A method for staining and observing leaf infection by blumeria graminis
By tearing off the epidermis of wheat leaves and eluting with Tritium X-100 solution, combined with low-concentration Coomassie brilliant blue staining, the problem of unclear observation in existing technologies has been solved, achieving a safe and rapid staining effect on leaves infected with powdery mildew.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG AGRICULTURAL UNIVERSITY
- Filing Date
- 2023-12-11
- Publication Date
- 2026-06-23
AI Technical Summary
Existing methods for observing wheat powdery mildew infection in leaves have several drawbacks: the decolorizing solution cannot completely remove interference from mesophyll cells and chloroplasts; the decolorizing solution is highly toxic; the reagent preparation is complex; the observation period is long; and the structure of powdery mildew haustoria and other structures within the cells cannot be clearly observed.
Leaves were fixed with double-sided tape and cotton thread, epidermal cells were peeled off, and the epidermal cell components were washed off with Tritium X-100 solution (a nonionic surfactant) and then stained and observed with low concentration Coomassie Brilliant Blue R250 staining solution.
This method enables safe, rapid, and clear observation of the epidermal cell structure of wheat powdery mildew-infected leaves, reducing experimental time and reagent toxicity while improving staining efficiency.
Smart Images
Figure CN117405475B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crop biology research, specifically to a staining observation method for wheat powdery mildew-infected leaves. Background Technology
[0002] Wheat powdery mildew (Blumeria graminis) is a significant global disease, causing substantial food security problems worldwide. Observing and studying the infection and development of the powdery mildew pathogen in host plant leaves is crucial for understanding the interaction between the pathogen and the host, conducting powdery mildew-resistant breeding, and providing a basis for morphological identification of the wheat powdery mildew pathogen. Furthermore, it provides an effective detection method for studying the occurrence patterns of wheat powdery mildew and for researching effective control and the mechanisms of action of fungicides. Therefore, providing a new, high-throughput, rapid, and safe method for staining and observing wheat powdery mildew pathogens is of paramount importance.
[0003] Currently, the observation of powdery mildew-infected leaves involves decolorizing the leaves with a decolorizing solution and then staining them with Coomassie Brilliant Blue R250. For example, Wold and Fric (Wolf G, Fric F. Arapid staining method for Erysiphe graminis f.sp. hordeiin and on whole barley leaves with a protein-specific dye. Phytopathology, 1981, 71(6):596-598.) used the "Coomassie Brilliant Blue tissue clearing staining" method: the sample leaves were decolorized in an ethanol-chloroform (75:25, v / v) solution containing 0.15% trichloroacetic acid, and the decolorizing solution needed to be changed frequently. The decolorized sample was then stained in a mixed solution of 15% trichloroacetic acid aqueous solution and 0.6% Coomassie Brilliant Blue R250 prepared with 99% methanol in equal volumes. In the published patent CN 101324494A (a staining method for identifying the pathogen of powdery mildew in melon), a decolorizing solution is prepared by mixing trichloroethanol and chloroform at a volume ratio of 3:1. This solution is then incubated at 70℃ for 30 minutes for 1-5 cycles. Afterward, the sample is stained in a staining solution prepared by mixing 0.15% trichloroacetic acid aqueous solution and 0.6% Coomassie Brilliant Blue R250 methanol solution at a volume ratio of 1:1 for 5-30 minutes. The sample is then rinsed with water and observed under a microscope. Li et al. (Li S, Lin D, Zhang Y, et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature, 2022, 602(7897).) used a decolorizing solution (1:1:7 (v / v / v) lactic acid / glycerol / water) to decolorize wheat leaves susceptible to powdery mildew for 48 hours, followed by staining with 0.6% Coomassie Brilliant Blue staining solution.
[0004] Existing methods often fail to completely remove the interference caused by mesophyll cells and chloroplasts in leaf decolorization treatment. Some decolorizing solutions contain chloroform, the reagent preparation is complex and has a certain degree of toxicity. In addition, the experimental cycle is long and it is impossible to directly stain and observe the powdery mildew haustoria located in leaf cells. Only transparent and blurry structures can be observed. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention discloses a staining observation method for wheat powdery mildew-infected leaves.
[0006] This invention provides a staining observation method for wheat powdery mildew-infected leaves, comprising the following steps:
[0007] (1) Preparation of the observation sample: Use double-sided tape, rubber band and cotton thread to fix the back of the blade to the acrylic plate with the back facing up;
[0008] (2) Inoculation: Inoculate evenly with powdery mildew spores;
[0009] (3) Sample preparation: Take the inoculated leaves and peel off the epidermis on the back of the leaves;
[0010] (4) Elution of sample cell components: Perform elution treatment of epidermal cell components;
[0011] (5) Staining observation: The samples for observation were prepared by Coomassie brilliant blue staining.
[0012] Preferably, the preparation of the observation sample in step (1) involves inserting an L-shaped acrylic plate into the edge of the seedling tray, with the short side facing up. Two 2mm wide double-sided adhesive strips are then pasted onto the long side of the acrylic plate, parallel to the bending marks. The leaf is then pasted onto the adhesive strip with its back side facing up, and secured to the long side of the acrylic plate with elastic bands and cotton thread for easy inoculation. It should be noted that the leaf can also be fixed with its front side facing up; in this case, the front epidermis is peeled off after inoculation.
[0013] Preferably, the inoculation in step (2) is carried out by blowing powder into a sealed inoculation box and allowing it to settle. Powdery mildew spores are blown in evenly and slowly at the top four corners of the inoculation box, and the box is immediately sealed and left to settle freely to ensure uniform inoculation of powdery mildew spores.
[0014] Preferably, the sample processing in step (3) involves using a blade to gently slice across the area near the end of the leaf on the front side when tearing off the epidermal cells, cutting off the remaining tissue except for the epidermal cells on the back side, then turning the leaf over, using tweezers to hold the end of the leaf, gently tearing off the epidermis along the cut, removing the end of the leaf, and immediately immersing the torn epidermis in phosphate buffer.
[0015] Preferably, the elution of sample cell components in step (4) is an elution treatment of epidermal cell components. Elution reagent A is 1% Tritium X-100 solution, which is a non-ionic cell surfactant. Elution reagent B is 10×PBS buffer. All added reagents must immerse the material.
[0016] More preferably, the epidermis is immersed in a 2 ml centrifuge tube containing 1.5 ml of 1% Tritium X-100, eluted on a shaker for 20 min, then the Tritium X-100 solution is removed, an equal volume of 10×PBS buffer is added, and eluted on a shaker for 5 min. This process is repeated three times with 10×PBS buffer.
[0017] Preferably, the staining observation in step (5) is performed by staining with 0.025% Coomassie Brilliant Blue R250 staining solution at room temperature for 2-4 hours.
[0018] More preferably, the preparation method of the Coomassie Brilliant Blue R250 staining solution in step (5) is as follows: 1g of Coomassie Brilliant Blue R250 water-soluble powder, 250ml of anhydrous ethanol, 35ml of glacial acetic acid, and deionized water are used to make up to 500ml to prepare a stock solution. When using, the stock solution is diluted 8 times with deionized water.
[0019] The results obtained through the above method of "powdery mildew infection - epidermal removal - washing - staining" (e.g.) Figure 3 Compared to the traditional staining method of "powdery mildew infection - prolonged leaf chlorosis - staining" (e.g.) Figure 6 Significant improvement. Figure 6 The results show the results of using the traditional long-term dechlorosis staining method. Although spores and hyphae can be observed, the powdery mildew fungal haustoria in the epidermal cells of wheat leaves cannot be clearly stained.
[0020] The beneficial effects of this invention are:
[0021] This invention provides a method that uses epidermal peeling and nonionic surfactants to treat leaves instead of traditional decolorizing solutions, significantly reducing experimental time. The reagents used avoid the health hazards to researchers associated with chloroform and other substances found in traditional decolorizing solutions. Simultaneously, the concentration of the staining solution has been optimized, with the concentration of Coomassie Brilliant Blue R250 solution reduced to 0.025%, minimizing dye loss. The treated samples allow for clear observation of the infection structures within the epidermal cells under a microscope. This method achieves safe, high-quality, and rapid staining and observation of wheat powdery mildew-infected leaves. Attached Figure Description
[0022] Figure 1 The illustration shows wheat leaves fixed to an acrylic plate with the back facing up, in preparation for spraying powdery mildew spores.
[0023] Figure 2 The illustration shows the process of tearing off the leaf epidermis.
[0024] Figure 3 The illustration shows Coomassie brilliant blue staining of leaf epidermal cells infected with powdery mildew, showing that the haustoria, haustorium, infection sites, and hyphae are stained blue.
[0025] Figure 4 The image shows the Coomassie Brilliant Blue staining results of leaf epidermal cells after elution with 1% Tritium X-100 solution.
[0026] Figure 5The image shows the Coomassie Brilliant Blue staining results of leaf epidermal cells without elution with 1% Tritium X-100 solution.
[0027] Figure 6 The image shows the results of Coomassie Brilliant Blue staining after decolorization using the traditional method. Detailed Implementation
[0028] Example 1: Sample preparation of wheat leaves infected with wheat powdery mildew.
[0029] 1.1 Preparation of Observation Samples
[0030] Wheat seeds were evenly planted on both sides of the seedling tray. When the seeds reached the two-leaf-one-heart stage, an L-shaped acrylic plate was inserted into the edge of the tray. Two 2mm wide double-sided tape strips were then attached to the long side of the acrylic plate, parallel to the creases. The leaves were then attached to the tape with their backs facing up, and secured to the long side of the acrylic plate with rubber bands and cotton thread. The seedling tray was then placed in a powdery mildew inoculation box. Wheat powdery mildew spores (E09) were slowly and evenly sprayed from the four corners of the top of the box. The box was immediately sealed and left to stand for 30 minutes, then incubated in an incubator at 25℃ and 70% humidity. Figure 1 The illustration shows the preparation of the sample.
[0031] 1.2 Sample Preparation
[0032] When it is time to collect samples, cut off the wheat leaves for analysis and fix the leaf base to the operating platform. Use wide-tipped forceps to turn the leaf so that the upper surface is facing up, and gently cut the leaf tip with a blade to cut off the tissue except for the lower epidermal cells; turn the leaf so that the lower surface is facing up; use wide-tipped forceps to hold the leaf tip and gently press it backward and downward to bend the cut. Use the tip of a pointed forceps or a scalpel blade to gently press the leaf below the cut to separate the epidermal cells from the mesophyll cells, and slowly peel the epidermis towards the operator; finally, trim the epidermis, remove the terminal part containing mesophyll cells, and immerse the epidermis in a 2ml centrifuge tube containing 1.5ml of 10×PBS buffer. Figure 2 The illustration shows the peeling of the leaf epidermis.
[0033] Example 2: Sample processing and observation of wheat leaves infected with wheat powdery mildew.
[0034] 2.1 Elution of sample cell components
[0035] Remove the PBS buffer from the centrifuge tube, add an equal volume of 1% Tritium X-100 solution, and elute on a shaker for 20 min. Then remove the Tritium X-100 solution, add an equal volume of 10×PBS buffer, and continue eluting cell fractions on a shaker for 5 min. Repeat the 10×PBS buffer treatment three times.
[0036] Treatment with 1% Tritium X-100 solution increases the cell membrane permeability of leaf epidermal cells, making it easier for substances affecting staining observation to be eluted by 10×PBS solution. 10×PBS solution helps maintain cell morphology, balance the cellular environment, and facilitate the elution of intracellular substances.
[0037] 2.2 Observation of Coomassie Brilliant Blue Staining
[0038] Remove the PBS buffer, add 1.5 ml of 0.025% Coomassie Brilliant Blue R250 staining solution, and stain for 2 hours. Transfer the stained leaves to a 2 ml centrifuge tube containing 1.5 ml of deionized water, rinse three times, and then prepare slides for observation. Figure 3 To illustrate the Coomassie brilliant blue staining of leaf epidermal cells infected with powdery mildew, the haustoria, haustorium, infection sites, and hyphae are shown to be stained blue. The preparation method for a 0.025% Coomassie brilliant blue R250 staining solution is as follows: 1g of water-soluble Coomassie brilliant blue R250 powder, 250ml of anhydrous ethanol, 35ml of glacial acetic acid, and dilute to 500ml with deionized water to prepare a stock solution. Before use, dilute the stock solution 8 times with deionized water.
[0039] The background of the stained samples treated with 1% Tritium X-100 solution was cleaner and clearer, showing a significant difference compared to the stained results without 1% Tritium X-100 solution treatment. Figure 4 The image shows the staining results of Coomassie Brilliant Blue R250 after treatment with 1% Tritium X-100 solution. Figure 5 The image shows the staining results of Coomassie Brilliant Blue R250 without treatment with 1% Tritium X-100 solution. Figure 6 The illustration shows the dyeing effect after traditional decolorization.
[0040] Traditional observation methods require lengthy destaining processes, are time-consuming, involve complex reagent preparation, and some reagents are toxic. Furthermore, after destaining, Coomassie brilliant blue staining only reveals extracellular epiphytic structures (hyphae and spores), offering poor results for observing haustoria, haustoriums, and infection sites. The method provided by this invention significantly reduces experimental time and provides more intuitive and clear staining results for haustoria, haustoriums, and infection sites. The reagents used are simpler to prepare and safer.
[0041] The above description is only a preferred embodiment of this patent. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of this patent, and these improvements and substitutions should also be considered within the scope of protection of this patent.
Claims
1. A staining observation method for wheat powdery mildew infection of leaves, characterized in that, Includes the following steps: (1) Preparation of the observation sample: Use double-sided tape, rubber band and cotton thread to fix the back of the blade to the acrylic plate with the back facing up; (2) Inoculation: Inoculate evenly with powdery mildew spores; (3) Sample preparation: Take the inoculated leaves and peel off the epidermis on the back of the leaves; (4) Elution of sample cell components: Immerse the epidermis in a centrifuge tube containing 1% Triton X-100 solution, elute on a shaker for 20 min, then remove the Triton X-100 solution, add 10×PBS buffer, elute on a shaker for 5 min, and repeat the elution with 10×PBS buffer three times. (5) Staining observation: The sample was prepared by staining with 0.025% Coomassie Brilliant Blue R250 staining solution at room temperature for 2-4 hours to obtain the sample for observation.
2. The method according to claim 1, characterized in that, In step (1), insert the short side of the L-shaped acrylic sheet into the edge of the seedling pot, and stick two 2mm wide double-sided tapes on the long side of the acrylic sheet in the direction parallel to the bending marks of the acrylic sheet. Stick the leaf with the back facing up on the double-sided tape, and use rubber bands and cotton thread to help fix it to the long side of the acrylic sheet.
3. The method according to claim 1, characterized in that, In step (2), the powdery mildew spores are blown into the four corners of the top of the inoculation box evenly and slowly, and then the inoculation box is immediately sealed and left to stand until the spores settle freely.
4. The method according to claim 1, characterized in that, In step (3), use a blade to gently cut across the area near the end of the leaf on the front side, cutting off the remaining tissue except for the epidermal cells on the back side. Then turn the leaf over, use tweezers to hold the end of the leaf, and gently tear off the epidermis along the cut. Remove the end of the leaf and immediately immerse the torn epidermis in phosphate buffer.
5. The method according to claim 1, characterized in that, In step (5), the Coomassie Brilliant Blue R250 staining solution is obtained by diluting the mother liquor. The preparation method of the mother liquor is as follows: 1g of Coomassie Brilliant Blue R250 water-soluble powder, 250ml of anhydrous ethanol, 35ml of glacial acetic acid, and dilute to 500ml with deionized water. When using, the mother liquor is diluted 8 times with deionized water.