Dyeing method and application of pathogenic fungi of southern corn rust disease

Abstract

The invention provides a dyeing method of corn southern rust pathogenic fungi, which comprises the following steps: dropwise adding a Tween-20 solution into a mature multi-stack puccinia striiformis spore stack, and standing to obtain a spore suspension; dropwise adding the spore suspension on a glass slide or a corn leaf, and placing the glass slide or the corn leaf on wet filter paper in a culture dish; then, placing in an illumination incubator for culturing to obtain germinated spores; and dropwise adding the ruthenium red dye liquor onto the germinated spores for dyeing, and then washing with water. The invention further provides application of the dyeing method in research of the corn southern rust disease and screening of corn southern rust disease resistant germplasm resources. The puccinia multiflora is dyed through the ruthenium red dye liquor, the safety problem of a traditional method is avoided, the dyeing time is greatly shortened, meanwhile, the dyeing effect is good, spores in all germination stages can be greatly reserved after treatment, and the integrity of a hypha structure is maintained; the research on the corn southern rust disease and the screening of corn southern rust disease resistant germplasm resources are facilitated.

Description

Technical Field

[0001] The invention relates to the field of plant germplasm resource screening, and in particular to a staining method and application of pathogenic fungi of southern corn rust. Background Art

[0002] Corn (Zea mays L.) is the largest food crop in my country, widely planted from the eastern coastal provinces to the western provinces, from Hainan Province in the south to Heilongjiang Province in the north. At the same time, corn ranks first in the world in terms of production and is an important raw material for many fields such as food, industry, and medicine.

[0003] Corn rust is an airborne disease that spreads worldwide and is one of the most common diseases in corn cultivation. Corn rust is divided into southern corn rust and common corn rust.

[0004] The pathogen of southern corn rust is Puccinia polysora Underw., an obligate parasitic fungus. Compared with common corn rust, southern corn rust has the characteristics of long transmission distance and strong infection ability, and the damage it causes to corn is more destructive and fatal. The pathogen mainly infects corn leaves. In severe cases, it will further infect the sheaths, bracts and other parts, causing the leaves to dry up, the photosynthesis function to be lost, the kernels to shrivel, and the yield to drop significantly.

[0005] The observation of the infection and structural characteristics of Puccinia multipilosa is the basis for the study of southern corn rust and the screening of corn rust-resistant germplasm resources. However, at present, the main staining method for Puccinia multipilosa is the triphenyl blue transparent staining method, which uses corrosive reagents during the staining and bleaching process, affecting the health of operators. At the same time, the operation cycle is long (bleaching is required), the staining effect is poor (the color is darker and the internal details of the fungus cannot be observed), and the ungerminated and unfirmly attached spores are easily washed off during the processing process, which mechanically damages the hyphae, making it difficult to observe the complete structures such as the appressorium and hyphae.

[0006] Therefore, it is urgent to develop a new method for staining the pathogenic fungus of southern corn rust so that it can be applied to the research of southern corn rust and the screening of southern corn rust-resistant germplasm resources. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a dyeing method and application of the pathogenic fungus of southern corn rust. It uses ruthenium red dye to dye multiple piles of rust fungi, avoids the safety problems caused by the use of phenol in traditional dyeing methods, and greatly reduces the time required for dyeing (no decolorization is required). At the same time, the dyeing effect is good. After treatment, spores at various germination stages can be largely retained, and the integrity of structures such as hyphae and appressorium can be maintained, which is helpful for the research of southern corn rust and the screening of southern corn rust-resistant germplasm resources.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A method for dyeing pathogenic fungi of southern corn rust comprises the following steps:

[0010] S1. Add Tween-20 solution to multiple piles of mature rust spores and let them stand to obtain a spore suspension;

[0011] S2, dropping the spore suspension onto a glass slide or corn leaf, and placing it on a moist filter paper in a culture dish; then, placing it in a light incubator for culturing to obtain germinating spores;

[0012] S3. Add ruthenium red dye solution to the germinated spores for staining, and then wash with water.

[0013] As one of the preferred embodiments of the present invention, in step S1, the mature multiple piles of Puccinia spores refer to: spores proliferating under the epidermis, breaking through the corn epidermis to be exposed, and forming powdery spore accumulations.

[0014] As one of the preferred embodiments of the present invention, in step S1, the mass concentration of the Tween-20 solution is 0.05-0.15%, and the amount of the solution added is 18-22 μL.

[0015] As one of the preferred embodiments of the present invention, in step S2, the amount of the spore suspension added is 8 to 12 μL.

[0016] As one of the preferred embodiments of the present invention, in step S2, the culture conditions are: temperature 26-28° C., humidity 70-80%, and light intensity 10000-12000 LUX.

[0017] As one of the preferred embodiments of the present invention, in step S3, the mass concentration of the ruthenium red dye solution is 0.06-0.1%, and the drop amount is 18-22 μL / cm 2 Germination of spores.

[0018] As one of the preferred embodiments of the present invention, in step S3, the dyeing time is 5 to 10 minutes.

[0019] As one of the preferred embodiments of the present invention, in step S3, water washing is performed by dripping ddH2O solution and the washing is repeated 2 to 3 times.

[0020] The invention discloses an application of the staining method of the pathogenic fungus of southern corn rust in the research of southern corn rust and the screening of germplasm resources resistant to southern corn rust.

[0021] As one of the preferred embodiments of the present invention, the difference in the growth state of conidia of the pathogenic fungus, Puccinia polystomata, on the surface of corn leaves is observed by the staining method to screen out corn germplasm resources resistant to southern rust.

[0022] Ruthenium red (CAS No.: 11103-72-3), English name Ruthenium Red (Ammoniated rutheniumoxychloride), molecular formula Ru3O2Cl6·14(NH3), molecular weight 786.35, appearance is red crystal. At present, there is no report on the staining of ruthenium red on Puccinia polysticta. The present invention has found through research that ruthenium red has a significant staining effect on Puccinia polysticta, the pathogen of southern corn rust, and can be applied to the staining observation method of corn leaves infected by Puccinia polysticta, and further screen the germplasm resources resistant to southern rust.

[0023] The advantages of the present invention compared to the prior art are:

[0024] (1) The present invention uses ruthenium red dye to dye multiple piles of Puccinia, avoiding the threat to the health of experimental operators caused by the use of phenol in traditional dyeing methods, and greatly reducing the time required for dyeing (no decolorization is required). At the same time, the dyeing effect is good, and after treatment, spores at various germination stages can be largely retained, and the integrity of structures such as hyphae and appressorium can be maintained;

[0025] (2) Based on the staining method of the present invention, a complete, efficient and safe multi-pile Puccinia staining observation is achieved, and the resistance level of the tested corn variety to southern rust can be quickly identified by further observing and comparing the growth of multiple piles of Puccinia inoculated on leaves of different strains and strains;

[0026] (3) The present invention is simple to operate, requires a small area, and greatly reduces the test period, which is helpful for the research of southern corn rust and the screening of southern corn rust-resistant germplasm resources, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a physical picture of the pile of mature ruptured spores raised on the leaves in Examples 1 to 3;

[0028] Figure 2 This is the ruthenium red staining of multiple piles of Puccinia spores cultured for 1 hour in Experimental Example 1 (40× field of view);

[0029] Figure 3 This is the ruthenium red staining of multiple piles of Puccinia spores cultured for 6 hours in Experimental Example 1 (20× field of view);

[0030] Figure 4 is a photograph of a culture dish containing a spore suspension in Experimental Example 2;

[0031] Figure 5 This is the result of ruthenium red staining of multiple piles of Puccinia odorifera on the detached leaves of the corn variety "Yanzi 7016" in Experimental Example 2 (10× field of view);

[0032] Figure 6 This is the result of ruthenium red staining of multiple piles of Puccinia oleracea on the detached leaves of the corn variety "B73" in Experimental Example 2 (10× field of view). DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below. The present embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagents and materials used in the following embodiments are commercially available reagents and materials conventional in the art unless otherwise specified. The experimental methods used in the following embodiments are conventional methods in the art unless otherwise specified, and will not be repeated.

[0034] Example 1

[0035] A method for dyeing southern corn rust pathogenic fungi of this embodiment comprises the following steps:

[0036] S1. In the mature piles of stem rust spores (spores increase under the epidermis, break the corn epidermis and form powdery spore accumulations, Figure 1 20 μL of 0.1% Tween-20 solution was added dropwise to the culture medium and allowed to stand for 1 min to obtain a spore suspension.

[0037] S2. Take 10 μL of spore suspension, drop it on a glass slide or corn leaf, and place it on a moist filter paper (Φ7 cm) in a culture dish (13 cm×13 cm); then, place it in a light incubator and culture it under the following conditions: temperature 27° C., humidity 75%, light intensity 11000 LUX, and germinate spores.

[0038] S3, take 0.08% ruthenium red dye solution and place it at 20 μL dye solution / cm 2 The amount of germinating spores was added dropwise onto the germinating spores and stained in the dark for 8 min; thereafter, ddH2O was added dropwise from one side of the leaf and the other side was absorbed with filter paper and repeated twice.

[0039] Example 2

[0040] A method for dyeing southern corn rust pathogenic fungi of the present embodiment comprises the following steps:

[0041] S1. In mature piles of rust spores (spores proliferate under the epidermis, break through the corn epidermis and form powdery spore accumulations, Figure 1 18 μL of 0.15% Tween-20 solution was added dropwise to the culture medium and allowed to stand for 1 min to obtain a spore suspension.

[0042] S2. Take 8 μL of spore suspension, drop it on a glass slide or corn leaf, and place it on a moist filter paper (Φ7 cm) in a culture dish (13 cm×13 cm); then, place it in a light incubator and culture it under the following conditions: temperature 26°C, humidity 70%, light intensity 10000LUX, and germinate spores.

[0043] S3, take 0.1% ruthenium red dye solution and place it at 18 μL dye solution / cm 2 The amount of germinating spores was added dropwise onto the germinating spores and stained in the dark for 5 min; thereafter, ddH2O was added dropwise from one side of the leaf and the other side was absorbed with filter paper and repeated twice.

[0044] Example 3

[0045] A method for dyeing southern corn rust pathogenic fungi of the present embodiment comprises the following steps:

[0046] S1. In the mature piles of stem rust spores (spores increase under the epidermis, break the corn epidermis and form powdery spore accumulations, Figure 1 22 μL of 0.05% Tween-20 solution was added dropwise to the culture medium and allowed to stand for 1 min to obtain a spore suspension.

[0047] S2. Take 12 μL of spore suspension, drop it on a glass slide or corn leaf, and place it on a moist filter paper (Φ7 cm) in a culture dish (13 cm×13 cm); then, place it in a light incubator and culture it under the following conditions: temperature 28° C., humidity 80%, light intensity 12000 LUX, and germinate spores.

[0048] S3, take 0.06% ruthenium red dye solution and place it at 22 μL dye solution / cm 2 The amount of germinating spores to be added was dropped onto the germinating spores and stained in the dark for 10 minutes; after that, ddH2O was added from one side of the leaf and the other side was absorbed with filter paper and repeated 3 times.

[0049] Experimental Example 1

[0050] This experimental example is used to verify the feasibility of the dyeing method of the present invention (taking Example 1 as an example):

[0051] (1) Preparation of spore suspension

[0052] 20 μL of 0.1% Tween-20 solution was added dropwise to multiple piles of mature stem rust spores (spores proliferated under the epidermis, broke through the corn epidermis and exposed to form powdery spore accumulations), and the mixture was allowed to stand for 1 minute to obtain a spore suspension.

[0053] (2) Sample preparation

[0054] 10 μL of spore suspension was dripped onto a coverslip and placed on a moist filter paper (Φ7 cm) in a culture dish (13 cm×13 cm); then, the culture dish was placed in a 27°C light incubator, 11000 LUX light, 75% humidity, and cultured to obtain germinated spores; during the culture process, ddH2O was sprayed every 6 h to ensure that there were always plump water droplets on the spore suspension on the coverslip.

[0055] (3) Dyeing

[0056] Prepare ruthenium red dye solution with a mass concentration of 0.08% and use it now.

[0057] Take 0.08% ruthenium red dye solution and place it on the surface of the plate at 20 μL dye solution / cm 2 The amount of germinated spores added was dropped onto spores at different germination stages and stained in the dark for 8 min; after that, ddH2O was added dropwise from one side of the leaf and the other side was absorbed with filter paper, and this was repeated twice.

[0058] (3) Observation:

[0059] When placed under an inverted microscope, multiple piles of Puccinia mycelium at different germination stages can be observed clearly and completely, such as Figure 2 , 3 shown.

[0060] Experimental Example 2

[0061] This experimental example is used to verify the application of the dyeing method of the present invention (taking Example 1 as an example) in the method for screening corn southern rust resistance germplasm resources, and the steps are as follows:

[0062] (1) Preparation of spore suspension

[0063] 20 μL of 0.1% Tween-20 solution was added dropwise to multiple piles of mature stem rust spores (spores proliferated under the epidermis, broke through the corn epidermis and exposed to form powdery spore accumulations), and the mixture was allowed to stand for 1 minute to obtain a spore suspension.

[0064] (2) Sample preparation

[0065] Take corn "B73" and corn "Yanzi 7016" seeds (existing varieties, seed number: 20231002628) with full and intact kernels, soak them in 2.0% H2O2 for 36 hours, and sow them after the corn seed embryo germinates and breaks through the seed coat, with the embryo facing up and placed 2 cm below the soil.

[0066] When the seeds to be identified grow to V14, fresh leaves are selected and cut into three rectangular pieces of 2.5 cm × 2.5 cm using a blade. 10 μL of spore suspension is dripped onto the front of each leaf group and placed on a moist filter paper (Φ7 cm) in a petri dish (13 cm × 13 cm). Figure 4 Next, the culture dish was placed in a 27°C light incubator, 11000LUX light, 75% humidity, and cultured for 24 hours. During the culture process, ddH2O was sprayed every 6 hours to ensure that there were always plump water droplets on the spore suspension on the corn leaves.

[0067] (3) Dyeing

[0068] Prepare ruthenium red dye solution with a mass concentration of 0.08% and use it now.

[0069] Place the corn leaves on a glass slide facing up and stain at 20 μL / cm 2 Add the amount of ruthenium red dye solution to the germinating spores, keep it away from light for 10 minutes in the dark, add ddH2O from one side of the leaf and absorb it with filter paper on the other side, repeat twice. Then, place it under an inverted microscope to observe the state of multiple piles of rust.

[0070] (3) Data Statistics

[0071] Randomly select "10×" field of view to take photos, and use Image J software to count the germination rate of multiple piles of rust in each photo, etc., to identify the resistance level of different varieties of corn to southern rust, and select the plants with the best resistance to southern rust. Among them, spore germination rate = (germination spore number / total spore number) × 100%.

[0072] Figure 5 The results of ruthenium red staining of multiple piles of Puccinia on the detached leaves of the corn variety "Yanzi 7016" (10× field of view). Figure 6 The following are the results of ruthenium red staining of multiple piles of Puccinia oleracea on detached leaves of the corn variety "B73" (10× field of view), and Table 1 shows the corresponding statistical germination rate results.

[0073] Table 1. Germination rate results of different corn varieties

[0074]

[0075] From the above results, it can be seen that compared with the "B73" variety, the "Yanzi 7016" variety has a certain inhibitory effect on the germination of multiple piles of stem rust.

[0076] In summary, the present invention uses ruthenium red dye to dye multiple piles of Puccinia, thereby avoiding the threat of phenol used in traditional dyeing methods to the health of experimental operators, and greatly reducing the time required for dyeing. At the same time, the dyeing effect is good. After treatment, spores in various germination stages can be largely retained, and the integrity of structures such as hyphae and attachment cells can be maintained. Based on the dyeing method of the present invention, complete, efficient and safe staining observation of multiple piles of Puccinia is achieved, and the growth of multiple piles of Puccinia inoculated on different strains and strain leaves can be further observed and compared to quickly identify the resistance level of the test variety of corn southern rust, which is helpful for the research on corn southern rust and the screening of corn southern rust resistant germplasm resources, and has great application value.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for dyeing the pathogenic fungus of southern corn rust, characterized in that: The steps include: S1. Add Tween-20 solution to multiple piles of mature rust spores and let them stand to obtain a spore suspension; S2, dropping the spore suspension onto a glass slide or corn leaf, and placing it on a moist filter paper in a culture dish; then, placing it in a light incubator for culturing to obtain germinating spores; S3. Add ruthenium red dye solution to the germinated spores for staining, and then wash with water.

2. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In the step S1, the mature piles of rust spores refer to: the spores proliferate under the epidermis, break the corn epidermis and expose to the outside, and form powdery spore accumulations.

3. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In the step S1, the mass concentration of the Tween-20 solution is 0.05-0.15%, and the amount of the solution added is 18-22 μL.

4. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In the step S2, the amount of the spore suspension added is 8 to 12 μL.

5. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In step S2, the culture conditions are: temperature 26-28° C., humidity 70-80%, and light intensity 10000-12000 LUX.

6. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In step S3, the mass concentration of the ruthenium red dye solution is 0.06-0.1%, and the amount of the solution added is 18-22 μL / cm 2 Germination of spores.

7. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In step S3, the dyeing time is 5 to 10 minutes.

8. The method for dyeing the southern corn rust pathogenic fungus according to claim 1, characterized in that: In step S3, water washing is performed by dripping ddH2O solution, and the washing is repeated 2 to 3 times.

9. Use of the staining method for the pathogenic fungus of southern corn rust according to any one of claims 1 to 8 in the study of southern corn rust and the screening of southern corn rust-resistant germplasm resources.

10. The use of the pathogenic fungus of southern corn rust according to claim 9, characterized in that: The staining method is used to observe the difference in the growth state of conidia of the pathogenic fungus, Puccinia multispinosa, on the surface of corn leaves, and to screen out corn germplasm resources resistant to southern rust.

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