Method for evaluating resistance of pear anthracnose

Abstract

The invention relates to the technical field of fruit trees, in particular to a pear anthracnose resistance evaluation method. The method comprises the following steps: (1) rejuvenating and culturing colletotrichum gloeosporioides; (2) inoculating and culturing pear fruit germplasm resource fruits by using the colletotrichum gloeosporioides obtained in the step (1); (3) measuring the diameter of the scab at the 9th day of inoculation, and calculating the average value of the diameter of the scab; and (4) according to the average value of the scab diameters of the 9th day obtained in the step (3), dividing the resistance of the pear germplasm resources to the anthracnose germs into five grades by a systematic clustering analysis method, and taking the five grades as a criterion for judging the resistance of the pear fruits to the anthracnose germplasm germplasm resources to the anthracnose germplasm germplasm resources. The pear anthracnose resistance evaluation method is not limited by the external environment, is short in evaluation period, low in cost and simple to operate, and can greatly improve the screening efficiency of pear fruit resistance germplasm resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pomology, in particular to a method for evaluating resistance of pear anthracnose. BACKGROUND

[0002] Pear anthracnose is mainly caused by Colletotrichum glorosporioides, which not only infects branches, leaves, but also harms fruits. Pear anthracnose occurs universally in all main pear producing areas, and the disease rate of serious disease can reach 70%, which causes serious economic losses. Chemical control is mainly used in production, and chemicals are used before the disease occurs. Long-term use of chemical fungicides can cause fruit pesticide residues and environmental pollution, etc. Therefore, it is urgent to select and breed disease-resistant varieties to fundamentally solve the problem.

[0003] Screening and evaluating disease-resistant germplasm resources are of great significance for the collection of breeding resources and the rational layout of cultivated varieties. At present, the resistance evaluation of pear anthracnose mainly uses artificial inoculation and field investigation methods. Among them, the field environment is complex and difficult to control, and it is easy to cause unstable results, so the in vitro inoculation screening method has certain advantages. At present, the indoor screening methods are also different. If fruit inoculation is used, and resistance grading evaluation is directly performed according to the size difference of disease spots, the consistency of inoculation conditions can be ensured, the evaluation period can be shortened, the cost is low, and the screening efficiency can be greatly improved. It provides a solid theoretical basis for pear variety improvement, disease-resistant breeding and scientific cultivation. SUMMARY

[0004] The purpose of the present application is to provide a method for evaluating the resistance of pear anthracnose.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a method for evaluating the resistance of pear anthracnose, comprising the following steps:

[0007] Step 1: Cultivation of pear anthracnose fungus

[0008] The preserved pear anthracnose fungus is inoculated on pear fruits for rejuvenation, and after isolation and culture, the rejuvenated pathogen cake is punched with a puncher, and cultured on a 90mm diameter potato agar plate. When the colony diameter grows to 2 / 3 of the plate, inoculation is performed.

[0009] Step 2: Inoculation of pear fruits

[0010] Select maturity, uniform size, no disease and mechanical injury of test fruit, 6 fruits per treatment, a total of 71 different germplasm resources. The test fruit was sterilized with 75% alcohol and dried, and the needle inoculation method was used. The inoculation needle was used to prick the symmetrical part of the pear fruit body, and the 4-6mm diameter puncher was used to punch the edge of the prepared anthracnose fungus, and the size of the fungus was ensured to be consistent. The fungus was pasted to the wound for inoculation, and the same size of PDA medium was used as a control for inoculation of the fruit. The inoculated fruit was placed in a plastic box lined with wet filter paper, the humidity was maintained at 90%-95%, and the fruit was cultured in a constant temperature incubator at 20℃±2℃.

[0011] Step 3: Measurement and statistics of pear fruit lesion

[0012] On the 6th and 9th day, the lesion diameter of the inoculated fruit was measured by the cross method using a digital vernier caliper, and the statistics were performed. The average value of the lesion diameter of each variety after inoculation represented the expansion of the anthracnose lesion.

[0013] Step 4: Determination of resistance grading evaluation standard

[0014] A large number of experimental studies have shown that there is a significant difference in the expansion of the lesion on the fruit after inoculation of different varieties of pear fruit with the same pathogen. According to the average diameter of the lesion on the 9th day after inoculation of the pathogen on the fruit of all test varieties, the resistance of different pear fruit varieties is divided into 5 grades by systematic cluster analysis method with the best euclidean distance of 8. The present application judges that 0< average lesion diameter≤10mm is a high resistance variety of anthracnose, 10mm<average lesion diameter≤15mm is a resistant variety of anthracnose, 15mm<average lesion diameter≤20mm is a moderately resistant variety of anthracnose, 20mm<average lesion diameter≤30mm is a susceptible variety of anthracnose, and average lesion diameter>30mm is a highly susceptible variety of anthracnose.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The pear anthracnose resistance evaluation method of the present application is not limited by external environment, has short evaluation period, low cost, and simple operation, and can greatly improve the screening efficiency of pear fruit resistance germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creating any inventive labor.

[0018] Figure 1This is the phylogenetic diagram of the hierarchical cluster analysis of lesion diameters on the 9th day after inoculation with pear anthracnose. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] A method for evaluating pear anthracnose resistance is provided, wherein the pear anthracnose resistance is evaluated according to the following steps:

[0022] 1. Cultivation of pear anthracnose pathogen

[0023] Inoculate pear fruit with preserved pear anthracnose bacteria. After the inoculated fruit develops disease, disinfect the surface surrounding the lesions with 75% alcohol. Then, sample 3-5 mm of fruit tissue from the border between the diseased and healthy areas and culture on potato dextrose agar (6.0 g potato extract, 20 g glucose, 20 g agar powder, 1000 mL distilled water, pH 5.6 ± 0.2) for 5 days to rejuvenate the pathogen. Then, use a hole punch to punch out a bacterial cake, transfer it to a 90 mm diameter potato agar plate, and inoculate when the plaque has grown to two-thirds of the plate.

[0024] 2. Pear Fruit Inoculation

[0025] Six representative fruits from different germplasm varieties were selected, with uniform maturity, size, and no pests, diseases, or mechanical damage. The test fruits were disinfected with 75% alcohol and air-dried. A 1mm-deep puncture was made on opposite sides of the fruit trunk using an inoculating needle. A 5mm-diameter punch was used to punch a mycelial cake from the edge of a cultured pear anthracnose pathogen colony. The mycelial surface was placed on the wound. A PDA culture cake of the same size was used as a control. The cake or culture cake was secured with transparent tape. The inoculated fruits were placed in a plastic box lined with moistened filter paper and sealed with plastic wrap. The humidity was maintained at 95% and the fruit was incubated in a constant-temperature incubator at 20±1°C. The diameter of the lesions was measured on the 6th and 9th day.

[0026] 3. Measurement and statistics of pear fruit lesion diameter

[0027] On the sixth and ninth day after inoculation, the diameter of the lesions on each fruit was measured using the cross-hatch method with a vernier caliper. The average diameter of the lesions on six fruits per variety represents the lesion expansion after inoculation with Colletotrichum spp. Table 1 shows the average lesion diameters of all tested varieties on the sixth and ninth day after inoculation with Colletotrichum spp.

[0028] Table 1 Evaluation of anthracnose resistance of different pear varieties

[0029]

[0030]

[0031]

[0032]

[0033] [1] Chen CX. Disease resistance identification and disease resistance screening of pear cultivar resources [D]. Nanjing Agricultural University, 2016.

[0034] [2] Sun JY, Chen CX, Gu C, et al. Identification and screening of pear cultivar resources resistance to anthracnose [J]. Journal of Fruit Tree Research, 2016, 33(S1): 184-195.

[0035] [3] Peng YH. Differentiation analysis of pathogenicity of Guignardia bidwellii on pear in southern China and in vitro determination of pear cultivar resistance [D]. Central China Agricultural University, 2020.

[0036] [4] Zhao M, Tao ST, Qi KJ, et al. Evaluation of pear fruit resistance to spread of G. bidwellii and Physalospora piricola [J]. Plant Protection, 2013, 39(04): 65-71.

[0037] 4. Determination of resistance grading evaluation criteria

[0038] A large number of experimental studies have shown that after inoculating different varieties of pear fruits with the same pathogenic bacteria, there are obvious differences in the expansion of disease spots on the fruits. In the test of the present application, 71 varieties were inoculated with G. bidwellii, and it was found that the difference in the diameter of the disease spot on the 9th day after inoculation was more obvious than that on the 6th day. Therefore, according to the average diameter of the disease spot on the 9th day after inoculating the fruits of all the tested varieties with pathogenic bacteria, the resistance of different pear fruit varieties and germplasm resources is divided into five grades by using the method of system clustering analysis, and the best Euclidean distance is 8. The average value of the diameter of the disease spot in each grade after inoculation of the two pathogenic bacteria is determined. Among them, the present application judges 0< average disease spot diameter≤10mm as high resistance to anthracnose, 10mm<average disease spot diameter≤15mm as resistance to anthracnose, 15mm<average disease spot diameter≤20mm as medium resistance to anthracnose, 20mm<average disease spot diameter≤30mm as susceptible to anthracnose, and average disease spot diameter>30mm as high susceptible to anthracnose, as shown in Table 1. Figure 1

[0039] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for evaluating resistance to anthracnose of Pyrus, characterized in that, It comprises the following steps: (1) rejuvenating and culturing anthracnose bacteria; (2) inoculating the anthracnose bacteria obtained in step (1) on the fruits of pear germplasm resources for culture; (3) measuring the lesion diameter on the 9th day after inoculation and calculating the average lesion diameter; (4) according to the average lesion diameter on the 9th day obtained in step (3), the resistance of pear germplasm resources to anthracnose bacteria is divided into 5 grades by system clustering analysis method, and is used as the determination standard of the resistance of pear to anthracnose: 0<average lesion diameter≤10mm is judged as high resistance variety, 10mm<average lesion diameter≤15mm is resistant variety, 15mm<average lesion diameter≤20mm is medium resistance variety, 20mm<average lesion diameter≤30mm is susceptible variety, and average lesion diameter>30mm is high susceptible variety.

2. The method for evaluating resistance to anthracnose of pear according to claim 1, wherein, The inoculation method in step (2) is: using a punch to take the cultured anthracnose bacteria for inoculation, and selecting fruits with consistent maturity, uniform size, no disease and insect pests and mechanical damage for needle inoculation.

3. The method for evaluating resistance to pear anthracnose according to claim 2, wherein The diameter of the punch is 4-6mm.

4. The method for evaluating resistance to pear anthracnose according to claim 1, wherein The culture temperature in step (2) is 18-22℃, and the humidity is 90%-95%.

5. The method for evaluating resistance to pear anthracnose according to claim 1, wherein At least 6 fruits of each pear germplasm resource are inoculated in step (2).

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