CN112725404B - Method for detecting activity of mycotoxin in poplar canker by using in vitro leaves - Google Patents

Method for detecting activity of mycotoxin in poplar canker by using in vitro leaves Download PDF

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CN112725404B
CN112725404B CN202011560337.XA CN202011560337A CN112725404B CN 112725404 B CN112725404 B CN 112725404B CN 202011560337 A CN202011560337 A CN 202011560337A CN 112725404 B CN112725404 B CN 112725404B
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leaves
poplar
toxin
sterile water
mycotoxin
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CN112725404A (en
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赵嘉平
张冰玉
张一南
李金鑫
李敏
邢军超
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Research Institute of Forestry New Technology of Chinese Academy of Forestry
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Abstract

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.

Description

Method for detecting activity of mycotoxin in poplar canker by using in-vitro leaves
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 can be used as a selective pressure to replace the pathogenic bacteria to treat host plant tissues or organs and 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. Methods for treating cutting plants, hydroponic branches, calluses, apples and pear fruits by toxins have been used for measuring the biological activity of the stem canker germ toxins, but in the above measuring methods, several days or more are required from experimental treatment to appearance of toxin damage phenotypes, and a large amount of experimental materials are consumed. Researches prove that at the early stage of infection of branch and stem fungal diseases such as canker pathogenic bacteria and dermatophyte of poplar, pathogenic fungi can obviously reduce physiological indexes such as net photosynthetic rate, stomatal conductance and the like of poplar and other plant leaf tissues, and the canker pathogenic toxin has 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 -2 s -1 Supplementing 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 populus canker fungi.
Further, the toxin of the poplar canker pathogenic bacteria is acetone precipitated pathogenic 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 described in detail below by way of examples, which are intended to illustrate the present invention, but not to limit 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 that these improvements and modifications also fall within the scope of protection 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) in sterile water, cutting off leaves from the joint of a petiole and a branch of the healthy poplar branch, 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 -2 s -1 Supplementing 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 -2 s -1 And (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 as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (2)

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 branches of poplar with mature leaves, soaking in filled sterile water, and storing in 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 -2 s -1 Supplementing light for 30 minutes;
(4) transferring the leaves into a treatment solution containing 5.00% of toxin, measuring the net photosynthetic rate of the leaves after 10min, 30min, 50min, 70min and 90min respectively, taking whether the net photosynthetic rate of the leaves in the toxin treatment solution is significantly different from the net photosynthetic rate of the sterile water control leaves or not and the time when the significant difference occurs as evaluation indexes, and measuring the time as the sunshine duration, wherein the 5.00% of toxin treatment solution is an aqueous solution containing 5.00% of poplar ulcer germ toxin, and the poplar ulcer germ toxin is germ toxin precipitated by acetone.
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: the determination of net photosynthetic rate adopts photosynthetic system determination, 3 seedlings are selected for each treatment, and 2 mature leaves are determined for each seedling.
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CN108660181A (en) * 2018-07-06 2018-10-16 福建省农业科学院植物保护研究所 A kind of detection method of kiwi berry bacterial canker bacterium pathogenicity

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Publication number Priority date Publication date Assignee Title
CN108660181A (en) * 2018-07-06 2018-10-16 福建省农业科学院植物保护研究所 A kind of detection method of kiwi berry bacterial canker bacterium pathogenicity

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* Cited by examiner, † Cited by third party
Title
Bacterial Canker of Tomato:Current Knowledge of Detection,Management,Resistance,and Interactions;Yusuf Sen等;《The American Phytopathological Society》;20141001;第99卷(第1期);第4-13页 *
利用溃疡菌毒素测定杨树品种的抗溃疡病性能;张晓刚等;《云南林业科技》;20030930(第3期);摘要，第72页右栏第2段，第73页左栏第1段 *
欧美杨溃疡病的危害损失评估;徐静伟;《中国优秀博硕士学位论文全文数据库（硕士） 农业科技辑》;20140515(第05期);第20页第1-3段，第23页第3-4段 *

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