Manufacturing method of plant disease specimen

Abstract

The invention discloses a manufacturing method of a plant disease specimen. The manufacturing method of the plant disease specimen provided by the invention comprises the following steps: collecting a fresh plant disease sample for pretreatment; carrying out vacuum freeze-drying treatment on the pretreated sample; embedding the sample subjected to vacuum freeze-drying treatment by using an epoxy resin crystal glue solution subjected to bubble removal to obtain the plant disease specimen. According to the method, the problems of early-stage shape fixation and later-stage preservation and maintenance of the specimens are solved, the morphological characteristics, colors and the like of host plants and pathogenic bacteria in the plant disease specimens in a natural state are reserved, the plant disease specimens can be massively preserved for a long time, and the use efficiency of experimental materials in circulation teaching is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of specimen preparation, and in particular relates to a method for preparing a plant disease specimen. Background Art

[0002] Plant disease specimens are important teaching materials in the experimental teaching of plant pathology in higher agricultural colleges. They are carriers and teaching aids for the experimental links of professional courses in plant protection and related majors, and are indispensable objects in teaching practice. There are many types of plant disease specimens, and plant disease specimens mainly show three aspects: 1) the color of the host plant and the morphological characteristics after being infected by pathogens; 2) the morphological characteristics of pathogenic microorganisms after infecting the host plant; 3) the morphological characteristics of the tissues where the two interact.

[0003] Common specimen preparation methods include bottle immersion specimens, wax leaf specimens, plastic specimens, etc. However, in the process of preparing plant disease specimens using these methods, it is very difficult to achieve the above three points at the same time.

[0004] Host plants are usually green, and this green color is produced by chlorophyll with magnesium atoms as the core structure. Chlorophyll exists in chloroplasts in cells. The chlorophyll structure is very unstable and easy to decompose. After decomposition, the color of the leaves loses its original green color. The conventional method of preserving plant disease specimens is to replace magnesium ions with relatively stable copper ions by boiling in water, thereby maintaining the green color of the host plants to a certain extent, but it cannot achieve the original green color; and this method has a good color preservation effect on some grass plants, but it is not suitable for woody branches, fruits, or thin leaves, young leaves; in addition, for some leaves with waxy or leathery surfaces, the color preservation effect is not good and they are easy to fall off.

[0005] It is difficult for wax leaf specimens to maintain their original color during the pressing process, and the pressed specimens lack a three-dimensional sense. Taking pear leaf disease specimens as an example, when picking fresh pear leaves to make wax leaf specimens, the wrinkled areas on the leaves will turn from green to black and cannot be pressed any further; if they are picked and left overnight before being pressed again, the entire leaf will turn black. This problem has long been an unsolved problem in the preservation of pear leaf disease specimens. At present, pear leaf disease specimens are all fresh specimens. In the teaching process, pear leaf disease specimens are also in a state of being unable to be preserved or distorted, which is extremely detrimental to teaching.

[0006] The preparation process of bottle-immersed specimens is complicated. The specimens are placed in specimen bottles with preservation liquid, and the immersion liquid needs to be replaced regularly. However, volatile immersion liquids are generally toxic, which is not good for human health and the environment. Plastic specimens are only suitable for leaf and petal diseases, and must be stored in a refrigerator, otherwise they are prone to discoloration and lack three-dimensionality.

[0007] In addition, the preservation of pathogens also has certain requirements. Pathogens are very small and each has a different morphology. For example, powdery mildew will have a layer of powder on the leaves. Pressing the specimens during preservation will destroy the morphological characteristics of the powdery mildew. Bottle-immersing specimens will cause the powdery mildew to fall off the host plant, and the morphological relationship between the pathogen and the host plant cannot be shown. In addition, traditional plant disease specimens are often in contact with air and are greatly affected by the environment. Dried specimens are prone to breakage, moisture, corruption, insect infestation or mildew, and require regular maintenance.

[0008] It can be seen that the existing specimen preparation process is difficult to solve the problem of fixing the specimen's early morphology and preserving and maintaining it in the later stage. In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a method for preparing plant disease specimens by using vacuum freeze-drying technology combined with crystal glue curing technology. The method solves the problems of long specimen morphology fixation time in the early stage and great difficulty in later preservation and maintenance. The prepared specimens have high fidelity and long preservation time, and are simple to prepare, save trouble and labor, are green and environmentally friendly, and improve the utilization efficiency of experimental materials in circular teaching.

[0010] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for preparing a plant disease specimen, comprising the following steps: S1. Collect fresh plant disease samples and pre-treat the samples; S2, subjecting the pretreated samples to vacuum freeze-drying treatment; S3. Using epoxy resin crystal glue to embed the sample after vacuum freeze-drying to obtain the plant disease glue specimen.

[0011] In step S1, the plant is selected from the genus Sabina, and specifically, the plant is selected from juniper, cypress, and arborvitae.

[0012] In step S1, the disease is rust of Yamada ( Gymnosporangium yamadai Miyabe and / or Asian Glue Rust ( Gymnosporangium asiaticum Miyabe) caused by the disease.

[0013] The plant disease samples are cockscomb-shaped or gum-shaped teliospore horns of apple cypress rust and / or pear cypress rust on juniper plants. The color of the cockscomb-shaped teliospore horns is dark brown or bright yellow.

[0014] In step S1, the climate conditions collected are: the ten-day average temperature is above 8.2°C, the daily average temperature is 10.6-11.6°C, and the rainfall is above 4 mm.

[0015] In the present invention, the pretreatment is performed according to the following operations: trimming branches and leaves of the collected samples and packaging them; the samples after the pretreatment immediately enter the next step.

[0016] In step S2, the freeze dryer is precooled first, and then the sample is subjected to vacuum freeze-drying treatment; the precooling conditions are: temperature is -45°C, time is 0.5h; the vacuum freeze-drying conditions are: vacuum degree is 1Pa, cold trap temperature is -50°C, drying time does not exceed 72h, and the degree of freeze-drying is based on the sample being extremely easy to break.

[0017] In step S3, the embedding is performed according to the following operations: preparing epoxy resin crystal glue on site, and evacuating the glue at room temperature; placing the sample in a mold, injecting glue twice, standing, demoulding, and obtaining a plant disease specimen; The epoxy resin crystal glue liquid is composed of glue A and glue B in a weight ratio of 3:1; The time for vacuuming the glue solution under the normal temperature condition is 1-3 minutes.

[0018] The two injections include: The first injection includes: injecting the glue liquid into the mold to cover the bottom layer of the mold, filtering, and standing for 12 hours; The second injection includes: filtering the glue liquid in the mold, injecting the glue liquid again until the liquid level reaches the top of the mold edge, standing until the drip glue is completely solidified, demolding, and obtaining the plant disease specimen. The filtration time is 3 minutes; the standing time is 12-24 hours.

[0019] In a second aspect, the present invention also provides plant disease specimens obtained by the preparation method.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts vacuum freeze-drying technology combined with crystal glue curing process to greatly shorten the specimen production efficiency and improve the authenticity. 1. The drying and curing time are greatly shortened. The present invention uses vacuum freeze-drying technology to dry the samples. By optimizing the pre-cooling and freeze-drying conditions, the drying time of the samples is greatly shortened. For plant tissues with low water content, such as winter spore horns or juniper branches and leaves with cockscomb-shaped undulations, drying is sufficient for 24 hours; for plant tissues with high water content, such as winter spore horns or apple leaves with gum flower shapes, the drying time does not exceed 72 hours. Compared with the traditional wax leaf tablet pressing method of drying for 7-10 days, the present invention significantly saves drying time. At the same time, the crystal glue curing process is used to solidify the specimens, which is simple to operate, saves time and effort, and can avoid the complicated processes of the traditional specimen tablet pressing process such as replacing the dry absorbent paper every day during the pressing process (the replacement process lasts for 7 days). 2. High fidelity. The present invention adopts a crystal glue curing process to achieve sample shaping, which can better maintain the original natural morphology, color and other characteristics of plant diseases, and can fully present the natural morphological characteristics of host plants and pathogens, meeting the needs of teaching and observation, and can be preserved for a long time, not easy to be damaged, and improves the use efficiency of experimental materials in circular teaching.

[0021] 3. Green and environmentally friendly. The specimen preparation method provided by the present invention does not require measures such as insect prevention, anti-corrosion, and easy breakage, nor does it use chemical agents, which is more in line with the development concept of specimen preparation technology.

[0022] In summary, the production method provided by the present invention has significantly improved production time, cost, effect and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of the teliospore horn specimen of juniper rust disease. In the picture: A represents the cockscomb-shaped undulating teliospore horn; B represents the gum-flower-shaped teliospore horn.

[0024] Figure 2 This is a photo of the teliospore horns of the juniper diseased with apple rust before collection; in the figure: A represents the cockscomb-shaped undulating teliospore horns; B represents the gum-flower-shaped teliospore horns.

[0025] Figure 3 These are photos of plant disease specimens prepared by traditional air-drying and vacuum freeze-drying. In the figure: A represents air-dried specimens; B represents vacuum freeze-dried specimens.

[0026] Figure 4 This is a comparison chart of the production time and effect of traditional plant specimen preparation and freeze-dried gel specimen preparation; A represents the traditional specimen preparation method; B represents the vacuum freeze-dried gel specimen. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources.

[0030] The sample materials used in the following examples are juniper branches with winter spore horns collected from March to May 2023 in the Summer Palace in Beijing.

[0031] Formation of teliospore horns: Temperature is the main reason affecting the development of hyphae and teliospore formation in the overwintering galls on Juniperus. When the average temperature in the spring is above 3.8-4.8℃ and the daily average temperature is 9.7-11.3℃, teliospores can form; in most years, Beijing has this temperature in mid-March, and the reference phenological period is the flowering of mountain peach, the budding of willow, and the budding of poplar. When the temperature rises in spring, the smooth galls protrude obviously, and the teliospores push a small crack from the protrusion. After the galls crack, the teliospore horns protrude and grow from the cracks, forming dark brown cockscomb-shaped undulating teliospore horns with dense teliospores. Spring rain is the key to the germination of teliospores. Early spring rain and heavy rainfall will cause serious damage. The average temperature in the ten-day period is above 8.2-8.3℃, and the average daily temperature is 10.6-11.6℃. When there is more than 4 mm of rainfall, the winter spore horns of the cockscomb-shaped undulating plants absorb water and swell to become orange-yellow gum-like, and the winter spores germinate. This temperature is available in some years in late March and most years in early April in Beijing. Figure 2 -A, after rain, the winter spore horns absorb water and swell and gel, forming an orange-yellow flower-like gelatinous substance such as Figure 2 -B.

[0032] The epoxy resin crystal glue used in the following embodiments is Indigo Epoxy, product number 10074830365466.

[0033] Example 1 1 Materials and Methods 1.1 Sample collection Time selection: According to suitable climatic conditions, collect the cockscomb-shaped undulating winter spore horns grown from galls with typical morphology and color and the gum-shaped winter spore horns that swell after rain as specimen samples.

[0034] 1.2 Sample pretreatment Collect samples of juniper branches with cockscomb-shaped undulating winter spore horns, properly trim the branches and leaves, hang labels, and pack them in sealing bags. Do not squeeze them to ensure the integrity of the entire branch, and proceed to the next step as soon as possible.

[0035] After the rain, the juniper branch samples with swollen gum-shaped winter spore horns should be labeled and placed in a moist fresh-keeping bag at 4°C to keep them moist. Do not squeeze them to ensure the integrity of the samples and proceed to the next step as soon as possible.

[0036] 1.3 Sample vacuum freeze drying The vacuum freeze dryer was precooled at -45℃ for 0.5h, and the freshly collected juniper branch samples with cockscomb-shaped undulating winter spore horns or gum-shaped winter spore horns were placed in the precooled freeze dryer to ensure the original morphological characteristics of the samples. A small bracket can also be used to hang the samples upside down or place them on the bracket for vacuum drying. The freeze drying parameters of the vacuum freeze dryer (model: SCIENTZ-18N) are vacuum degree of 1Pa and cold trap temperature of -50℃. The vacuum machine was turned on, and after 24h of freeze drying, the plant disease specimens were dried.

[0037] 1.4 Preparation of crystal glue specimen 1) Preparation and bubble removal of crystal glue: Prepare the epoxy resin crystal glue liquid in the weight ratio of 3:1 between A and B glue, and use it immediately after preparation; stir it evenly after preparation, put it into the vacuum freeze dryer, do not turn on the refrigerator, and vacuum it at room temperature for 1 to 3 minutes. In this process, you can see a lot of bubbles coming out of the glue, which is the gas in the glue; turn off the vacuum dryer, and the bubbles in the glue disappear quickly. At this time, the vacuumed glue can be used for the first injection. The bubbles can be removed well after vacuum filtration. The prepared glue is bright and transparent without bubble influence. The mixing of A and B glue is an exothermic process. When preparing a large volume of crystal glue mixture, it is easy to solidify in a short time, so it should be prepared and used immediately.

[0038] 2) Inject the glue into the mold (mold inner diameter 6.0cm×8.0cm×2.0cm) until it covers the bottom layer, about 10-15ml; Take the freeze-dried juniper branch sample with cockscomb-shaped undulating winter spore horns (the sample is about 6.5cm high and 3.5cm in diameter) and put it into the mold, inject the glue liquid, cover the bottom of the mold, inject the height until the sample floats slightly, and vacuum filter at room temperature for 1-3 minutes. This step is to allow the specimen and the drip glue to contact more fully and avoid the generation of bubbles. Especially fungal tissues, such as winter spore horns, are uneven and have small gaps between structures. The glue concentration is high and cannot penetrate smoothly. During the solidification process of the glue, these tissues are squeezed, resulting in bubbles, which remain in the crystal glue.

[0039] The mixed glue solution after vacuum filtration is introduced into the mold where the sample is placed and left to stand at room temperature for 12 hours or overnight. After the glue solution solidifies, the mixed crystal glue solution is vacuum filtered for 3 minutes. The second glue injection height is to the top of the mold edge and left at room temperature for 12 hours. After the glue solution is completely solidified, it can be demoulded to form a specimen. Figure 1 -A.

[0040] Example 2 The difference from Example 1 is that the sample is a rubber flower-shaped teliospore horn.

[0041] According to the method of Example 1, the glaze-shaped winter spore horns are first vacuum-freeze-dried and then crystal-dried for storage. Figure 1 -B.

[0042] Comparative Example 1 The difference from Example 1 is that the sample specimens are only stored at room temperature after being air-dried.

[0043] Comparative Example 2 The difference from Example 2 is that the specimen is not preserved by dripping glue after vacuum freeze-drying.

[0044] Test Case 1. Fidelity and preservation time 1) The undulating teliospore horn in the shape of a cockscomb After the complete specimen of winter spores with cockscomb-shaped undulations was made and stored at room temperature for 10 months, the fine structure of the sealed sample was clearly visible, without shrinkage, corruption or discoloration, and the shape and color of the fresh state were maintained. However, the sample specimen prepared in comparative example 1, which was air-dried and stored at room temperature, turned yellow, had no fresh morphological characteristics, and the material became brittle and was very easy to break ( Figure 3 -A).

[0045] 2) Gum-shaped teliospore horn The difference in the part of the glaze-shaped winter spore horns is particularly obvious: the glaze-shaped winter spore horns preserved by dripping glue after vacuum freeze-drying maintain the original shape, that is, the color is yellow-brown and flower-shaped, and the plant tissue after vacuum freeze-drying maintains the original tender green. However, in the specimens prepared in Comparative Example 2 that were not preserved by dripping glue after vacuum freeze-drying, the plant tissue faded seriously, the glaze-shaped winter spore piles turned white, and no longer had the original plump yellow-brown color. The shape was also significantly different from the fresh samples or the samples preserved by dripping glue, and it was very easy to break and shatter ( Figure 3 -B).

[0046] It was also found that specimens that were not freeze-dried in vacuo but directly preserved by dripping glue would show serious problems such as yellowing and loss of green color of plant tissues, and pathogenic tissues losing their original brown-yellow color and turning white.

[0047] This shows that the present invention combines the vacuum freeze-drying technology with the crystal glue dripping technology, and the specimens produced maintain the original natural morphology, color and other characteristics of the host plants and pathogens, and can present the natural morphological characteristics of the host plants and pathogens in an all-round and three-dimensional manner, which can meet the needs of plant pathology teaching, scientific research and observation; while traditional plant disease specimens are made by wax leaf preparation or air drying, and the specimens made are not easy to preserve and are very easy to turn yellow, fade and break.

[0048] 2. Production efficiency The vacuum freeze drying time of the specimen in Example 1 does not exceed 24 hours, and the preparation time of the glue dripping and standing step does not exceed 36 hours. In the traditional wax leaf pressing method, the drying time requires 7 to 10 days, the later pressing process also requires 7 days, and the absorbent paper needs to be replaced every day. It can be seen that the specimen preparation method provided by the present invention greatly improves the production efficiency.

[0049] The specimen preparation method provided by the present invention is significantly different from the currently used tablet pressing method in terms of time, cost, effect, etc. Figure 4 shown.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing plant disease specimens, comprising the following steps: S1. Collect plant disease samples and pre-treat the samples; S2, subjecting the sample pretreated in step S1 to vacuum freeze-drying; S3. Using epoxy resin crystal glue to embed the sample processed in step S2 to obtain the plant disease glue specimen.

2. The method for preparing plant disease specimens according to claim 1, characterized in that: In step S1, the plant is selected from the genus Sabina.

3. The method for preparing plant disease specimens according to any one of claims 1 or 2, characterized in that: In step S1, the disease is caused by Puccinia yamada and / or Puccinia asiatica.

4. The method for preparing plant disease specimens according to any one of claims 1 to 3, characterized in that: In step S1, the plant disease sample is cockscomb-shaped or gum-shaped winter spore horns of apple cypress rust and / or pear cypress rust on Sabina plants.

5. The method for preparing plant disease specimens according to any one of claims 1 to 4, characterized in that: The climatic conditions for the data collection are: the ten-day average temperature is above 8.2°C, the daily average temperature is 10.6~11.6°C, and the rainfall is above 4mm.

6. The method for preparing plant disease specimens according to any one of claims 1 to 5, characterized in that: In step S1, the pretreatment is performed according to the following operations: trimming branches and leaves of the collected samples and packaging; The sample after the pretreatment immediately enters the next step.

7. The method for preparing a plant disease specimen according to any one of claims 1 to 6, characterized in that: In step S2, the freeze dryer is precooled, and then the sample is subjected to vacuum freeze drying; The precooling conditions are: temperature of -45°C and time of 0.5h; The conditions of the vacuum freeze-drying treatment are: vacuum degree of 1 Pa, cold trap temperature of -50°C, drying time not exceeding 72h, and the degree of freeze-drying is based on the sample being extremely easy to break.

8. The method for preparing plant disease specimens according to any one of claims 1 to 7, characterized in that: In step S3, the embedding is performed according to the following operations: preparing epoxy resin crystal glue on site, and evacuating the glue at room temperature; placing the sample in a mold, injecting glue twice, standing, demoulding, and obtaining a plant disease specimen; The epoxy resin crystal glue liquid is composed of glue A and glue B in a weight ratio of 3:1; The time for vacuuming the glue solution under the normal temperature condition is 1-3 minutes.

9. The method for preparing plant disease specimens according to claim 8, characterized in that: The two injections include: The first injection includes: injecting the glue liquid into the mold to cover the bottom layer of the mold, filtering, and standing for 12 hours; The second glue injection includes: filtering the glue liquid in the mold, injecting the glue liquid again until the liquid level reaches the top of the edge of the mold, standing until the drip glue is completely solidified, demoulding, and obtaining the plant disease specimen.

10. The plant disease specimen obtained by the preparation method according to any one of claims 1 to 9.

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