Method for evaluating resistance of apple ring spot and anthracnose

Abstract

The invention relates to the technical field of fruit tree science, in particular to a method for evaluating resistance of apple ring spot and anthracnose. Apple fruits are inoculated with physalospora piricola or colletotrichum gloeosporioides and cultured; and at the 10th day of culture, measuring the scab diameter of the apple ring spot bacteria or the apple colletotrichum gloeosporioides, and dividing resistance grades by a systematic clustering analysis method. The apple ring spot and 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 apple resistance germplasm resources.

Description

Technical Field

[0001] The present invention relates to the technical field of pomology, and particularly to a method for evaluating the resistance to apple ring rot and anthracnose. Background Art

[0002] Botryosphaeria berengeriana and Colletotrichum gloriosporioides are two important fungal diseases during the growth and storage of apples. They infect both branches and fruits. In recent years, these two diseases have occurred commonly in all major apple-producing areas across the country. In production, chemical control is mainly adopted, but the efficacy is limited. Long-term use of chemical fungicides will also cause problems such as the decline in fruit quality and environmental pollution. Therefore, it is urgent to breed disease-resistant varieties through breeding to fundamentally solve the problem.

[0003] Screening and evaluating disease-resistant germplasm resources is of great significance for variety breeding and the rational layout of cultivated varieties. At present, there is no unified standard for the resistance evaluation of apple ring rot and anthracnose. The disease resistance evaluation of these two diseases mainly adopts methods such as inoculation on field branches or leaves and inoculation on in vitro branches or leaves in the laboratory, which have problems such as environmental condition limitations, long evaluation periods, and complex operations. If artificial inoculation can be directly carried out on in vitro fruits indoors, after incubation for the same time, the resistance level can be directly evaluated according to the difference in lesion diameter, which can ensure the consistency of inoculation conditions, shorten the evaluation period, have low costs, and greatly improve the screening efficiency. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for evaluating the resistance to apple ring rot and anthracnose. The method for evaluating the resistance to apple ring rot and anthracnose of the present invention is not restricted by the external environment, has a short evaluation period, low costs, and is simple to operate, and can greatly improve the screening efficiency of apple disease-resistant germplasm resources.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for evaluating the resistance to apple ring rot and anthracnose, comprising the following steps:

[0007] 1) Inoculate Botryosphaeria berengeriana or Colletotrichum gloriosporioides on apple fruits and culture.

[0008] 2) On the 10th day of culture, measure the lesion diameter of Botryosphaeria berengeriana or Colletotrichum gloriosporioides, and divide the resistance level by the systematic cluster analysis method.

[0009] When the average diameter of the lesion caused by the apple ring rot pathogen ≤ 15 mm, the apple variety is judged as a highly resistant variety to ring rot; when 15 mm < average diameter of the lesion ≤ 33 mm, the apple variety is judged as a resistant variety to ring rot; when 33 mm < average diameter of the lesion ≤ 42 mm, the apple variety is judged as a susceptible variety to ring rot; when the average diameter of the lesion > 42 mm, the apple variety is judged as a highly susceptible variety to ring rot;

[0010] When the average diameter of the lesion caused by the apple anthracnose pathogen ≤ 11 mm, the apple variety is judged as a highly resistant variety to anthracnose; when 11 mm < average diameter of the lesion ≤ 17 mm, the apple variety is judged as a resistant variety to anthracnose; when 17 mm < average diameter of the lesion ≤ 27 mm, the apple variety is judged as a susceptible variety to anthracnose; when the average diameter of the lesion > 27 mm, the apple variety is judged as a highly susceptible variety to anthracnose.

[0011] Preferably, the apple ring rot pathogen or the apple anthracnose pathogen in step 1) is rejuvenated before inoculation.

[0012] Preferably, the inoculation method in step 1) is acupuncture.

[0013] Preferably, holes with a needle insertion depth of 0.5 - 1 mm are made on opposite sides of the equatorial part of the apple fruit.

[0014] Preferably, the culture conditions in step 1) include: environmental humidity of 90 - 95% and temperature of 19 - 21 °C.

[0015] Preferably, the apple ring rot pathogen or the apple anthracnose pathogen is rejuvenated on the apple fruit.

[0016] Advantages of the present invention:

[0017] A large number of experimental studies have shown that after inoculating the same pathogen on apple fruits of different varieties, there are obvious differences in the expansion of lesions on the fruits. According to the average diameter of the lesions on the 10th day after inoculating the pathogen on the fruits of all tested varieties, the present invention divides the resistance of different apple variety germplasms into 4 grades by the systematic cluster analysis method, and respectively determines the corresponding grade evaluation ranges for the two pathogens. Among them, in this application, when the average diameter of the lesion caused by the ring rot pathogen ≤ 15 mm, it is judged as a highly resistant variety to ring rot; when 15 mm < average diameter of the lesion ≤ 33 mm, it is judged as a resistant variety to ring rot; when 33 mm < average diameter of the lesion ≤ 42 mm, it is judged as a susceptible variety to ring rot; when the average diameter of the lesion > 42 mm, it is judged as a highly susceptible variety to ring rot; in this application, when the average diameter of the lesion caused by the anthracnose pathogen ≤ 11 mm, it is judged as a highly resistant variety to anthracnose; when 11 mm < average diameter of the lesion ≤ 17 mm, it is a resistant variety to anthracnose; when 17 mm < average diameter of the lesion ≤ 27 mm, it is a susceptible variety to anthracnose; when the average diameter of the lesion > 27 mm, it is a highly susceptible variety to anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0019] Figure 1 It is a dendrogram of the hierarchical cluster analysis of the lesion diameter on the 10th day after inoculation with Botryosphaeria berengeriana f. sp. piricola;

[0020] Figure 2 It is a dendrogram of the hierarchical cluster analysis of the lesion diameter on the 10th day after inoculation with Colletotrichum gloeosporioides Penz. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a method for evaluating the resistance of apples to ring rot and anthracnose, including the following steps:

[0022] 1) Inoculate Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides Penz. on apple fruits and culture;

[0023] 2) On the 10th day of culture, measure the lesion diameter of Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides Penz., and divide the resistance level by the hierarchical cluster analysis method;

[0024] When the average lesion diameter of Botryosphaeria berengeriana f. sp. piricola ≤ 15 mm, it is determined that the apple variety is highly resistant to ring rot; when 15 mm < average lesion diameter ≤ 33 mm, it is determined that the apple variety is resistant to ring rot; when 33 mm < average lesion diameter ≤ 42 mm, it is determined that the apple variety is susceptible to ring rot; when the average lesion diameter > 42 mm, it is determined that the apple variety is highly susceptible to ring rot;

[0025] When the average lesion diameter of Colletotrichum gloeosporioides Penz. ≤ 11 mm, it is determined that the apple variety is highly resistant to anthracnose; when 11 mm < average lesion diameter ≤ 17 mm, it is determined that the apple variety is resistant to anthracnose; when 17 mm < average lesion diameter ≤ 27 mm, it is determined that the apple variety is susceptible to anthracnose; when the average lesion diameter > 27 mm, it is determined that the apple variety is highly susceptible to anthracnose.

[0026] Inoculate Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides on apple fruits and culture them. In the present invention, the Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides is preferably rejuvenated before inoculation. In the present invention, it is preferred to inoculate the stored Botryosphaeria berengeriana f. sp. piricola and Colletotrichum gloeosporioides on apple fruits for rejuvenation, punch out mycelial discs of the rejuvenated pathogenic bacteria, and culture them on a potato agar plate with a diameter of 90 mm. When the colony grows to 2 / 3 of the petri dish, inoculation is carried out. In the present invention, the inoculation method is preferably acupuncture. In the present invention, 6 representative fruits of different apple varieties are selected, with consistent maturity, uniform size, and no pests, diseases, or mechanical damage. Disinfect the test fruits with 75% alcohol and let them dry, then inoculate the mycelial discs of the well-cultured Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides by acupuncture. The inoculated fruits are placed in a plastic box lined with moist filter paper, maintaining the humidity at 90% - 95%, and placed in a constant temperature incubator at 20°C ± 1°C for cultivation and observation. In the present invention, it is preferred to puncture holes with a depth of 0.5 - 1 mm on the opposite sides of the equatorial part of the apple fruit. In the present invention, the cultivation conditions preferably include: environmental humidity of 90 - 95%, and temperature of 19 - 21°C. In the present invention, on the 5th day and 10th day respectively, the "cross method" is used to measure the diameter of the lesion on the inoculated fruits and conduct statistics. The average value of the lesion diameter after inoculation of each variety represents the expansion of the lesion caused by the corresponding pathogenic fungus. In the present invention, the Botryosphaeria berengeriana f. sp. piricola or Colletotrichum gloeosporioides is preferably rejuvenated on apple fruits.

[0027] To further illustrate the present invention, the following examples are used to describe the present invention in detail, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] A method for evaluating the resistance of apples to ring rot and anthracnose, the steps are as follows:

[0030] 1. Cultivation of Botryosphaeria berengeriana f. sp. piricola and Colletotrichum gloeosporioides

[0031] Inoculate the stored Botryosphaeria berengeriana f. sp. piricola and Colletotrichum gloeosporioides on apple fruits. After the inoculated fruits become diseased, surface disinfect the area around the lesion with 75% alcohol, and then take 3 - 5 mm of fruit tissue from the border between the diseased and healthy parts and culture it on a potato dextrose agar medium (6.0 g of potato extract powder, 20 g of glucose, 20 g of agar powder, 1000 mL of distilled water, pH 5.6 ± 0.2) for 3 - 5 days to obtain rejuvenated pathogenic bacteria. Then, punch out mycelial discs and transfer them to a potato agar plate with a diameter of 90 mm for cultivation. When the colony grows to 2 / 3 of the petri dish, inoculation is carried out.

[0032] 2. Apple fruit inoculation

[0033] Select 6 representative fruits from different apple varieties, with consistent maturity, uniform size, and no pests, diseases, or mechanical damage. Disinfect the test fruits with 75% alcohol and let them dry. Use an inoculation needle to create small holes with a needle penetration depth of 0.5 - 1 mm on opposite sides of the equatorial part of the fruit. Use a punch with a diameter of 5 mm to punch out a mycelial cake from the front edge of the colony of Botryosphaeria berengeriana or Colletotrichum gloeosporioides cultured in a Petri dish No. 1. Attach the mycelial surface to the small hole and fix it with transparent tape. Place the inoculated fruits in a plastic box lined with moist filter paper and seal it with plastic wrap to maintain a humidity of 90% - 95%. Incubate them in a constant temperature incubator at 20 ± 1°C. Investigate the lesion diameter on the 5th day and 10th day respectively.

[0034] 3. Measurement and Statistics of Apple Fruit Lesion Diameter

[0035] On the 5th day and 10th day after fruit inoculation, use the "cross - method" and measure the lesion diameter of each inoculated fruit with a vernier caliper. The average value of the lesion diameters of 6 fruits of each variety represents the lesion expansion situation of the variety after inoculation with Botryosphaeria berengeriana or Colletotrichum gloeosporioides. The average diameter results of the lesions caused by inoculation with Botryosphaeria berengeriana or Colletotrichum gloeosporioides on the 5th day and 10th day of all tested varieties of fruits are shown in Table 1 and Table 2.

[0036] Table 1 Evaluation of the Resistance of Different Apple Varieties to Botryosphaeria berengeriana

[0037]

[0038]

[0039] Table 2 Evaluation of the Resistance of Different Apple Varieties to Colletotrichum gloeosporioides

[0040]

[0041]

[0042] 4. Determination of the Resistance Classification Evaluation Criteria

[0043] A large number of experimental studies have shown that there are obvious differences in the expansion of lesions on the fruits of different apple varieties after inoculation with the same pathogen. In the tests of this application, 45 varieties were inoculated with Botryosphaeria berengeriana de Not. and Colletotrichum gloeosporioides Penz., respectively. Statistical analysis found that the difference in lesion diameter on the 10th day after inoculation was more obvious than that on the 5th day. Therefore, according to the average diameter of the lesions on the 10th day after inoculation of the fruits of all tested varieties, this application classified the resistance of different apple variety germplasms into 4 grades by the method of systematic cluster analysis, and determined the range of the average diameter of the lesions in each grade after inoculation with the two pathogens. Among them, this application judged that the apple variety with an average lesion diameter of Botryosphaeria berengeriana de Not. ≤ 15 mm was a highly resistant variety to Botryosphaeria berengeriana de Not., 15 mm < average lesion diameter ≤ 33 mm was a resistant variety to Botryosphaeria berengeriana de Not., 33 mm < average lesion diameter ≤ 42 mm was a susceptible variety to Botryosphaeria berengeriana de Not., and the average lesion diameter > 42 mm was a highly susceptible variety to Botryosphaeria berengeriana de Not., as Figure 1 shown; this application judged that the apple variety with an average lesion diameter of Colletotrichum gloeosporioides Penz. ≤ 11 mm was a highly resistant variety to Colletotrichum gloeosporioides Penz., 11 mm < average lesion diameter ≤ 17 mm was a resistant variety to Colletotrichum gloeosporioides Penz., 17 mm < average lesion diameter ≤ 27 mm was a susceptible variety to Colletotrichum gloeosporioides Penz., and the average lesion diameter > 27 mm was a highly susceptible variety to Colletotrichum gloeosporioides Penz., as Figure 2 shown.

[0044] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for evaluating the resistance to apple ring rot and anthracnose, characterized in that, It includes the following steps: 1) Inoculate the apple ring rot pathogen or the apple anthracnose pathogen onto apple fruits and culture them; 2) On the 10th day of culture, measure the lesion diameter of the apple ring rot pathogen or the apple anthracnose pathogen, and divide the resistance levels by the systematic clustering analysis method; When the average lesion diameter of the apple ring rot pathogen ≤ 15 mm, determine that the apple variety is a highly resistant variety to ring rot; when 15 mm < average lesion diameter ≤ 33 mm, determine that the apple variety is a resistant variety to ring rot; when 33 mm < average lesion diameter ≤ 42 mm, determine that the apple variety is a susceptible variety to ring rot; when the average lesion diameter > 42 mm, determine that the apple variety is a highly susceptible variety to ring rot; When the average lesion diameter of the apple anthracnose pathogen ≤ 11 mm, determine that the apple variety is a highly resistant variety to anthracnose; when 11 mm < average lesion diameter ≤ 17 mm, determine that the apple variety is a resistant variety to anthracnose; when 17 mm < average lesion diameter ≤ 27 mm, determine that the apple variety is a susceptible variety to anthracnose; when the average lesion diameter > 27 mm, determine that the apple variety is a highly susceptible variety to anthracnose.

2. The method according to claim 1, wherein In step 1), the apple ring rot pathogen or the apple anthracnose pathogen is rejuvenated before inoculation.

3. The method according to claim 1, wherein The inoculation method in step 1) is acupuncture.

4. The method according to claim 3, characterized in that, Puncture holes with a depth of 0.5 - 1 mm on the opposite sides of the equatorial part of the apple fruit.

5. The method according to claim 1, characterized in that, The culture conditions in step 1) include: environmental humidity of 90 - 95% and temperature of 19 - 21 °C.

6. The method according to claim 2, wherein Rejuvenate the apple ring rot pathogen or the apple anthracnose pathogen on the apple fruit.

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