Rapid and efficient pear and apple rot resistance evaluation method
By optimizing the methods for evaluating resistance to pear and apple rot diseases and adopting the contact inoculation method, the problems of low efficiency and complex operation in existing technologies have been solved, enabling rapid and accurate large-scale resistance evaluation that is suitable for standardized evaluation in different laboratories.
Patent Information
- Application Number
- CN202511711024.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-20
- Publication Date
- 2026-01-06
AI Technical Summary
Existing methods for evaluating resistance to pear and apple rot are inefficient, complex to operate, and their results are easily affected by interference, making them difficult to apply on a large scale. Traditional methods, such as the hole-punching method, the needle-pricking method, and the field evaluation method, are time-consuming, have large errors, and are costly.
By optimizing the parameters of the tested branches, the culture conditions of the pathogen, and the inoculation method, a standardized contact inoculation method was established. This method includes selecting healthy, disease-free branches, optimizing the pathogen culture and inoculation device, using sealed container culture to avoid drilling or pricking, and directly infecting the mycelium through the branch cut.
It enables rapid and efficient resistance evaluation, and can complete the inoculation of 100-120 materials in 2 hours, which is more than 10 times more efficient than traditional methods. It is suitable for large-scale germplasm resource screening, and the results are accurate and reduce operational errors. It is applicable to replication in different laboratories.
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Figure CN121272004A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of horticultural plant disease resistance evaluation technology, specifically a rapid and efficient method for evaluating resistance to pear and apple rot diseases. Background Technology
[0002] Composed of black rot dermatitis fungi ( Valsa pyri pear rot disease Valsa mali Apple rot (Atrophic rot) is a major and devastating disease affecting the global pear and apple industries, with an incidence rate of 30% to 80% in major producing areas. The pathogen infects the bark of fruit tree branches and trunks, causing bark rot, hindering nutrient transport, and in severe cases, killing the entire tree, resulting in reduced yields or even the destruction of the orchard.
[0003] Currently, the control of rot disease mainly relies on chemical control and agricultural management. However, long-term use of chemical agents can easily lead to increased drug resistance in pathogens. Traditional scraping methods are labor-intensive and prone to secondary infections. Due to their environmental friendliness and the sustainability of resistance, the breeding of disease-resistant varieties has become one of the most effective control methods. An efficient and accurate resistance evaluation system is a prerequisite for disease-resistant breeding.
[0004] Existing methods for evaluating resistance to pear and apple rot diseases have significant shortcomings: The perforation method requires drilling holes in branches and inoculating them with fungal cakes; only 8-10 samples can be evaluated in 2 hours, resulting in a large workload, long processing time, and the fungal cakes easily detach from the holes, leading to data loss. The needle-pricking method relies on manual control of the pricking depth, resulting in large operational errors; young branches are easily damaged by non-pathogenic factors such as dehydration and cracking, interfering with the results. The spore suspension inoculation method is difficult and time-consuming to cultivate in vitro, making it difficult to scale up. The field evaluation method has a testing period of several months to several years, is affected by environmental factors such as temperature, humidity, and pathogen concentration, requires large-scale planting to ensure accurate results, and is costly and inefficient.
[0005] Therefore, there is an urgent need to develop a simple, efficient, accurate, and suitable method for evaluating resistance to rot diseases that is suitable for large-scale germplasm screening. Summary of the Invention
[0006] To address the shortcomings of existing methods for evaluating resistance to pear and apple rot diseases mentioned in the background section—namely, low efficiency, complex operation, susceptibility to interference, and difficulty in scaling up—this invention provides the following technical solution: a rapid and efficient method for evaluating resistance to pear and apple rot diseases. This invention establishes a standardized evaluation system by optimizing the parameters of the tested branches, pathogen culture conditions, and inoculation methods. The specific steps are as follows: 1. Preparation of test materials S1. Selection and treatment of test branches: Selection criteria: Select healthy, disease-free branches of uniform length and consistent water content from pear or apple germplasm resource nurseries / experimental bases; prioritize one-year-old branches (excessive branch age will lead to faster disease rate and affect resistance differentiation); control the thickness of the branch base to 0.4-0.8 cm (excessive or insufficient thickness will significantly affect the spread of lesions, and this range can ensure the stability of results). Storage conditions: Use the branches immediately after harvesting. If short-term storage is required, place them in a low-temperature environment of 4℃ for ≤5 days (storage for more than 5 days will cause the branches to lose water, reduce cell vitality, and significantly reduce the longitudinal diameter of lesions. For example, the longitudinal diameter of lesions on 'Yaluxiang' branches decreased by 53.3% after 20 days of storage compared to 0 days). Pretreatment: Rinse the branches with sterile water, air dry, disinfect the surface with 75% alcohol, and then cut them into 15cm long sections (to ensure consistency of inoculation sites and facilitate lesion measurement).
[0007] S2, Pathogen culture: Strain activation: Using pear rot pathogen ( Valsa pyri strain Vp-P-002) or the pathogen of apple rot ( Valsa mali strain Vm-A-003 was inoculated into PDA (potato dextrose agar) medium; Culture conditions: Place in a 25℃ constant temperature incubator for 3 days for activation culture (the pathogen mycelium is most active after 3 days of culture, the disease rate is the fastest, and the longitudinal diameter of the lesion is the largest; after more than 3 days of culture, the pathogen activity decreases and the lesion expansion rate is significantly reduced), to obtain pathogen plates (9cm in diameter, covered with mycelium layer).
[0008] S3, Contact inoculation: Preparation of inoculation device: Take a sealed jar with a diameter of 10cm and a height of 30cm, and place a layer of moistened sterile gauze at the bottom of the jar (to maintain the humidity inside the jar and prevent the branches from dehydrating). Inoculation procedure: Place the pathogen plate prepared in step S2 into a sealed container, and place the 15cm branch segment pretreated in step S1 upright on the mycelial layer of the pathogen plate, ensuring that the bottom of the branch segment is in complete contact with the mycelial layer (no need to punch holes or needle, infection can be achieved directly through the branch segment cut to contact the mycelium). Culture conditions: After sealing the container and covering it, place it in a constant temperature incubator at 25℃ for 3 days (within 3 days, the lesions will expand stably and the differences in resistance between different varieties can be accurately distinguished; if the culture time is too short, the lesions will not expand sufficiently, and if it is too long, it will easily lead to the aggravation of branch rot).
[0009] S4. Resistance Evaluation and Grading Index determination: After the culture was completed, the branch segments were removed and the longitudinal diameter of the lesions was measured with a ruler. Each treatment was set up with 3 biological replicates. The results are expressed as "mean ± standard deviation". Resistance grading: Based on the longitudinal diameter of the lesion, the resistance to pear / apple rot is divided into 5 levels. The resistance grading criteria are as follows: lesion longitudinal diameter ≤ 6.0 cm is highly resistant (HR), 6.1~10.0 cm is resistant (R), 10.1~14.0 cm is moderately resistant (MR), 14.1~18.0 cm is susceptible (S), and >18.0 cm is highly susceptible (HS).
[0010] This invention provides a rapid and efficient method for evaluating resistance to pear and apple rot diseases, which has the following beneficial effects: 1. No need for drilling or preparing mycelium cakes, simplifying the operation steps, 100-120 material inoculations can be completed in 2 hours, which is more than 10 times more efficient than the traditional drilling method, making it suitable for rapid screening of large-scale germplasm resources (such as hundreds of seedlings). 2. The lesion expansion rate was highly correlated with that of the traditional punching method (correlation coefficient r=0.978 for pear varieties and r=0.989 for apple varieties, P<0.01), which can accurately reflect the inherent resistance of the varieties and avoid the data loss problem caused by the shedding of the fungal cake; 3. Only conventional equipment such as sealed containers and PDA tablets are required. Key parameters (branch age, pathogen culture time, culture temperature) are clearly defined, and the results can be replicated in different laboratories, reducing operational errors. 4. Applicable to the evaluation of rot resistance of different varieties, germplasm resources and hybrid offspring of pear and apple, providing technical support for the breeding of disease-resistant varieties, the discovery of resistance genes and green prevention and control. Attached Figure Description
[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0012] Figure 1 This is a schematic diagram of the contact method in this invention; Figure 2 This is a graph showing the correlation analysis between different inoculation methods of pear branches in embodiments of the present invention; Figure 3 This is a graph showing the correlation analysis between different inoculation methods for apple branches in embodiments of the present invention; Figure 4 This is a diagram illustrating the effect of different branch ages and lignification degrees on the pathogenicity of pear rot in an embodiment of the present invention. Figure 5 This is a diagram showing the effect of branch thickness and storage time on inoculation effect in an embodiment of the present invention; Figure 6This is a diagram illustrating the effect of the pathogen's growth cycle on the inoculation effect and the resistance evaluation of *Pyrus pyrifolia* seedlings in this embodiment of the invention. Detailed Implementation
[0013] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.
[0014] Example 1: Materials and Methods 1. Inoculation materials and test strains The pear varieties used were Huangguan pear, Nanguo pear, Zhutou pear, Hongba pear, Yuluxiang, Zaoyu, Ruan'er pear, Apple pear, Xinli No. 7, Ugly pear, Latai pear, Zaosu pear, Duli G-03, Yuluxiang branches, and 164 Duli seedlings, all obtained from the germplasm resource nursery of Wuwei Forestry Academy. Apple varieties Huashuo, Venus Gold, Ruixiang Red, David Gala, Ruixue, and Aifei branches were obtained from the Tianshui Experimental Base of the College of Horticulture, Gansu Agricultural University. Healthy branches of uniform length and consistent moisture content were selected as inoculation materials for this invention.
[0015] Culture of pathogens causing pear and apple rot: Apple rot pathogen strain Vm-A-003 and pear rot pathogen strain Vp-P-002 were isolated in our laboratory and identified as typical pathogens causing apple and pear rot, respectively. The strains were inoculated onto PDA (Potato Dextrose Agar) medium and activated at 25°C for 3 days before being used to inoculate experimental materials.
[0016] 2. Inoculation method 2.1 Drilling method Branches were washed with sterile water and dried, then disinfected with 75% alcohol before being cut into 15 cm sections. Holes were punched (using a sterile 5 mm diameter punch to remove the bark phloem) and girdled (using a sterile scalpel to make a circumferential cut around the branch). After culturing the pear rot pathogen strain on PDA medium for 3 days, mycelial blocks were extracted from the outermost edge using a 5 mm punch and inoculated at the wound sites. The inoculated branches were placed in white dishes (the bottom of the dish was lined with sterile gauze dripped with sterile water, and the rim was covered with plastic wrap) and incubated at 25°C for 3 days before measuring the longitudinal diameter of the lesions. This experiment was performed in triplicate.
[0017] 2.2 Contact Method The branches were washed with sterile water and dried. After disinfection with 75% alcohol, they were cut into 15 cm sections. The pear rot pathogen strain was cultured on PDA medium for 3 days, then placed in a sealed jar (10 cm in diameter and 30 cm in height, with a moistened sterile gauze at the bottom and a sealed lid). The treated branches were placed upright on the PDA-cultured rot pathogen petri dishes, ensuring full contact. After culturing at 25℃ for 3 days, the longitudinal diameter of the lesions was measured. Figure 1 This experiment was performed in three biological replicates.
[0018] 3. Analysis of the influence of branch development factors and branch storage time on inoculation effect The effects of branch age, location, and thickness, as well as the storage time of branch segments, on inoculation efficacy were determined. Branch age: 15 cm segments from the base of one-year-old, two-year-old, and three-year-old branches within the same branch group were used for inoculation. Location: One-year-old branches were divided into three segments based on length: base, middle, and top, for inoculation. Storage time: 15 cm segments from the base of one-year-old branches with a base thickness of approximately 0.8 cm were used for inoculation. Thickness: 15 cm segments from the base of branches with base thicknesses of 0.8 cm, 0.6 cm, and 0.4 cm, respectively, were used for inoculation. Each treatment segment was inoculated using the contact method, and the longitudinal diameter of lesions was measured 3 days post-inoculation. All experiments were performed in triplicate.
[0019] 4. Analysis of the impact of pathogen culture cycle on inoculation effect The pathogen was activated on PDA plates. Mycelial cakes were collected using a 5 mm diameter punch and inoculated onto PDA plates for culture. Colony plates were collected at 3, 5, 7, and 9 days after inoculation for further inoculation. Basal segments of one-year-old branches with similar base thickness were selected and inoculated using the contact method. The longitudinal diameter of lesions was measured 3 days after inoculation. This experiment was performed in triplicate.
[0020] 5. Data Statistics Experimental data were processed, analyzed, and plotted using Excel, Origin64, and SPSS 27. All data are expressed as "mean ± standard deviation".
[0021] Example 2 Results and Analysis 1. The contact method can accurately evaluate the resistance of pear resources to rot disease. One-year-old branch segments from 12 pear varieties with varying resistance in the field were selected, and the spread of lesions after inoculation using the punching and contact methods was statistically analyzed for each variety. Figure 2A, B). Three days after inoculation using the hole-punch method, the longitudinal diameter of the lesions in the 12 varieties, from largest to smallest, was as follows: 'Hongba Pear', 'Hongqie Pear', 'Yuluxiang', 'Xiangshui Pear', 'Huangguan Pear', 'Chou Pear', 'Latai Pear', 'Nanguo Pear', 'Zaoyu', 'Dongguo Pear', 'Duli G-03', and 'Zhutou Pear'. Among them, 'Hongqie Pear' and 'Hongba Pear' showed weak resistance to pear rot, with lesion longitudinal diameters of 10.92±0.2 and 9.95±0.03, respectively; 'Zhutou Pear' and 'Duli G-03' showed strong resistance to pear rot, with lesion longitudinal diameters of 3.31±0.12 and 4.09±0.26, respectively. Figure 2 A, B). Three days after inoculation using the contact method, the longitudinal diameter of lesions in the 12 varieties, from largest to smallest, was the same as that of the perforation method, with significant differences among varieties. Among them, 'Red Eggplant Pear' and 'Red Ba Pear' showed weak resistance to pear rot, with lesion longitudinal diameters of 15.03±0.07 and 13.54±0.18, respectively; 'Pig Head Pear' and 'Du Pear G-03' showed strong resistance to pear rot, with lesion longitudinal diameters of 5.18±0.12 and 6.53±0.26, respectively. Compared with the perforation method, the longitudinal diameter of lesions in the same variety was significantly reduced after inoculation using the contact method, but the resistance performance of each variety was similar to that of the perforation method. Figure 2 A, B). Further correlation analysis showed a high correlation between the two vaccination methods, with a correlation coefficient as high as 0.978 ( ). P <0.01)( Figure 2 C). The above results indicate that the contact inoculation method with pear rot pathogens can accurately evaluate the rot resistance of different pear varieties.
[0022] 2. The contact method can accurately evaluate the resistance of apple resources to rot disease. One-year-old branch segments from six apple varieties with varying resistance in the field were selected, and the spread of lesions after inoculation using the punching and contact methods was statistically analyzed for each variety. Figure 3 AC). Three days after inoculation using the hole-punch method, the longitudinal diameter of the lesions in the six varieties, from largest to smallest, was as follows: 'Ruixianghong', 'Huashuo', 'Venus Gold', 'David Gala', 'Ruixue', and 'Aifei'. Among them, 'Ruixianghong' and 'Huashuo' showed weak resistance to apple rot, with lesion longitudinal diameters of 14.87±0.83 and 13.16±0.39, respectively; 'Ruixue' and 'Aifei' showed relatively strong resistance to apple rot, with lesion longitudinal diameters of 7.45±0.56 and 6.24±0.38, respectively. Figure 3AC). Three days after inoculation using the contact method, the longitudinal diameter of lesions in the six varieties, from largest to smallest, was the same as that of the perforation method, with significant differences among varieties. Among them, 'Ruixianghong' and 'Huashuo' showed weak resistance to pear rot, with lesion longitudinal diameters of 18.25±0.25 and 16.53±0.26, respectively; 'Ruixue' and 'Aifei' showed strong resistance to pear rot, with lesion longitudinal diameters of 11.18±0.26 and 10.29±0.19, respectively. Compared with the perforation method, the longitudinal diameter of lesions in the same variety was significantly reduced after inoculation using the contact method, but the resistance performance of each variety was similar to that of the perforation method. Figure 3 Further correlation analysis showed a high correlation between the two vaccination methods, with a correlation coefficient as high as 0.989 (AC). P <0.01)( Figure 3 D). The above results indicate that the contact inoculation method with apple rot pathogens can accurately evaluate the rot resistance of different apple varieties.
[0023] 3. Effects of different branch ages and lignification degrees on inoculation effectiveness Three pear varieties with different resistance levels were selected: the resistant variety 'Duli-G03', the moderately resistant variety 'Zaosu', and the susceptible variety 'Yuluxiang'. The effects of branch age and lignification degree on inoculation efficacy were analyzed. Three days after inoculation with the canker pathogen, significant differences were observed in the disease incidence rates of one-year-old, two-year-old, and three-year-old branches of the same variety. The disease incidence rate was fastest in three-year-old branches, followed by two-year-old branches, with one-year-old branches showing the slowest rate. For example, after inoculation of 'Yuluxiang' branches, the longitudinal diameter of lesions on three-year-old, two-year-old, and one-year-old branches were 20.18±0.39, 17.34±0.67, and 15.50±0.39 mm, respectively. Figure 4 (A, B). Significant differences in the disease incidence rate of lesions were observed after inoculation of branch segments of the same variety with different degrees of lignification. The basal branch segments showed the fastest disease incidence rate, followed by the middle segments, and the top segments showed the slowest. For example, after inoculation of early-ripening pear branch segments, the longitudinal diameters of lesions at the base, middle, and top were 12.59±0.33, 11.67±0.19, and 9.97±0.1 mm, respectively. Figure 4 (C, D). The above results indicate that the age and lignification degree of branches have a significant impact on the disease rate.
[0024] 4. Effects of branch thickness and storage time on inoculation effectiveness The effects of branch thickness and storage time on inoculation efficacy were analyzed for the three varieties. Significant differences were found between the branches with the largest (0.8 cm) and smallest (0.4 cm) basal diameters after inoculation with branches of different thicknesses across all three varieties. For example, the longitudinal diameter of lesions after inoculation with a 0.8 cm branch was 5.38 ± 0.29 mm, while that with a 0.4 cm branch was 5.99 ± 0.48 mm. However, compared to resistant and moderately resistant varieties, the inoculation efficacy of susceptible varieties was more significantly affected by branch thickness. Three days after inoculation with branches of 0.4 cm, 0.6 cm, and 0.8 cm basal diameters, the longitudinal diameters of lesions in 'Yuluxiang' were 10.22 ± 0.54, 12.24 ± 0.39, and 16.16 ± 0.27 mm, respectively, while those in 'Duli-G03' were 6.29 ± 0.48, 5.71 ± 0.21, and 5.38 ± 0.29 mm, respectively. Figure 5 A, B). Furthermore, 3 days after inoculation with the rot pathogen, significant differences were observed in the longitudinal diameter of lesions in the same variety of branches at 0, 5, 10, and 20 days of storage. The longitudinal diameter of lesions decreased with prolonged storage time for 'Yuluxiang', 'Zaosu', and 'Duli-G03'. Figure 5 (C, D). For example, the longitudinal diameter of lesions on one-year-old branches of the same batch of *Haworthia cooperi* after inoculation at 0, 5, 10, and 20 days of storage were 15.80±0.26, 12.35±0.41, 9.08±0.52, and 7.38±0.50, respectively. These results indicate that branch thickness and storage time significantly affect the disease rate.
[0025] 5. The impact of pathogen growth cycle on inoculation efficacy One-year-old branch segments from three pear varieties with varying resistance in the field were selected to analyze the effect of pathogen culture for 3, 5, 7, and 9 days on inoculation efficacy. Figure 6 (A, B) Three days after inoculation with the rot pathogen, significant differences were observed in the disease incidence rates of branch segments of the same variety on plates cultured for 3, 5, 7, and 9 days. The disease incidence rate was fastest at 3 days, followed by 5 and 7 days, with the slowest rate at 9 days. For example, the longitudinal diameter of lesions on one-year-old branches of *Pyrus pyrifolia* inoculated with the pathogen at 3, 5, 7, and 9 days were 15.56 ± 0.55, 11.08 ± 0.22, 6.89 ± 0.56, and 4.04 ± 0.27, respectively. The disease incidence rates of one-year-old branches of *Pyrus pyrifolia* and *Pyrus pyrifolia* also decreased with increasing pathogen culture time. These results indicate that the pathogen culture period significantly affects the disease incidence rate.
[0026] In the evaluation and breeding of resistance resources, rapid and efficient evaluation of a large number of germplasm resources or hybrid seedlings is crucial for improving research efficiency and shortening the research cycle. This invention improves upon the traditional perforation method, further analyzing the effects of factors such as branch age, lignification degree, thickness, storage time, and pathogen culture time on the inoculation effect. Ultimately, a more efficient inoculation method—the contact method—is proposed. This method eliminates the need for perforation of branches, increasing the number of germplasm resources evaluated in the same timeframe by more than 10 times compared to the traditional perforation method. It enables rapid and accurate evaluation of the rot resistance of a large number of germplasm resources or hybrid seedlings.
[0027] In summary, this invention establishes a rapid and efficient resistance evaluation system that can be used for large-scale evaluation of resistance to rot disease in pear and apple germplasm resources or hybrid progeny. Multiple factors can affect the inoculation effect, among which the storage time of branches and the pathogen culture cycle have the most significant impact.
[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A rapid, efficient method for evaluating resistance to fire blight in pear and apple, characterized by, The method comprises the following steps: S1, test branch preparation: selecting healthy, uniform length, uniform moisture content of pear or apple branches, washing with sterile water, disinfecting with 75% alcohol, and cutting into 15 cm long branches; S2, Pathogen culture: Culture the pear rot pathogen... Valsa pyri Or the pathogen of apple rot Valsa mali Inoculate onto PDA medium and activate culture at 25°C for 3 days to obtain pathogen plates; S3, inoculation by contact method: take a sealed jar, put the pathogenic bacteria plate of step S2 into the sealed jar, and then put the branch of step S1 vertically on the mycelium layer of the pathogenic bacteria plate, so that the bottom of the branch is in complete contact with the mycelium layer, seal and place in a 25℃ constant temperature incubator for 3 days; S4, resistance evaluation: after the culture is finished, the longitudinal diameter of the lesion of the branch is measured, and the resistance of the pear or apple to the rot disease is graded according to the longitudinal diameter of the lesion.
2. The rapid, efficient method for evaluating resistance to fire blight of pear and apple according to claim 1, characterized in that, In step S1, the branch is an annual branch, and the base diameter of the branch is 0.4-0.8 cm; the branch is treated immediately after being harvested, and if it needs to be stored, it is placed in a 4℃ environment, and the storage time is ≤5 days.
3. The rapid, efficient method for evaluating resistance to fire blight of pear and apple according to claim 1, characterized in that, In step S2, the pathogen of the fire blight is Valsa pyri Strain Vp-P-002, the pathogen of the fire blight is Valsa mali Strain Vm-A-003.
4. The rapid, efficient method for evaluating resistance to fire blight of pear and apple according to claim 1, characterized in that, In step S3, the size of the sealed jar is 10 cm in diameter and 30 cm in height; the degree of wetness of the sterile gauze is that the gauze is completely water-absorbed but no water drops.
5. The rapid, efficient method for evaluating resistance to fire blight of pear and apple according to claim 1, characterized in that, In step S4, the resistance grading standard is: the longitudinal diameter of the lesion ≤6.0 cm is highly resistant (HR), 6.1-10.0 cm is resistant (R), 10.1-14.0 cm is moderately resistant (MR), 14.1-18.0 cm is susceptible (S), and >18.0 cm is highly susceptible (HS).
6. The rapid, efficient method for evaluating resistance to Valsa mali according to any one of claims 1 to 5, characterized in that, In steps S1-S4, 3 biological replicates are set for each treatment, and the longitudinal diameter of the lesion is represented by "mean ± standard deviation".