CN111466293B - Rapid inoculation and identification method for citrus canker - Google Patents
Rapid inoculation and identification method for citrus canker Download PDFInfo
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- CN111466293B CN111466293B CN202010288022.8A CN202010288022A CN111466293B CN 111466293 B CN111466293 B CN 111466293B CN 202010288022 A CN202010288022 A CN 202010288022A CN 111466293 B CN111466293 B CN 111466293B
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Abstract
A rapid inoculation and identification method for citrus canker comprises the following steps: (1) collecting sample leaves; (2) culturing pathogenic bacteria of citrus canker; (3) identifying the sample leaf inoculated canker; (4) rooting culture of sample leaves; (5) and (5) culturing and managing sample leaves. Aiming at the defects and shortcomings of the existing citrus canker resistance identification, the invention provides a culture and identification mode through culturing and inoculating canker leaves in vitro to root, supplementing nutrition and moisture, prolonging the growth and observation period. The method has the advantages of simple operation, high efficiency and rapidness in inoculation and identification, reliable result and more intuition.
Description
Technical Field
The invention relates to a method for detecting citrus canker, in particular to a method for quickly inoculating and identifying the citrus canker.
Background
The citrus is the first fruit in the world, and the citrus canker is a worldwide quarantine disease which seriously hinders the development of the citrus industry, and the prevention and control are difficult.
The citrus canker is caused by Xanthomonas citri pathogenic variant (Xanthomonas citri subsp. citri), the growth temperature is 5-36 ℃, the optimum growth temperature of canker is 25-30 ℃, and the death temperature is 55-65 ℃. The Citrus canker (Citrus bacterial canker disease) has the defects of difficult control and incapability of eradicating, the pathogen transmission capacity is strong, and once the Citrus canker disease occurs in a new area, a large amount of manpower and material resources are needed for control, so that great economic loss is caused. The breeding of disease-resistant varieties is the only effective method for preventing and treating the ulcer disease, wherein the simplest and most intuitive method for primarily screening the disease-resistant varieties is to inoculate leaves for the ulcer disease, and the resistance can be basically distinguished through the symptom expression of the leaves.
In the process of breeding disease-resistant varieties, a large number of disease resistance identification tests are required, and the traditional citrus anti-ulcer disease identification mode is divided into in vitro identification and in vivo identification. Generally, the inoculation method can be divided into injection method and spraying method according to the study of the internal or external resistance of the organism, wherein the injection method mainly explores the resistance in the organism, and the spraying method mainly explores the resistance on the surface of the organism, so the injection method is usually adopted for in vitro and living body identification and screening of disease-resistant varieties. The two identification modes of the in vitro and the living body have different defects, for example, in vitro identification is that leaves are separated from a mother tree, the nutrition and water supply of the leaves is insufficient, the disease is unstable, and the leaves are easy to dry and die; the in vivo identification has stable disease, but the operation is inconvenient, the required culture environment occupies a large area, the requirement of inoculated nursery stocks is large, and the management of water, fertilizer and plant diseases and insect pests is complex.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provide a simple, efficient, rapid, visual and reliable-result rapid inoculation and identification method for citrus canker.
The technical scheme adopted by the invention for solving the technical problems is as follows: a rapid inoculation and identification method for citrus canker comprises the following steps:
(1) collecting sample leaves: selecting leaves from the branch tips of suspected infected plants, sampling and marking the leaves to obtain sample leaves;
(2) culturing pathogenic bacteria of citrus canker: selecting single bacterial colonies of the citrus canker, adding a liquid LB culture medium for culturing, and suspending and diluting the obtained culture solution to obtain a pathogen heavy suspension liquid;
(3) and (3) identifying the sample leaf inoculated canker: wiping the front and back surfaces of the sample blade obtained in the step (1) by alcohol cotton, turning the blade to make the blade back upward, performing puncture treatment at the middle part of two side veins after alcohol is volatilized, and then injecting the pathogen heavy suspension liquid obtained in the step (2) into mesophyll tissues for inoculation to obtain an inoculated blade;
(4) rooting culture of sample leaves: placing the inoculated leaves obtained in the step (3) into a rooting culture tray, thoroughly watering the leaves with sterile water, covering petioles of the leaves with wet cotton strips soaked by a rooting agent to keep sufficient water and promote the petioles to root; putting the culture tray into a semi-closed self-sealing bag, periodically checking, and timely irrigating a rooting agent if the cotton strips with the leafstalks are dry;
(5) and (3) sample leaf culture management: and (4) after the sample leaves obtained in the step (4) root, supplementing a water-soluble compound fertilizer every week, supplementing nutrition and water to the sample leaves cultured for the roots, identifying the sample leaves with the canker, storing the sample leaves in a constant-temperature culture chamber, regularly observing the disease condition of the sample leaves, and storing the sample leaves until the canker spreads to the leaf erosion if the sample leaves are infected with the canker.
Further, in the step (1), the sample leaf is a tender leaf which reaches the maximum leaf area but does not turn green completely.
Further, in the step (2), the culture conditions are 25-28 ℃, and the shaking culture is carried out in a constant temperature shaking table at 150-200 r/min for 18-20 h.
Further, in the step (3), the puncturing treatment is that at least 3 positions are selected from the middle of the two side veins for puncturing, and the needle head part penetrates through the leaf tissue.
Further, in the step (3), the injection amount of the pathogen re-suspended bacteria liquid during inoculation is 10 muL, and the concentration of the injection is 10 muL3-106 cfu/mL, the complete infiltration of mesophyll tissue between two side veins is taken as the standard for inoculation.
Further, in the step (3), the residual re-suspended bacterium liquid on the leaf surface is wiped dry by absorbent paper after inoculation.
Further, in the step (5), the culture conditions of the constant temperature culture chamber are that the temperature is 25-28 ℃ and the humidity is 80% -90%.
The inoculation method of the invention has the advantages and positive effects that: the method can be used for quickly and efficiently inoculating the canker of the citrus excised leaves, and the excised leaves are subjected to rooting culture in a constant-temperature culture chamber, so that the environment and the growth are proper, the influence of environmental factors can be reduced, the stable morbidity is ensured, the inherent resistance level of the citrus identification material can be more objectively reflected, and the identification result is more reliable; the required occupied space is small, the operability is strong, the test device is suitable for testing large sample capacity, and the working strength is obviously reduced; after inoculation, plants can be observed for a long time, stable and typical citrus canker infection-resistant symptoms can be seen, and the method has positive significance for quickly and efficiently screening disease-resistant materials.
Drawings
FIG. 1 is a graph showing the effect of rooting culture of different citrus varieties on leaves for 40 days in example 1 of the present invention;
FIG. 2 is a graph showing the effect of rooting culture of leaves for 150 days in example 1 of the present invention;
FIG. 3 is a schematic representation of the inoculation of examples 1 and 2 of the present invention;
FIG. 4 is a graph of the results of 40 days of incubation of control susceptible crystal sugar oranges in example 2 of the present invention;
FIG. 5 is a graph showing the identification results of 60 days of rooting culture of the inoculated leaves in example 2 of the present invention.
Detailed Description
The invention is further illustrated by the following examples and figures.
The chemical reagents used in the examples of the present invention, unless otherwise specified, are commercially available in a conventional manner.
Example 1
The operation steps of this embodiment are as follows:
(1) collecting samples: picking 3 tender leaves which reach the maximum leaf area but do not turn green completely from the plant shoot tips, putting the tender leaves into a self-sealing bag, and marking a sample; in each identification, disease-resistant variety of Citrus citri C-05 and susceptible variety of Crystal sugar orange are used as controls;
(2) and (3) pathogenic bacteria culture: selecting ulcer single colony from the preserved solid culture medium plate, adding 20 mL liquid LB culture medium, culturing at 28 deg.C under shaking at 200 r/min in constant temperature shaking table for 20 h, and diluting the pathogenic bacteria suspension to 10 with sterile water3、104、105、106cfu/ml；
(3) And (3) identifying the sample inoculation ulcer disease: after taking the sample back to the laboratory, the front and back surfaces of the blade are wiped by alcohol cotton, the blade is turned to be upward in the shape of the back of the blade, after the alcohol is volatilized, a small hole (not penetrating the blade) is slightly punched at the middle part of the veins at two sides by a 1 mL syringe needle, and then the heavy suspension liquid is injected into mesophyll tissues by a syringe. The amount injected per pointIs 10 mu L, and takes the complete infiltration of mesophyll tissues between two side veins as the standard. Referring to FIG. 3, each leaf was injected at 5 sites from small to large concentration, and the concentration of the injection was 103、104、105、106cfu/ml and sterile water as controls; after injecting the bacterial liquid with each concentration, wiping the residual heavy suspension bacterial liquid on the leaf surface by using absorbent paper;
(4) rooting culture of leaves: placing the inoculated leaves into a rooting culture tray, filling two layers of sterile toilet paper at the bottom of the tray, covering a layer of filter paper on the uppermost layer, thoroughly watering with sterile water, covering petioles of the leaves with wet cotton strips soaked by a rooting agent to keep sufficient moisture and promote the petioles to root; putting the culture tray into a self-sealing bag (the mouth of the self-sealing bag is sealed by a half), checking once every week, timely watering a rooting agent if the cotton part of the leafstalk is dry, and keeping the culture tray moist because too much water cannot be accumulated in the tray;
(5) culturing and managing: after the leaves root, 1000 times of water-soluble compound fertilizer is added every week; after the in vitro leaves are rooted, the nutrition and the moisture of the leaves subjected to rooting culture are timely supplemented, the leaves subjected to ulcer disease identification can be stored for several months until the ulcer germs are spread to the erosion of the leaves, and the leaves are required to be uniformly placed in a constant-temperature culture room with the temperature of 28 ℃ and the humidity of 80 percent, and the disease occurrence condition of the leaves is regularly observed.
And (4) displaying a rooting culture result: referring to fig. 1, the rooting ability and rooting time of different citrus varieties such as pomelo, lemon and crystal sugar orange are different, and most of the citrus varieties can be stored for one month under normal management by adopting the rooting culture method; referring to fig. 2, some varieties such as leaves of the citrus C-05 are easy to root, can absorb nutrition and moisture from the outside, can be stored for three months all the time, and can still grow after being transplanted to soil after rooting, so that the citrus C-05 is very suitable as a material for the inoculation identification of citrus canker, and lays a foundation for the rapid and efficient canker inoculation.
Example 2
(1) Collecting samples: picking 3 tender leaves which reach the maximum leaf area but do not turn green completely from the plant shoot tips, putting the tender leaves into a self-sealing bag, and marking a sample; in each identification, disease-resistant variety of Citrus citri C-05 and susceptible variety of Crystal sugar orange are used as controls;
(2) and (3) pathogenic bacteria culture: selecting ulcer single colony from the preserved solid culture medium plate, adding 20 mL liquid LB culture medium, culturing at 28 deg.C under shaking at 200 r/min in constant temperature shaking table for 20 h, and diluting the pathogenic bacteria suspension to 10 with sterile water3、104、105、106cfu/ml；
(3) And (3) identifying the sample inoculation ulcer disease: after taking the sample back to the laboratory, the front and back surfaces of the blade are wiped by alcohol cotton, the blade is turned to be upward in the shape of the back of the blade, after the alcohol is volatilized, a small hole (not penetrating the blade) is slightly punched at the middle part of the veins at two sides by a 1 mL syringe needle, and then the heavy suspension liquid is injected into mesophyll tissues by a syringe. The injection amount of each point is about 10 mu L, and the complete infiltration of mesophyll tissues between two side veins is taken as the standard; referring to FIG. 3, each leaf was injected at 5 sites from small to large concentration, the injection concentration was 103、104、105 、106cfu/ml and sterile water as controls; after injecting the bacterial liquid with each concentration, wiping the residual heavy suspension bacterial liquid on the leaf surface by using absorbent paper;
(4) rooting culture of leaves: placing the inoculated leaves into a rooting culture tray, filling two layers of sterile toilet paper at the bottom of the tray, covering a layer of filter paper on the uppermost layer, thoroughly watering with sterile water, covering petioles of the leaves with wet cotton strips soaked by a rooting agent to keep sufficient moisture and promote the petioles to root; putting the culture tray into a self-sealing bag (the mouth of the self-sealing bag is sealed by a half), checking once every week, timely watering a rooting agent if the cotton part of the leafstalk is dry, and keeping the culture tray moist because too much water cannot be accumulated in the tray;
(5) culturing and managing: after the leaves root, 1000 times of water-soluble compound fertilizer is added every week; after the in vitro leaves are rooted, the nutrition and the moisture of the rooted and cultured leaves are timely supplemented, the leaves which are identified to have canker disease can be stored for 3 months until the canker disease is diffused to the erosion of the leaves, and the leaves are required to be uniformly placed in a constant-temperature culture room with the temperature of 28 ℃ and the humidity of 85 percent, and the disease occurrence condition of the leaves is regularly observed.
Symptoms after inoculation appeared: after 15 days after inoculation, adventitious roots of citrus petioles can grow out through rooting culture, and then each variety of leaves take roots successively; referring to fig. 4 and 5, after the rooting culture of the leaves is continuously observed for 60 days, the control crystal sugar orange leaf inoculation area shows obvious disease-sensitive typical symptoms such as crater bulges, yellow halos and the like, the control lemon C-05 leaf inoculation area shows a necrosis disease-resistant symptom, and the citrus inbred group leaf inoculation areas all show typical necrosis symptoms and belong to disease resistance; 439 plants of the lemon inbred population show the disease symptoms of raised crater and yellow halo, and 15 plants show the disease symptoms of necrosis; 644 Shatian pomelo and lemon hybrid groups show disease symptoms of crater bulge and yellow halo, and 204 Shatian pomelo and lemon hybrid groups show disease resistance symptoms of necrosis. Has positive significance for quickly and efficiently screening disease-resistant materials.
Claims (7)
1. A rapid inoculation and identification method for citrus canker is characterized in that: the method comprises the following steps:
(1) collecting sample leaves: selecting leaves from the branch tips of suspected infected plants, sampling and marking the leaves to obtain sample leaves;
(2) culturing pathogenic bacteria of citrus canker: selecting a single bacterial colony of the citrus canker, adding a liquid LB culture medium for culturing, and suspending and diluting the obtained culture solution to obtain a pathogen heavy suspension solution;
(3) and (3) identifying the sample leaf inoculated canker: wiping the front and back surfaces of the sample blade obtained in the step (1) by alcohol cotton, turning the blade to make the blade back upward, performing puncture treatment at the middle part of two side veins after alcohol is volatilized, and then injecting the pathogen heavy suspension liquid obtained in the step (2) into mesophyll tissues for inoculation to obtain an inoculated blade;
(4) rooting culture of sample leaves: placing the inoculated leaves obtained in the step (3) into a rooting culture tray, thoroughly watering the leaves with sterile water, covering petioles of the leaves with wet cotton strips soaked by a rooting agent to keep sufficient water, and promoting the petioles to root; putting the culture tray into a semi-closed self-sealing bag, periodically checking, and timely irrigating a rooting agent if the cotton strips with the leafstalk are dry;
(5) and (3) sample leaf culture management: and (4) after the sample leaves obtained in the step (4) root, supplementing a water-soluble compound fertilizer every week, supplementing nutrition and water to the sample leaves cultured for the roots, identifying the sample leaves with the canker, storing the sample leaves in a constant-temperature culture chamber, regularly observing the disease condition of the sample leaves, and storing the sample leaves until the canker spreads to the leaf erosion if the sample leaves are infected with the canker.
2. The method for rapidly inoculating and identifying the citrus canker according to claim 1, wherein: in the step (1), the sample leaf is a tender leaf which reaches the maximum leaf area but does not turn green completely.
3. The method for rapidly inoculating and identifying the citrus canker according to claim 1, wherein: in the step (2), the culture conditions are 25-28 ℃, and the shaking culture is carried out in a constant temperature shaking table at 150-.
4. The method for rapidly inoculating and identifying the citrus canker according to claim 1, wherein: in the step (3), the puncturing treatment is as follows: selecting at least 3 points in the middle of the two side veins for acupuncture, and enabling the needle head to partially penetrate through the leaf tissue.
5. The method for rapidly inoculating and identifying the citrus canker according to claim 1, wherein: in the step (3), the injection amount of the pathogen re-suspended bacteria liquid during inoculation is 10 muL, and the concentration is 10 muL3-106 cfu/mL, the complete infiltration of mesophyll tissue between two side veins is taken as the standard for inoculation.
6. The method for rapidly inoculating and identifying the citrus canker according to claim 1, wherein: and (3) wiping the residual resuspended bacterial liquid on the leaf surface with absorbent paper after inoculation.
7. The method for rapidly inoculating and identifying citrus canker according to any one of claims 1 to 6, wherein: in the step (5), the culture conditions of the constant-temperature culture chamber are that the temperature is 25-28 ℃ and the humidity is 80-90%.
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| CN109136327A (en) * | 2018-10-08 | 2019-01-04 | 华中农业大学 | A method of identification citrus plant Resistance To Root Rot Disease |
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