阅读说明：本技术 一种利用离体叶片检测杨树溃疡病真菌毒素活性的方法 (Method for detecting activity of mycotoxin in poplar canker by using in-vitro leaves ) 是由 赵嘉平 张冰玉 张一南 李金鑫 李敏 邢军超 于 2020-12-25 设计创作，主要内容包括：本发明涉及一种利用离体叶片检测杨树溃疡病真菌毒素活性的方法,包括采集带有成熟叶片的杨树健康枝条,浸泡于装满无菌水中,避光保存；在无菌水中,从所述杨树健康枝条的叶柄与枝条连接处剪下叶片,将所述杨树健康枝条的叶柄向下插入所述无菌水中；将所述叶柄固定在离心管中并置于光照度为200μmol m～(-2) s～(-1)补光30分钟；测定叶片净光合速率。以毒素处理液中叶片的净光合速率与无菌水对照叶片的净光合速率之间是否出现显著差异及出现显著差异的时间作为评定杨树溃疡病真菌毒素活性的指标。该方法不受自然天气的影响,是一种方便快捷、准确的评定杨树溃疡病真菌毒素活性的方法。(The invention relates to a method for detecting the activity of mycotoxin in poplar canker by utilizing in vitro leaves, which comprises the steps of collecting healthy branches of poplar with mature leaves, soaking the branches in sterile water, and storing the branches in a dark place; cutting off leaves from the joint of the petiole and the branch of the healthy poplar branch in sterile water, and downwards inserting the petiole of the healthy poplar branch into the sterile water; fixing the petiole in a centrifuge tube and placing the centrifuge tube under the illumination of 200 mu mol m ‑2 s ‑1 Supplementing light for 30 minutes; and (4) measuring the net photosynthetic rate of the leaves. Whether a significant difference occurs between the net photosynthetic rate of the leaves in the toxin treatment solution and the net photosynthetic rate of the sterile water control leaves and the time when the significant difference occurs are taken as indexes for evaluating the activity of the mycotoxin of the poplar canker disease. The method is not affected by natural weather, and is a convenient, rapid and accurate method for evaluating the activity of mycotoxin in the poplar canker.)

1. A method for detecting the activity of mycotoxin in poplar canker by utilizing in vitro leaves is characterized by comprising the following steps:

(1) collecting healthy poplar branches with mature leaves, soaking the branches in sterile water, and storing the branches in a dark place;

(2) cutting off leaves from the joint of the petiole and the branch of the healthy poplar branch in sterile water, and downwards inserting the petiole of the healthy poplar branch into the sterile water;

(3) fixing the petiole in a centrifuge tube and placing the centrifuge tube under the illumination of 200 mu mol m^-2s^-1Supplementing light for 30 minutes;

(4) transferring the leaves into a treatment solution containing 5.00% toxin, measuring the net photosynthetic rate of the leaves after 10min, 30min, 50min, 70min and 90min respectively, and evaluating the indexes of the activity of the mycotoxin in the poplar ulcer disease.

2. The method for detecting the mycotoxin activity of poplar canker using excised leaves according to claim 1, wherein the method comprises the following steps: and (3) taking whether the net photosynthetic rate of the leaves in the toxin treatment solution is obviously different from the net photosynthetic rate of the sterile water control leaves or not and the time when the obvious difference appears as evaluation indexes, and determining the time as the sunshine time.

3. The method for detecting the mycotoxin activity of poplar canker using excised leaves according to claim 1, wherein the method comprises the following steps: the toxin treatment solution with the concentration is an aqueous solution containing 5.00 percent of toxin of the poplar canker pathogen.

4. The method for detecting the mycotoxin activity of poplar canker using excised leaves as claimed in claim 3, wherein the method comprises the following steps: the poplar ulcer germ toxin is acetone precipitated germ toxin.

5. The method for detecting the mycotoxin activity of poplar canker using excised leaves according to claim 1, wherein the method comprises the following steps: the determination of the net photosynthetic rate adopts the net photosynthetic rate determined by a photosynthetic system, 3 seedlings are selected for each treatment, and 2 mature leaves are determined for each seedling.

Technical Field

The invention belongs to the technical field of ulcer disease mycotoxin activity detection, and relates to a method for detecting poplar ulcer disease mycotoxin activity by using in-vitro leaves.

Background

The poplar canker is the most important disease of branches and trunks of trees in China and commonly occurs in various poplar survival areas in China. The canker mainly harms poplar seedlings and young trees, but persistent disasters caused by the canker of the poplar widely occur on perennial poplar trees, and the development of the artificial poplar forest industry and the construction of ecological public welfare forest in China is seriously influenced.

Poplar canker is a branch disease caused by canker fungi (Botryosphaeria dothidea). Research shows that the pathogenic fungi can produce mycotoxin in the growth process or the interaction process with host, and the mycotoxin can inhibit host enzyme, nucleic acid metabolism, protein synthesis and the like, so that membrane permeability can be increased, liquid flow of stems can be reduced, transpiration can be disturbed, and leaves can be wilted. The toxin is an important pathogenic factor of most pathogenic bacteria, and is considered to be capable of replacing the pathogenic bacteria to treat host plant tissues or organs as a selective pressure to screen toxin-resistant plants. Bioassays are the most traditional and most straightforward methods for determining pathogenic mycotoxins. Bioassay generally takes damage of toxins to plants as an index, and is performed by adopting various materials such as complete plants, seeds, seedlings, partial organs, callus tissues, plant cells or organelles and the like, however, the above methods generally have the defects of long experimental period, time and labor waste in material preparation and the like. The method for treating cuttage plants, hydroponic branches, calluses, apples and pear fruits by toxins is already used for measuring the biological activity of the branch canker germ toxins, but in the measuring method, the time from experimental treatment to appearance of toxin damage phenotypes is usually several days or longer, and a large amount of experimental materials are consumed. Research proves that pathogenic fungi can obviously reduce physiological indexes such as net photosynthetic rate, stomatal conductance and the like of poplar and other plant leaf tissues in the early infection stage of branch and trunk fungal diseases such as poplar canker pathogen, dermatophytosis and the like, and the canker pathogen toxin is shown to have photosynthetic inhibition activity. Based on the above findings, there is a need for a method for determining the biological activity of toxin of populus canker pathogen based on photosynthetic parameter determination.

Disclosure of Invention

In view of the above, the invention provides a method for detecting the activity of mycotoxin of poplar canker by using in vitro leaves, which is simple, quick, accurate and practical, is not limited by natural weather, can be used for measuring under any weather condition, and provides an effective method for quickly and accurately identifying the activity of mycotoxin of poplar canker, quickly screening canker-resistant poplar varieties and accelerating the disease-resistant breeding process.

The invention provides a method for utilizing the activity of mycotoxin of poplar canker in vitro leaves, which comprises the following steps:

(1) collecting healthy poplar branches with mature leaves, soaking the branches in sterile water, and storing the branches in a dark place;

(2) cutting off leaves from the joint of the petiole and the branch of the healthy poplar branch in sterile water, and downwards inserting the petiole of the healthy poplar branch into the sterile water;

(3) fixing the petiole in a centrifuge tube and placing the centrifuge tube under the illumination of 200 mu mol m^-2s^-1Supplementing light for 30 minutes;

(4) transferring the leaves into a treatment solution containing 5.00% toxin, measuring the net photosynthetic rate of the leaves after 10min, 30min, 50min, 70min and 90min respectively, and evaluating the indexes of the activity of the mycotoxin in the poplar ulcer disease.

Further, whether a significant difference occurs between the net photosynthetic rate of the leaf blade in the toxin-treating fluid and the net photosynthetic rate of the sterile water control leaf blade and the time at which the significant difference occurs were taken as evaluation indexes, and the measurement time was the sunshine duration.

Further, the toxin treatment solution with the concentration is an aqueous solution containing 5.00% of toxin of the poplar canker pathogen.

Further, the poplar ulcer germ toxin is acetone precipitated germ toxin.

Further, the net photosynthetic rate was measured using a photosynthetic system, 3 seedlings were selected for each treatment, and 2 mature leaves were measured per seedling.

Drawings

FIG. 1 is a flow chart of the method for detecting the activity of mycotoxin in poplar canker by using in vitro leaves in the invention;

FIG. 2 shows the difference between the net photosynthetic rates of Xinjiang poplar leaves treated with 5.00% toxin-treating solution and the control leaves treated with sterile water.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention. The protection scope of the present invention shall be subject to the scope defined by the claims. It should be noted that the preferred embodiments should not be construed as limiting the invention, and that it is possible for those skilled in the art to make several improvements and modifications to the invention without departing from the principle of the invention, and these improvements and modifications also fall within the scope of the invention.

As shown in fig. 1, the present invention comprises the steps of:

(1) collecting healthy poplar branches with mature leaves, soaking the branches in sterile water, and storing the branches in a dark place;

(2) cutting off leaves from the joint of the petiole and the branch of the healthy poplar branch in sterile water, and downwards inserting the petiole of the healthy poplar branch into the sterile water;

(3) fixing the petiole in a centrifuge tube and placing the centrifuge tube under the illumination of 200 mu mol m^-2s^-1Supplementing light for 30 minutes;

(4) transferring the leaves into a treatment solution containing 5.00% toxin, measuring the net photosynthetic rate of the leaves after 10min, 30min, 50min, 70min and 90min respectively, and evaluating the indexes of the activity of the mycotoxin in the poplar ulcer disease.

The present invention will be further described with reference to the following examples, wherein the plants are mainly exemplified by Populus alba var. pyramidalis Bge, and the canker fungi of Populus trees are mainly exemplified by the bacterial strain CZA of Botryosphaeriadoothiae, without limiting the scope of the present invention: .

1. CZA plate culture of ulcer germs

The fungus material is canker bacterial strain CZA preserved in water physiology research room of Chinese Lin academy of sciences. The strain CZA was activated on 2.0% PDA medium (2.0% potato extract, 2.0% glucose, 1.5% agar) and cultured at 25 ℃ in the dark.

2. Liquid culture of ulcer bacteria CZA

After the strain is cultured for 7 days by PDA, the strain cake with the diameter of about 4mm is taken out and inoculated into a triangular flask with the capacity of 500ml and containing 300ml of sterilized potato glucose culture solution (PD), 5 blocks of strain cake are inoculated into each flask, and the strain cake is shake-cultured for 10 days under the conditions of 25 ℃, 110r/min and dark.

3. Ulcer bacteria CZA toxin extraction

Filtering the cultured bacteria solution with 0.2 μm microporous membrane under aseptic condition, vacuum rotary evaporating the filtrate to 30mL at 38-40 deg.C with rotary evaporator, adding 150mL acetone 3 times, and shaking at room temperature to extract l h. And then removing the acetone, carrying out vacuum rotary evaporation on the remaining insoluble part by using a rotary evaporator to remove the acetone, dissolving with sterile water to a constant volume of 300mL to obtain a germ toxin stock solution, and storing in a refrigerator at 4 ℃ for later use.

4. Preparation of toxin treatment solution

Preparing an aqueous solution containing 5.00 percent of the toxin of the poplar canker pathogen according to the volume ratio.

5. Outdoor sampling

The healthy branches of Xinjiang poplar with 2 mature leaves were taken and rapidly soaked in a bucket filled with sterile water and taken back to the laboratory in the dark.

6. Indoor sample processing

In the liquid, the leaves were cut from the petiole to shoot junction and the petiole was inserted down into a 5ml centrifuge tube in water. The centrifugal tube with the opening facing upwards is filled with water, the centrifugal tube and the petioles inside are taken out, and the petioles are fixed in the centrifugal tube by adhesive tape. Placing the centrifuge tube vertically on a test tube rack, and placing the centrifuge tube on a test tube rack under the illumination of 200 mu mol m^-2s^-1And (5) supplementing light for 30 minutes under the self-made light supplementing frame.

7. Toxin treatment fluid treatment and photosynthetic parameter determination

And (3) rapidly transferring the leaves subjected to light supplement into 5.00% toxin treatment solution, and measuring the net photosynthetic rate of the leaves after 10min, 30min, 50min, 70min and 90min respectively. The determination of the net photosynthetic rate adopts a Li-6400 photosynthetic system (LI-COR, Lincoln, USA), the determined net photosynthetic rate comprises net photosynthetic rate, transpiration rate, stomatal conductance and the like, 3 seedlings are selected in each treatment, and 2 mature leaves are determined for each seedling.

8. Data analysis

And (4) performing average number calculation and difference significance test on the measured photo-biological data by adopting SPSS software. As shown in FIG. 2, the difference between the net photosynthetic rates of the Xinjiang poplar leaves treated with the 5.00% toxin-treating solution and the control leaves treated with sterile water is shown.

Statistical results show that the net photosynthetic rate of leaves in 5.00% toxin treatment solution is kept unchanged from 10min to 30min after treatment. After 50min of toxin treatment solution treatment, the net photosynthetic rate mean value is 15.14 +/-6.86, and the net photosynthetic rate mean value is reduced compared with the previous 3 measurements, but the difference of the photosynthetic rate among different leaves is large, although the inhibition of the photosynthesis by the toxin treatment is shown, the difference is not significant from the control. After 70min of toxin treatment solution treatment, the net photosynthetic rate of the leaves is very significantly lower than that of the control leaves (ANOVA, P < 0.01). Therefore, 5.00% of the mycotoxin of the poplar canker disease has a significant influence on the net photosynthetic rate of the poplar leaves after the detached leaves are treated for 70min, which shows that the mycotoxin of the poplar canker disease has activity and strong activity.

The method has the obvious characteristics of economy, rapidness, accuracy and easy obtaining of materials, and can be applied to the aspects of performance measurement of branch and stem ulcer germ toxins, the measurement of the anti-ulcer germ capacity of different poplar varieties or clones, the rapid biological measurement of different components in the separation process of related toxins such as ulcer germ toxins and the like.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.