CN111471741A - Method for rapidly identifying ring spot resistance of pear trees - Google Patents

Abstract

The invention provides a method for rapid identification of pear ring disease resistance, belonging to the technical field of plant disease resistance identification evaluation and resource screening. The method of the invention includes the steps of culturing the pathogen of pear ring rot, leaf treatment, inoculation of isolated leaves, etc. The isolated leaves are inoculated with the pathogen of pear ring rot, and the disease time and incidence rate of leaves of different varieties after inoculation with the pathogen are observed and compared. to identify the resistance of different varieties to this disease. The invention has the advantages of early and multiple identification, shortens the identification period of disease resistance (it only takes 6-8 days to complete a batch of identification), improves the accuracy of identification results, and saves manpower and material resources for identification , to achieve high-throughput identification. In addition, the disease conditions of this method are artificially controllable, and the results of resistance identification are relatively accurate and consistent. This method can scientifically, efficiently and accurately identify the resistance of pears to ringworm.

Description

A method for rapid identification of pear ring disease resistance

technical field

The invention belongs to the technical field of plant disease resistance identification and evaluation and resource screening, and in particular relates to a method for rapid identification of pear ring disease resistance.

Background technique

Pear ring disease is one of the main diseases of pear trees in my country, which can cause peeling of branches and trunks, weakening of tree vigor, and fruit rot before and after harvesting. The occurrence and prevalence of the disease are closely related to meteorological conditions, the degree of occurrence of branch ring disease, and the growth period of the parasite. Current research shows that the disease is mainly transmitted through rainwater, causing large-scale disease in pear orchards, and seriously jeopardizing the yield and quality of pears. Since the main damage parts of pear ring rot are the trunk and fruit of pear, the pathogenic bacteria are not easy to observe during the incubation period and the initial symptoms of the disease, and early identification is difficult. The most effective solution is to explore disease-resistant genetic resources and cultivate resistant varieties. Establishing an accurate, reproducible and rapid method for identification of pear ring disease resistance is an important basis for screening resistant resources.

The method for identifying the resistance to ring scab disease of fruit trees disclosed in the prior art generally adopts a field inoculation identification method or a disease garden resistance identification method. The method of disease resistance identification is to plant different varieties of plants in the disease-carrying nursery with serious disease, measure the incidence rate and disease degree of different varieties, calculate the disease index, and compare the susceptibility and disease resistance of different varieties to diseases. The inoculation identification method is mainly used to isolate the pathogen, inoculate the hyphae or spores on healthy branches or fruits, cultivate under the same conditions, and calculate the disease index according to the incidence rate and disease degree of a specific part to identify the disease resistance of the plant. . The indicators identified include the onset time of fruit or two- to three-year-old branches and stems, the severity of the disease site, and changes in other symptoms of the tree. The results of resistance identification in disease gardens are easily affected by the density distribution of regional colonies and site environmental factors. The identification results of different identification times will be quite different, and it is also difficult to carry out high-throughput resistance identification.

Field inoculation identification is also a field identification method, which solves the limitations of disease garden identification to a certain extent, but it is still affected by the age of the inoculated tree, planting environment, and climatic conditions, and the error is large, and it is not easy to observe the time and time of the disease occurrence of pear branch cadres. The process and the degree of morbidity, the operation is complex, the cost is high, and the accuracy is poor. The field identification test generally covers a large area, requires a long identification period, requires a lot of manpower to handle and manage, and the test cost is high.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a method for rapid identification of pear ring disease resistance, which is simple to operate, low in cost, fast, high in accuracy and suitable for identification of pear ring disease resistance.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for quickly identifying the resistance of pear tree ring striae disease, comprising the following steps: (1) cutting pear leaves with a handle, and after sterilizing, wrapping moist sterile absorbent cotton at the cut; Lay wet sterile gauze, place a water barrier at the midline of the sterile gauze; spread the blade on the water barrier, make the sterile absorbent cotton contact with the sterile gauze, and get Pretreatment of isolated leaves;

(2) Utilize the plants with rot disease in the field to isolate and purify the rot pathogenic bacteria in the PDA medium, and after culturing for 5 to 7 days, take a bacterial cake with a diameter of 5 mm along the periphery of the colony;

(3) Symmetrically puncture 6 holes on both sides of the main veins of the pretreated isolated leaves, inoculate the bacterial cake on the back puncture holes of the pretreated isolated leaves, and remove the bacterial cake after culturing in the dark for 2 d , continue to cultivate in the dark for 5 days, calculate the disease grade, disease index and divide the disease resistance according to the average diameter of the lesions;

Among them, the classification criteria of the disease grade are as follows:

Grade 0: no lesions;

Grade 1: The diameter of the lesion is within the interval of (0, 5mm];

Grade 3: The diameter of the lesion is within the interval of (5mm, 8mm];

Grade 5: The diameter of the lesion is within the interval of (8mm, 12mm];

Grade 7: The diameter of the lesion is within the interval of (12mm, 15mm];

Grade 9: The diameter of the lesion is greater than 15mm;

Use the disease index to measure the degree of disease incidence:

Use disease index DI to classify disease resistance: when the disease index is 0, it is high resistance/immunity; when the disease index value is in the (0, 20) interval, it is disease resistance; when the disease index value is in the (20, 40] interval When the disease index value is in the (40, 60] interval, it is moderately susceptible; when the disease index value is in the (60, 80] interval, it is susceptible; when the disease index value is in the (80, 100] interval. Inner time is high sense;

There is no temporal sequence between steps (1) and (2).

Preferably, the sterilization in step (1) includes sterilizing the leaves with an aqueous sodium hypochlorite solution with a mass percentage of 0.1% for 10 minutes, and then rinsing with sterile water for 3 times.

Preferably, the leaves are selected from leaves with the same physiological state in the middle of annual shoots.

Preferably, in step (2), the pathogenic bacteria of rotundus include various physiological races of Botryosphaeria dothidea.

Preferably, the temperature of the culture in step (2) is 25°C.

Preferably, in step (2), the peripheral mycelium is to be stretched to the edge of the petri dish, and the fungus cake is punched out with a sterile hole punch.

Preferably, in the step (3), the puncture is performed at both sides of the main vein parallel to the midline of the main vein and at the upper and lower quartiles.

Preferably, the temperature of the dark culture in step (3) is 25°C, and the relative humidity is 80-100%.

The invention provides a method for rapidly identifying the resistance of pear ring scab disease, including the steps of culturing the pear ring scab pathogen, leaf treatment, inoculation of in vitro leaves and the like. Observe and compare the disease time and incidence rate of leaves of different varieties after inoculation with pathogens to identify the resistance of different varieties to this disease. Compared with the existing pear tree ring disease resistance identification technology, the invention breaks the limitation of the traditional method for identification of ring disease resistance, and has the advantages of early and multiple identification. The method of the present invention shortens the identification period of disease resistance (it takes only 6-8 days to complete a batch of identification), improves the accuracy of identification results, saves manpower and material resources for identification, and realizes high-throughput identification , In addition, the disease conditions of this method are artificially controllable, and the resistance identification results are relatively accurate and consistent, and the method can scientifically, efficiently and accurately identify the resistance of pears to ringworm.

Description of drawings

Fig. 1 is the normal colony morphology of rot fungus;

Figure 2 shows the disease situation on the front of the leaves after inoculation of rot.

Detailed ways

The invention provides a method for quickly identifying the resistance of pear tree ring striae disease, comprising the following steps: (1) cutting pear leaves with a handle, and after sterilizing, wrapping moist sterile absorbent cotton at the cut; Lay wet sterile gauze, place a water barrier at the midline of the sterile gauze; spread the blade on the water barrier, make the sterile absorbent cotton contact with the sterile gauze, and get Pretreatment of isolated leaves;

(2) Utilize the plants with rot disease in the field to isolate and purify the rot pathogenic bacteria in the PDA medium, and after culturing for 5 to 7 days, take a bacterial cake with a diameter of 5 mm along the periphery of the colony;

(3) Symmetrically puncture 6 holes on both sides of the main veins of the pretreated isolated leaves, inoculate the bacterial cake on the back puncture holes of the pretreated isolated leaves, and remove the bacterial cake after culturing in the dark for 2 d , continue to cultivate in the dark for 5 days, calculate the disease grade, disease index and divide the disease resistance according to the average diameter of the lesions;

Among them, the classification criteria of the disease grade are as follows:

Grade 0: no lesions;

Grade 1: The diameter of the lesion is within the interval of (0, 5mm];

Grade 3: The diameter of the lesion is within the interval of (5mm, 8mm];

Grade 5: The diameter of the lesion is within the interval of (8mm, 12mm];

Grade 7: The diameter of the lesion is within the interval of (12mm, 15mm];

Grade 9: The diameter of the lesion is greater than 15mm;

Use the disease index to measure the degree of disease incidence:

Use disease index DI to classify disease resistance: when the disease index is 0, it is high resistance/immunity; when the disease index value is in the (0, 20) interval, it is disease resistance; when the disease index value is in the (20, 40] interval When the disease index value is in the (40, 60] interval, it is moderately susceptible; when the disease index value is in the (60, 80] interval, it is susceptible; when the disease index value is in the (80, 100] interval. Inner time is high sense;

There is no temporal sequence between steps (1) and (2).

In the present invention, the pear tree stalked leaves are cut, and after sterilization, moist aseptic absorbent cotton is wrapped at the cut; the moist aseptic gauze is flattened on a metal tray, and a water barrier is placed at the midline of the aseptic gauze; The leaves are laid flat on the water barrier, and the sterile absorbent cotton is brought into contact with the sterile gauze to obtain pretreated isolated leaves. In the present invention, the leaves with the same physiological state in the middle of the annual branches are preferably cut, and the petiole length is not less than 4 cm. In the present invention, the cut leaves are sterilized, and the sterilization preferably includes sterilizing the pear tree leaves with an aqueous sodium hypochlorite solution with a mass percentage of 0.1% for 10 minutes, and then rinsing with sterile water for 3 times. The present invention preferably also includes pruning petioles under sterile water when testing the ring disease resistance among pear tree varieties, so that the petiole lengths of all pear tree varieties are the same, and the petiole length is not less than 2 cm. The present invention wraps the moist sterile absorbent cotton at the notch, and the wrapping is preferably done under the surface of sterile water. In the present invention, when the blade is pretreated, the blade does not directly contact the gauze, and the petiole absorbent cotton directly contacts the sterile gauze wetted at the bottom of the tray.

The invention utilizes the plants with rota disease in the field to separate and purify the rota pathogenic bacteria in a PDA medium, and after culturing for 5-7 days, a bacterial cake with a diameter of 5 mm is taken along the periphery of the colony. In the present invention, the epidermis or leaves of plants with rot disease are preferably collected from the field, and the rot pathogenic bacteria are obtained by separating and purifying in PDA medium. The method of separation and purification is not particularly limited in the present invention, and conventional methods for separation and purification of pathogenic bacteria in the art can be used. In the present invention, the obtained rotavirus pathogen is cultured for 5-7 days, and the culture is preferably carried out in a constant temperature incubator, and the temperature is preferably set at 25°C. In the present invention, preferably, after culturing for 5-7 days, after the peripheral mycelium stretches to the edge of the petri dish, a bacterial cake with a diameter of 5 mm is taken along the periphery of the colony. In the present invention, the fungus cake is preferably punched out at a position where the density of the mycelium is similar with a sterile puncher. The pathogenic bacteria of ringworm of the present invention preferably include various physiological races of Botryosphaeria dothidea, specifically including Lw018, WUHAN-WANG, WZ-279, PG45 and the like.

After obtaining the pretreated isolated leaves and the fungus cake, the present invention punctures 6 holes symmetrically on both sides of the main veins of the pretreated isolated leaves, and inoculates the fungus cake on the back puncture holes of the pretreated isolated leaves, After culturing in the dark for 2 d, remove the bacterial cake, continue cultivating in the dark for 5 d, and calculate the disease grade, disease index and disease resistance according to the average diameter of the lesions;

Among them, the classification criteria of the disease grade are as follows:

Grade 0: no lesions;

Grade 1: The diameter of the lesion is within the interval of (0, 5mm];

Grade 3: The diameter of the lesion is within the interval of (5mm, 8mm];

Grade 5: The diameter of the lesion is within the interval of (8mm, 12mm];

Grade 7: The diameter of the lesion is within the interval of (12mm, 15mm];

Grade 9: The diameter of the lesion is greater than 15mm;

Use the disease index to measure the degree of disease incidence:

Use disease index DI to classify disease resistance: when the disease index is 0, it is high resistance/immunity; when the disease index value is in the (0, 20) interval, it is disease resistance; when the disease index value is in the (20, 40] interval When the disease index value is in the (40, 60] interval, it is moderately susceptible; when the disease index value is in the (60, 80] interval, it is susceptible; when the disease index value is in the (80, 100] interval. High sense when inside.

In the present invention, sterile toothpicks are preferably used to puncture 6 holes symmetrically on both sides of the main vein of the leaf. In the present invention, the bacteria cake is inoculated at the back puncture of the pretreated isolated leaves, and the bacteria cake is removed after culturing in the dark for 2 d, and the culturing is continued in the dark for 5 days. The temperature of the dark cultivation is preferably 25° C. , the relative humidity is preferably 80 to 100%.

In the present invention, after the inoculation, preferably, the disease symptoms of pear leaves are observed at 6h, 12h, 24h and 48h after the inoculation, and the diameter and incidence of disease spots at different time points are counted. The diameter of the lesion in the present invention is preferably the mean value of the horizontal and vertical diameters of each leaf lesion.

The method for rapid identification of pear ring disease resistance provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

1. For identification of pear tree materials and pathogens

In the embodiment, the materials to be tested are Longquan Crisp, Dangshan Crispy Pear, Xiangcha, Jimi and Huali No. 2. The verticillium used was physiological race WZ-279, which was isolated from the diseased spots of three-year-old shoots of susceptible pear plants, and the strain was cultured and stored on potato dextrose agar (PDA) medium.

2. Cultivation of the pathogen of rotundus and preparation of the infective body

Transfer the preserved strain to a new medium for activation and cultivation, and grow to form a new colony. When the mycelium spreads to the medium, use an aseptically treated hole punch to punch a bacterium cake with a diameter of 5mm (Figure 1). .

3. Preparation of test pear leaves

Select healthy pear trees of the same age, and cut off two fully expanded mature leaves adjacent to the middle of the current year branches with petioles, rinse with clean water and blot dry, and sterilize them with a 0.1% sodium hypochlorite solution by mass. For 10 minutes, washed three times with sterile water, blotted dry, then trimmed the petiole length to the same length under sterile water, and wrapped sterile absorbent cotton in the water to keep moisturizing. Spread the moistened sterile gauze on a stainless steel tray, place a plastic water barrier, and then place the middle of the leaf on the water barrier, so that the petiole end wrapped with absorbent cotton touches the gauze.

4. Inoculation

Use sterilized toothpicks to puncture 6 holes symmetrically at the midline of the main vein of the leaf and at the upper and lower quartiles, and place the mycelium of the mycelium dipped in a small amount of sterile water on the surface of the mycelium to face the leaf, and place it on the far side of the leaf. At the puncture hole on the axial surface, a sterile PDA medium cake that was not inoculated with pathogenic bacteria was used as a control to simulate inoculation.

5. Cultivation

The top of the tray was sealed with plastic wrap, the relative humidity was kept at about 80%, the tray was placed in a constant temperature incubator at 25°C, and the bacteria cake and medium cake were removed after culturing in the dark for two days, and continued to cultivate in the dark for five days.

6. Investigation of leaf disease and statistical analysis

After 7 days of co-cultivation, the mean values of the horizontal and vertical diameters of each leaf lesion were measured and calculated, and the mean diameter was used to calculate the disease grade of each leaf, the disease index of each material, and the disease resistance of each material was calculated. Incidence grades are classified and counted according to the following indicators:

Grade 0: no lesions;

Grade 1: The diameter of the lesion is within the interval of (0, 5mm];

Grade 3: The diameter of the lesion is within the interval of (5mm, 8mm];

Grade 5: The diameter of the lesion is within the interval of (8mm, 12mm];

Grade 7: The diameter of the lesion is within the interval of (12mm, 15mm];

Grade 9: The diameter of the lesion is greater than 15mm;

Use the disease index to measure the degree of disease incidence:

Use disease index DI to classify disease resistance: when the disease index is 0, it is high resistance/immunity; when the disease index value is in the (0, 20) interval, it is disease resistance; when the disease index value is in the (20, 40] interval When the disease index value is in the (40, 60] interval, it is moderately susceptible; when the disease index value is in the (60, 80] interval, it is susceptible; when the disease index value is in the (80, 100] interval. High sense when inside.

7. Result analysis

After 7 days of inoculation treatment, the isolated leaves of Huali No. 2, Longquansu and Dangshansu all had larger lesions, while the lesions of the leaves of Xiangchao were smaller, and the lesions of the leaves of Jimi were the smallest, and the degree of infection of the leaves was significantly different ( figure 2). The specific disease investigation results are shown in Table 1 below. Among the five participating materials, Longquansu and Huali No. 2 have low resistance levels and are highly susceptible materials; Dangshansu pear is the susceptible material; Xiangcha and Jimi are moderately resistant. disease material. The results indicated that the resistance of different pear varieties to ring striae can be distinguished by in vitro leaf identification method.

Table 1 Identification results of in vitro leaf resistance of five tested pear varieties

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. a method for quickly identifying pear ring disease resistance, is characterized in that, comprises the following steps: (1) cut the leaf with handle of pear tree, wrap moist aseptic absorbent cotton at the notch after sterilization; spread wet aseptic gauze on a metal tray, and place a water barrier at the midline of the aseptic gauze; The blade is laid flat on the water barrier, and the sterile absorbent cotton is brought into contact with the sterile gauze to obtain pretreated isolated blades; (2) Utilize the plants with rot disease in the field to isolate and purify the rot pathogenic bacteria in the PDA medium, and after culturing for 5 to 7 days, take a bacterial cake with a diameter of 5 mm along the periphery of the colony; (3) Symmetrically puncture 6 holes on both sides of the main veins of the pretreated isolated leaves, inoculate the bacterial cake on the back puncture holes of the pretreated isolated leaves, and remove the bacterial cake after culturing in the dark for 2 d , continue to cultivate in the dark for 5 days, calculate the disease grade, disease index and divide the disease resistance according to the average diameter of the lesions; Among them, the classification criteria of the disease grade are as follows: Grade 0: no lesions; Grade 1: The diameter of the lesion is within the interval of (0, 5mm]; Grade 3: The diameter of the lesion is within the interval of (5mm, 8mm]; Grade 5: The diameter of the lesion is within the interval of (8mm, 12mm]; Grade 7: The diameter of the lesion is within the interval of (12mm, 15mm]; Grade 9: The diameter of the lesion is greater than 15mm; Use the disease index to measure the degree of disease incidence:

Use disease index DI to classify disease resistance: when the disease index is 0, it is high resistance/immunity; when the disease index value is in the (0, 20) interval, it is disease resistance; when the disease index value is in the (20, 40] interval When the disease index value is in the (40, 60] interval, it is moderately susceptible; when the disease index value is in the (60, 80] interval, it is susceptible; when the disease index value is in the (80, 100] interval. Inner time is high sense; There is no temporal sequence between steps (1) and (2). 2 . The method according to claim 1 , wherein the sterilization in step (1) comprises sterilizing the blade with an aqueous sodium hypochlorite solution with a mass percentage of 0.1% for 10 minutes, and then rinsing with sterile water for 3 times. 3 . 3 . The method according to claim 2 , wherein the leaves are selected from leaves with the same physiological state in the middle of annual shoots. 4 . 4 . The method according to claim 1 , wherein the pathogenic bacteria of rotundus in step (2) comprise each physiological race of Botryosphaeria dothidea. 5 . 5 . The method according to claim 1 , wherein the temperature of the culture in step (2) is 25° C. 6 . 6 . The method according to claim 1 , wherein in step (2), the peripheral mycelium is stretched to the edge of the petri dish, and the fungus cake is punched out with a sterile punch. 7 . 7. The method according to claim 1, characterized in that, in step (3), the positions of the puncture are at both sides of the main vein parallel to the midline of the main vein and at the upper and lower quartiles. 8 . The method according to claim 1 , wherein the temperature of the dark culture in step (3) is 25° C. and the relative humidity is 80-100%. 9 .

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