple and rapid method for observing the vacuoles of Oidium heveae, comprising the following steps:

[0035] (1) 0.04% trypan blue solution preparation. Weigh 0.02 g of trypan blue into a triangular flask with an analytical balance; use a graduated cylinder to measure 50 mL of distilled water and add it to the triangular flask containing trypan blue, and shake well.

[0036] (2) 0.05% Congo red solution preparation. Weigh 0.025 g of Congo red into a triangular flask with an analytical balance; use a graduated cylinder to measure 25 mL of distilled water and add it to the triangular flask containing Congo red, and shake well; measure 25 mL of absolute ethanol and add it to the above-mentioned Congo red aqueous solution and mix well again.

[0037] (3) Use transparent tape to paste on the newly picked, rubber tree leaf parts with Oidium heveae, gently press with your hand, so that the transparent tape is tightly bonded with the leaf, avoiding the transparent tape from appearing creases or hollow;

[0038] (4) Then hold one end of the transparent tape with your hand, gently tear the transparent tape with Oidium heveae from the rubber leaf, and cut the transparent tape with Oidium heveae into 1.5 cm x 2.0 cm size with scissors;

[0039] (5) Put the 1.5 cm x 2.0 cm transparent tape with Oidium heveae into a beaker, pour the above-mentioned 0.04% trypan blue solution into it, gently stir with tweezers to let the Oidium heveae contact with the 0.04% trypan blue solution, soak for 20 min, and rinse with distilled water for 2 times;

[0040] (6) Move the above-mentioned transparent tape with Oidium heveae to the beaker containing 0.05% Congo red solution with tweezers, gently stir to let the Oidium heveae spores contact with the Congo red solution, soak for 15 min;

[0041] (7) After treatment, place the transparent tape with Oidium heveae in a new container, rinse with distilled water for 2 times;

[0042] (8) Use a pipette to take 100 μL of distilled water onto a glass slide, place the above-mentioned transparent tape with Oidium heveae in the distilled water, cover the slide, flatten it, select the clear Oidium heveae spores in the field of view, first find the target to be observed under low power, then adjust the microscope to high power to observe the vacuoles and take pictures. The results are shown in Table 1 and Figure 1 .

[0043] Example 2

[0044] A simple and rapid method for observing the vacuoles of Oidium heveae, comprising the following steps:

[0045] (1) Preparation of 0.03% trypan blue solution. Weigh 0.015 g of trypan blue using an analytical balance and place it in an Erlenmeyer flask; measure 50 mL of distilled water using a graduated cylinder and add it to the Erlenmeyer flask containing trypan blue, and shake well.

[0046] (2) Preparation of 0.038% Congo red solution. Weigh 0.019 g of Congo red using an analytical balance and place it in an Erlenmeyer flask; measure 25 mL of distilled water using a graduated cylinder and add it to the Erlenmeyer flask containing Congo red, and shake well; measure 25 mL of anhydrous ethanol and add it to the above Congo red aqueous solution, and mix well again.

[0047] (3) Use transparent tape to stick to the newly picked leaves with rubber tree powdery mildew. Press gently with your hand to make the transparent tape adhere tightly to the leaves and avoid creases or air bubbles in the transparent tape.

[0048] (4) Then, hold one end of the transparent tape with your hand and gently peel the transparent tape with rubber tree powdery mildew off the rubber leaf. Cut the transparent tape with rubber tree powdery mildew into 1.5cm×2.0cm pieces with scissors.

[0049] (5) Place a 1.5cm×2.0cm transparent tape with rubber tree powdery mildew attached into a beaker, pour in the above 0.03% trypan blue solution, gently stir with tweezers to ensure that the rubber tree powdery mildew is fully in contact with the 0.03% trypan blue solution, soak for 30 minutes, and rinse twice with distilled water.

[0050] (6) Use tweezers to transfer the transparent tape with rubber tree powdery mildew after the above treatment into a beaker containing 0.038% Congo red solution, gently stir to allow the rubber tree powdery mildew spores to fully contact the Congo red solution, and soak for 22 minutes.

[0051] (7) After treatment, place the transparent tape with rubber tree powdery mildew in a new container and rinse it twice with clean water.

[0052] (8) Using a pipette, draw 100 μL of distilled water onto a glass slide. Place the transparent tape with rubber tree powdery mildew attached flat in the distilled water, cover with a coverslip, and flatten. Select rubber tree powdery mildew spores with a clear field of view. First, locate the target under low magnification, then adjust the microscope to high magnification to observe the vacuoles and take photos. The results are shown in Table 1 and... Figure 2 .

[0053] Example 3

[0054] A simple and rapid method for observing powdery mildew vacuoles on rubber trees includes the following steps:

[0055] (1) 0.02% trypan blue solution preparation. 0.01 g of trypan blue was weighed into a flask with an analytical balance; 50 mL of distilled water was measured with a graduated cylinder and added to the flask containing the trypan blue, and shaken thoroughly.

[0056] (2) 0.025% Congo red solution preparation. 0.0125 g of Congo red was weighed into a flask with an analytical balance; 25 mL of distilled water was measured with a graduated cylinder and added to the flask containing the Congo red, and shaken thoroughly; 25 mL of anhydrous ethanol was measured and added to the above-mentioned Congo red aqueous solution and shaken again.

[0057] (3) The transparent tape was pasted on the leaf part of the freshly picked rubber tree powdery mildew, and the transparent tape was tightly adhered to the leaf by gently pressing with the hand to avoid creases or hollows in the transparent tape;

[0058] (4) Then one end of the transparent tape was pinched with the hand, and the transparent tape with rubber tree powdery mildew was gently torn off from the rubber leaf, and the transparent tape with rubber tree powdery mildew was cut into 1.5 cm x 2.0 cm in size with scissors;

[0059] (5) The 1.5 cm x 2.0 cm transparent tape with rubber tree powdery mildew was placed in a beaker, and the above-mentioned 0.02% trypan blue solution was poured into the beaker, and the rubber tree powdery mildew was gently stirred with tweezers to make the rubber tree powdery mildew fully contact with the 0.02% trypan blue solution, and soaked for 40 min, and rinsed with distilled water once;

[0060] (6) The transparent tape with rubber tree powdery mildew after the above-mentioned treatment was moved into the beaker containing the 0.025% Congo red solution with tweezers, and the rubber tree powdery mildew spores were gently stirred to make them fully contact with the Congo red solution, and soaked for 30 min;

[0061] (7) The transparent tape with rubber tree powdery mildew after the treatment was placed in a new container and rinsed with clean water once;

[0062] (8) 100 μL of distilled water was sucked onto a glass slide with a pipette, and the above-mentioned transparent tape with rubber tree powdery mildew was placed flat in the distilled water, covered with a cover glass, and flattened, and the rubber tree powdery mildew spores with clear field of view were selected, the target to be observed was found under low magnification, and then the microscope was adjusted to high magnification to observe the vacuoles and take pictures for recording. The results are shown in Table 1 and Figure 3 .

[0063] Comparative Example 1

[0064] Traditional vacuole staining observation method. Stained with neutral red staining solution reagent (Beijing Solaybao Technology Co., Ltd., item number G1314). The leaves with powdery mildew were cut into small pieces and placed in a 5 mL centrifuge tube, and an appropriate amount of distilled water was added. The powdery mildew spores on the leaves were eluted by vortexing for 3 min, and the supernatant was transferred to a 2 mL centrifuge tube and centrifuged at 5000g for 10 min. The supernatant was removed, and distilled water was added to repeat the previous step once. The distilled water was removed, and 1.5 mL of neutral red staining solution was added to ensure full coverage of the powdery mildew spores. After 5 min of staining, microscopic observation was performed. The results are shown in Table 1 and Figure 4 .

[0065] Comparative Example 2

[0066] Comparative Example 2 differs from Example 1 in that only the lactic acid phenol cotton blue staining solution was used for 15 min. Preparation method of lactic acid phenol cotton blue staining solution: 0.125 grams of cotton blue and 50 grams of crystal phenol were weighed separately, added to 50 mL of distilled water, and then 50 mL of lactic acid was added. The results are shown in Table 1 and Figure 5 .

[0067] Comparative Example 3

[0068] Comparative Example 3 differs from Example 1 in that only 0.05% basic fuchsin was used for 15 min. Preparation method of 0.05% basic fuchsin: 0.025 g of basic fuchsin was weighed, added to 25 mL of distilled water, and then 25 mL of absolute ethanol was added and mixed. The results are shown in Table 1 and Figure 6 .

[0069] Comparative Example 4

[0070] Comparative Example 4 differs from Example 1 in that only 0.05% Congo red solution was used for 15 min. The results are shown in Table 1 and Figure 7 .

[0071] Comparative Example 5

[0072] Comparative Example 5 differs from Example 1 in that only 0.04% trypan blue solution was used. The results are shown in Table 1 and Figure 8 .

[0073] Comparative Example 6

[0074] Comparative Example 6 differs from Example 1 in that the transparent tape with rubber tree powdery mildew was gently peeled off the rubber leaf, and the tape with rubber tree powdery mildew was cut into 1.5 cm x 2.0 cm pieces with scissors without any staining solution treatment. The results are shown in Table 1 and Figure 9 .

[0075] Observation method: after the spores of Oidium heveae were treated, the target to be observed was found under low power microscope, and then the microscope was adjusted to high power to observe the integrity (whether broken), shape, boundary (whether clear) of vacuoles, and the contrast between vacuoles and non-targets (whether the vacuoles of Oidium heveae were easy to observe), and the observation results are shown in Table 1.

[0076] Table 1

[0077] Treatment group Vesicle integrity Vesicle shape Vesicle border Vesicle to non-target contrast Example 1 Intact Circular Clear Contrast apparent Example 2 Intact Circular Clear Contrast apparent Example 3 Intact Circular Clear Contrast apparent Comparative Example 1 Intact Shrunk, deformed Not clear Contrast not apparent Comparative Example 2 Broken \ \ Contrast not apparent Comparative Example 3 Broken \ \ Contrast not apparent Comparative Example 4 Intact Circular Clear Contrast somewhat apparent Comparative Example 5 Intact Circular Not clear Contrast not apparent Comparative Example 6 Intact Not easily observable Not easily observable Contrast not apparent

[0078] Note: "\" represents that the vacuole is broken and not observed.

[0079] From the results in Table 1, it can be seen that after the treatment of the present application, the vacuoles are not broken, the shape is not affected, the boundary is clear, the contrast between the vacuoles and the non-targets is obvious, and the shape and quantity of the vacuoles are easy to observe under a microscope, which solves the problem that in the prior art, the shape of the vacuoles of Oidium heveae is obviously affected after being dyed with neutral red, and irregular deformation and shrinkage occur, and the boundary between the vacuoles is not clear, which increases the difficulty of counting the number of vacuoles and observing the shape of the vacuoles.

[0080] Among them, examples 1-3 and comparative example 1 show that the vacuoles of the spores of Oidium heveae treated by the present application do not appear irregular deformation or shrinkage, the boundary is clear, and the contrast with the non-targets is obvious, which is convenient for observation.

[0081] Examples 1-3 and comparative examples 2 and 3 show that the vacuoles are complete and not broken, which indicates that the structure of the vacuoles of the spores of Oidium heveae is not damaged by the present application.

[0082] Examples 1-3 and comparative example 4 show that the contrast between the vacuoles and the non-targets is more obvious, and in addition, treating with the vital biological dye trypan blue first and then with Congo red is beneficial to easily distinguishing the activity of the spores of Oidium heveae, and can unify the state of the spores of Oidium heveae to be observed, which is convenient for subsequent statistical analysis.

[0083] Examples 1-3 and comparative example 5 show that the present application can not only distinguish the activity of the spores of Oidium heveae, but also the boundary of the vacuoles is clear.

[0084] Examples 1-3 and comparative example 6 show that the vacuoles of the present application are complete and not broken, the vacuoles are round, the boundary of the vacuoles is clear, the contrast between the vacuoles and the non-targets is obvious, and it is easy to observe under a microscope.

[0085] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and, while certain modifications are discussed, it is desired to be protected in accordance with the spirit and scope of the application. Therefore, the application is not limited to the specific embodiments shown and described, but only by the scope of the appended claims, unless otherwise specified.

Claims

1. A simple and rapid method for observing the vesicles of Erysiphe communis on rubber trees, characterized in that, The method comprises the following steps: (1) Paste the transparent tape on the leaf part of the freshly picked rubber tree with powdery mildew, and gently press the transparent tape with hands to make the transparent tape tightly adhere to the leaf, so as to avoid the transparent tape from being creased or hollow; (2) Then, hold one end of the transparent tape with hands, and gently tear the transparent tape with rubber tree powdery mildew from the rubber leaf, and cut the transparent tape with rubber tree powdery mildew into a size of 1.5 cm*2.0 cm by using scissors; (3) Put the transparent tape with rubber tree powdery mildew of 1.5 cm*2.0 cm into a beaker, immerse in 0.02-0.04% trypan blue solution for 20-40 min, then rinse with distilled water for 1-2 times, and then immerse in 0.025-0.05% Congo red solution for 15-30 min; (4) After the above treatment, put the transparent tape with rubber tree powdery mildew into a new container, and rinse with distilled water for 1-2 times; (5) Take 100 μL of distilled water on a glass slide by using a pipette, place the transparent tape with rubber tree powdery mildew in the distilled water, cover the glass slide, press the glass slide, select the rubber tree powdery mildew spores with clear field of view, find the target to be observed under a low-power microscope, then adjust the microscope to a high-power microscope to observe the vacuoles, and take a photo for record.

Images