Lily gray mold resistance identification method and application thereof

Abstract

The invention discloses a lily gray mold resistance identification method which comprises the following steps: (1) disinfecting lily leaves to be identified, punching to prepare leaf discs, puncturing the centers of the leaf discs, putting the leaf discs on water agar, inoculating spore suspension of botrytis cinerea strains, and culturing; (2) calculating the morbidity after the morbidity condition is stable according to the morbidity condition of the statistical record; and (3) solving membership function values of indexes corresponding to the lily varieties according to the morbidity obtained in the step (2), and carrying out lily gray mold resistance sequencing according to the membership function values. According to the method, only the lily leaf wafers are used for inoculation, so that the cost is low. During inoculation, the leaf discs are inoculated with conidium suspension liquid, so that the test result is more accurate. The morbidity result can be counted 5 days after inoculation, and the efficiency is high. The spore suspension is consistent in inoculation and moisturizing effects; and moreover, the spore liquid is inoculated by using the leaf disc, so that the space is saved, and technical support is provided for screening gray mold resistant lily varieties.

Description

Technical Field

[0001] This invention belongs to the technical fields of plant protection and crop genetics and breeding. Specifically, it relates to a method for identifying resistance to gray mold in lilies. Background Technology

[0002] Gray mold is a global plant disease. In lilies, gray mold, also known as leaf blight or fire blight, can damage the leaves, stems, and petals, causing leaves to dry out, stems to turn black, flowers to rot brown, and bulbs to stop growing. It is the most common, widespread, and serious fungal disease affecting lilies. Gray mold thrives in low-temperature, high-humidity environments and spreads rapidly, making it difficult to control. A large-scale outbreak can cause significant economic losses to lily cultivation, production, and transportation.

[0003] Gray mold in plants is caused by the fungus *Botrytis cinerea*, a necrotrophic fungus with a broad host range. Lily gray mold is primarily caused by *Botrytis cinerea* and *Botrytis elliptica*, with *Botrytis elliptica* being a specialized pathogen in lilies. *Botrytis cinerea* has a wide host range, successfully infecting plants throughout their entire growth cycle, causing severe gray mold. The pathogen not only exhibits obvious symptoms on living plants but can also remain latent in post-harvest plant products for extended periods. Therefore, it is considered one of the most serious pathogens affecting fruit, vegetable, and flower production and post-harvest care, and is the second most prevalent fungal plant pathogen globally.

[0004] Botrytis cinerea produces conidia that spread to plant surfaces via airflow, rainwater, and agricultural activities. It can initially infect plants through wounds caused by mechanical damage, stress-damaged organs, senescent organs, and natural pores.

[0005] Therefore, the inoculation material used in this invention is a leaf disc from a healthy, intact, and undamaged fresh lily leaf, inoculated with 4 μl of spore suspension (1×10⁻⁶). 6 The cfu / ml was used to identify resistance to gray mold in lilies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for identifying resistance to gray mold in lilies. This method uses leaf discs of healthy, intact, and undamaged fresh lily leaves for inoculation. The spores are diluted with PDB culture medium. When the inoculation amount is 4 μl, the incidence rate is relatively consistent, and there are significant differences among different resistant varieties, providing technical support for screening lily varieties resistant to gray mold.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A method for identifying resistance to gray mold in lilies, comprising three stages: explant disinfection and inoculation with spore suspension, determination of disease incidence, and evaluation of gray mold resistance using a membership function method. The method specifically includes the following steps:

[0009] (1) Disinfect the leaves of the lily to be identified, and make leaf discs by punching holes. After puncturing the center of the leaf discs, place them on 0.4% water agar and inoculate them with a spore suspension of Botrytis cinerea. Use PDB medium as a control. Then place them in a 23℃ light incubator for 0-48 hpi dark treatment, followed by a photoperiod of 12 h light per day.

[0010] (2) Based on the statistical records of the incidence, the incidence rate is calculated after the incidence rate stabilizes. The average value is taken after repeated calculations to obtain the incidence rate of each variety.

[0011] Incidence rate = (Number of diseased leaf discs - Number of diseased leaf discs in the control group) / Number of inoculated discs × 100%

[0012] (3) Calculate the membership function value of the corresponding incidence rate index for each lily variety based on the incidence rate obtained in step (2). Sort the lily gray mold resistance according to the membership function value: Level I high resistance: 0.80≤u(Xi)≤1; Level II low resistance: 0.50≤u(Xi)<0.80; Level III low susceptibility: 0.30≤u(Xi)<0.50; Level IV high susceptibility: 0≤u(Xi)<0.30. Here, Xi represents the incidence rate of the i-th variety.

[0013] Furthermore, the leaf blades are made into leaf discs with a diameter of 1 cm using a perforator.

[0014] Furthermore, the concentration of the spore suspension of the *Botrytis cinerea* strain is 1 × 10⁻⁶. 6 cfu / ml.

[0015] Furthermore, the inoculation amount of the botrytis cinerea spore suspension is 4 μl.

[0016] Furthermore, the preparation process of the spore suspension of the *Botrytis cinerea* strain is as follows:

[0017] (a) Botrytis cinerea was inoculated onto a solid culture medium and cultured at 22°C in the dark for 2 weeks to produce conidia;

[0018] (b) Prepare the liquid culture medium. Pour 10-30 ml into the solid culture medium prepared in step (a). Spores and hyphae are scraped from the surface of the medium. Filter the hyphae to obtain the spore solution, and dilute it with the liquid culture medium to obtain a spore concentration of 1×10⁻⁶. 6 A spore suspension of cfu / ml.

[0019] Furthermore, the solid culture medium is 1 / 2 PDA; the liquid culture medium is PDB.

[0020] Furthermore, in step (2), the disease incidence was stable 5 days after the spore suspension was inoculated onto the detached leaf discs, and the disease incidence at 5 dpi was used as the final disease incidence result.

[0021] Furthermore, mature functional leaves were selected for identification during the lily bud stage.

[0022] Furthermore, the aforementioned botrytis strain is Botrytis cinerea.

[0023] The beneficial effects of this invention are:

[0024] (1) The method described in this invention uses only lily leaf discs for inoculation, which is low in cost.

[0025] (2) When inoculating, leaf discs are inoculated with conidial suspension, and the test results are more accurate.

[0026] (3) The disease incidence results can be counted 5 days after vaccination, which is highly efficient.

[0027] (4) The spore suspension of the present invention has a consistent moisturizing effect when inoculated; and the use of leaf discs to inoculate the spore solution saves space. Attached Figure Description

[0028] Figure 1 These are gray mold spores on the culture medium.

[0029] Figure 2 To observe the spore suspension under a microscope.

[0030] Figure 3 Different concentrations of spore suspension were used for inoculation.

[0031] Figure 4 The disease progression after inoculation with spore solution.

[0032] Figure 5 The infection phenotypes of five lily varieties were observed.

[0033] Figure 6 The diameter of leaf circular lesions varies.

[0034] Figure 7 The change in the conductivity of the blade disc.

[0035] Figure 8 Changes in SOD activity in leaf discs.

[0036] Figure 9 This represents the change in CAT activity in leaf discs. Specific Implementation

[0037] The specific embodiments of the present invention will be further described in detail below with reference to figures and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the reagents and equipment used in the following examples are commercially available or commonly used in the art, and the methods used in the following examples are commonly used methods in the art.

[0038] Example 1

[0039] A method for identifying resistance to gray mold in lilies, using the lily variety 'Mumen' in this embodiment, specifically includes the following steps:

[0040] (1) Cultivating microbial strains

[0041] Potato dextrose agar (PDA) medium purchased from Beijing Solarbio Science & Technology Co., Ltd. was used to prepare a 1 / 2 PDA medium. Following the instructions, it was sterilized at 115℃ for 20 min, cooled to approximately 55℃, and poured into petri dishes for later use. Botrytis cinerea (a type of gray mold) was inoculated onto the 1 / 2 PDA medium and cultured at 22℃ in the dark for 2 weeks to produce conidia (such as...). Figure 1 (As shown).

[0042] (2) Obtaining spore suspension

[0043] Prepare PDB medium using potato glucose broth (PDB) medium purchased from Beijing Solarbio Technology Co., Ltd., and sterilize at 121℃ for 20 min according to the instructions.

[0044] Pour 15 ml of the prepared PDB medium into half of the PDA medium that will produce conidia. Use a sterilized inoculation loop to scrape spores and hyphae from the surface of the medium. Filter the solution through gauze to remove hyphae debris and obtain a clear liquid. The spore suspension should be prepared fresh before use. Count the spores in the prepared spore suspension using a hemocytometer and dilute the spore suspension to a concentration of 1×10⁻⁶ using PDB liquid medium. 6 CFU / ml (Results of spore suspension observed under a microscope are as follows) Figure 2 (As shown).

[0045] (3) Disinfect the explants and determine the inoculation spore suspension

[0046] Prepare 0.4% water agar by weighing 4 g of agar powder, diluting it to 1 L with distilled water, and sterilizing it at 121℃ for 20 min.

[0047] Mature functional leaves from the bud stage of the lily 'Wooden Door' were harvested. Surface impurities were washed away with tap water, followed by disinfection with 70% alcohol for 8 seconds, and then rinsed with distilled water. The leaves were dried with filter paper. Using a sterile punch with a diameter of 1 cm, avoiding the central vein, the leaves were punched into small round pieces. A dissecting needle was used to puncture the center of each round piece, creating a natural wound conducive to bacterial infection. The leaf rounds were then placed in a petri dish containing 0.4% water agar. Using a pipette, 4 μl of different concentrations (1×10⁻⁶) were pipetted into the petri dish. 4 cfu / ml, 1×10 5 cfu / ml, 1×10 6A spore suspension (cfu / ml) was dropped onto the puncture sites on leaves. The control group was inoculated with 4 μl of PDB medium. After inoculation, the cells were placed in a 23°C light incubator and incubated in the dark for 0–48 hpi. The photoperiod was then 12 h of light per day. Disease incidence was observed and recorded every 24 h, and the optimal inoculation concentration was determined based on the disease incidence. Inoculation was then carried out at the optimal concentration.

[0048] During the infection process, it was found that the inoculation concentration was 1×10⁻⁶. 6 After 2-3 days, the wounds turned brown and obvious lesions appeared in the CFU / ml spore suspension. These lesions continued to expand, allowing for detailed observation of the disease's dynamic changes. The inoculation concentration was 1×10⁻⁶. 4 cfu / ml, 1×10 5 No obvious lesions appeared on the leaf discs after 5 days when the cfu / ml spore suspension was applied, indicating that the spore suspension at this concentration could not successfully infect the leaf discs. Therefore, the optimal infection concentration was determined to be 1×10⁻⁶. 6 cfu / ml. (e.g.) Figure 3 (As shown) This method selects 1×10 6 Inoculation was performed at a concentration of cfu / ml.

[0049] (4) Determination of incidence rate

[0050] Leaf disc inoculation at a concentration of 4 μl (1×10⁻⁴) 6 Spore suspensions of CFU / ml were used to record the incidence rate. Based on the statistically recorded incidence, the incidence rate was calculated after the disease stabilized. The average of three replicates was taken as the incidence rate for each variety. In this method, the observation period was 5 days after inoculation of detached leaf discs with spore suspension, at which point the disease stabilized. The incidence rate at 5 dpi was used as the final incidence rate result (e.g., 5 days after inoculation). Figure 4 (As shown).

[0051] Incidence rate = (Number of diseased leaf discs - Number of diseased leaf discs in the control group) / Number of inoculated discs × 100%

[0052] (5) Evaluation of gray mold resistance in lilies using the membership function method

[0053] The membership function method was used to evaluate the resistance of different lily germplasm resources to gray mold. The formula is as follows: u(Xi) = (Xi - Xmin) / (Xmax - Xmin) ① u(Xi) = 1 - (Xi - Xmin) / (Xmax - Xmin) ②

[0054] Indicators positively correlated with resistance to gray mold in lilies were calculated using formula ①, while those negatively correlated were calculated using formula ②. u(Xi) is the membership function value of the i-th indicator for each lily variety, u(Xi)∈[0,1]; Xi represents the measured value of the i-th indicator for each lily variety; Xmax and Xmin are the maximum and minimum values ​​of the i-th indicator for all tested varieties.

[0055] Based on the above incidence rates, the membership function values ​​of the corresponding incidence rate indicators for each lily variety were calculated. Based on the membership function values, the resistance to gray mold in lilies was ranked as follows: Level I: High resistance: 0.80 ≤ u(Xi) ≤ 1; Level II: Low resistance: 0.50 ≤ u(Xi) < 0.80; Level III: Low susceptibility: 0.30 ≤ u(Xi) < 0.50; Level IV: High susceptibility: 0 ≤ u(Xi) < 0.30. The conclusions on the strength of gray mold resistance were then drawn.

[0056] Example 2

[0057] A method for identifying resistance to gray mold in lilies. In this embodiment, 19 cut lily varieties were selected (see Table 1), including 'Siberia', 'Wooden Door', 'Competition', and 'White Paradise'.

[0058] Table 1 Information on 19 Lily Germplasm Resources

[0059]

[0060] Specifically, the following steps are included:

[0061] (1) Cultivating microbial strains

[0062] Potato dextrose agar (PDA) medium purchased from Beijing Solarbio Science & Technology Co., Ltd. was used to prepare a 1 / 2 PDA medium. The medium was sterilized at 115℃ for 20 min according to the instructions, cooled to approximately 55℃, and poured into petri dishes for later use. Botrytis cinerea (a type of gray mold) was inoculated onto the 1 / 2 PDA medium and cultured at 22℃ in the dark for 2 weeks to produce conidia.

[0063] (2) Obtaining spore suspension

[0064] Prepare PDB medium using potato glucose broth (PDB) medium purchased from Beijing Solarbio Technology Co., Ltd., and sterilize at 121℃ for 20 min according to the instructions.

[0065] Pour 15 ml of the prepared PDB medium into half of the PDA medium that will produce conidia. Use a sterilized inoculation loop to scrape spores and hyphae from the surface of the medium. Filter the solution through gauze to remove hyphae debris and obtain a clear liquid. The spore suspension should be prepared fresh before use. Count the spores in the prepared spore suspension using a hemocytometer and dilute the spore suspension to a concentration of 1×10⁻⁶ using PDB liquid medium. 6 cfu / ml.

[0066] (3) Disinfect the explants and inoculate them with spore suspension

[0067] Prepare 0.4% water agar by weighing 4 g of agar powder and diluting to 1 L with distilled water. Sterilize at 121℃ for 20 min. Collect mature functional leaves from lily buds, wash away surface impurities with tap water, disinfect with 70% alcohol for 8 s, rinse with distilled water, and dry the leaf surface with filter paper. Using a sterile punch (1 cm diameter), avoid the central vein and punch the leaves into small round discs. Use a dissecting needle to puncture the center of each disc to create a natural wound that facilitates bacterial infection. Place the leaf discs in a petri dish containing 0.4% water agar. Use a pipette to extract 4 μl of spore suspension (1×10⁻⁶). 6 (cfu / ml) was dripped onto the puncture sites on the leaves. The control group was inoculated with 4 μl of PDB medium. After inoculation, the plants were placed in a 23℃ light incubator and in the dark for 0-48 hpi. The photoperiod was 12 h light per day thereafter. Disease incidence was observed and recorded every 24 h.

[0068] (4) Determination of incidence rate

[0069] Leaf disc inoculation at a concentration of 4 μl (1×10⁻⁴) 6 Spore suspensions of CFU / ml were used to record the incidence rate. Based on the statistically recorded incidence, the incidence rate was calculated after the disease stabilized. The average of three replicates was taken as the incidence rate for each variety. In this method, the observation period was 5 days after inoculation of detached leaf discs with spore suspension, at which point the disease stabilized. The incidence rate at 5 dpi was used as the final incidence rate result.

[0070] Incidence rate = (Number of diseased leaf discs - Number of diseased leaf discs in the control group) / Number of inoculated discs × 100%

[0071] (5) Evaluation of gray mold resistance in lilies using the membership function method

[0072] The membership function method was used to evaluate the resistance of different lily germplasm resources to gray mold. The formula is as follows: u(Xi) = (Xi - Xmin) / (Xmax - Xmin) ① u(Xi) = 1 - (Xi - Xmin) / (Xmax - Xmin) ②

[0073] Indicators positively correlated with resistance to gray mold in lilies were calculated using formula ①, while those negatively correlated were calculated using formula ②. u(Xi) is the membership function value of the i-th indicator for each lily variety, u(Xi)∈[0,1]; Xi represents the measured value of the i-th indicator for each lily variety; Xmax and Xmin are the maximum and minimum values ​​of the i-th indicator for all tested varieties.

[0074] Based on the above incidence rates, the membership function values ​​of the corresponding incidence rate indicators for each lily variety were calculated. The resistance to gray mold was then ranked according to the membership function values: Level I: Highly resistant: 0.80 ≤ u(Xi) ≤ 1; Level II: Lowly resistant: 0.50 ≤ u(Xi) < 0.80; Level III: Lowly susceptible: 0.30 ≤ u(Xi) < 0.50; Level IV: Highly susceptible: 0 ≤ u(Xi) < 0.30. The conclusion regarding the strength of gray mold resistance was then drawn (see Table 2). Among them, 'Bakadi', 'Yellow Surrey', 'Black Beauty', and 'Siberia' are highly resistant varieties, while 'Candy', 'Zambezi', and 'Pink Flight' are highly susceptible varieties.

[0075] Table 2. Evaluation of resistance to gray mold in lilies after inoculation with detached leaf disc spore suspension.

[0076]

[0077] Example 3

[0078] Based on the evaluation results of Example 2, five lily varieties with different resistance were selected: 'Robinna', 'Praffindo', 'Sorbonne', 'Competition', and 'Parazor'. The changes in the size of lesions after leaf disc inoculation were recorded, and relevant physiological indicators were measured to verify the accuracy of the disease resistance results in the disease incidence assessment.

[0079] (1) Statistics on lesion diameter

[0080] The diameter of the lesions was statistically analyzed using the cross-multiplication method and ImageJ image processing software.

[0081] (2) REC determination

[0082] Eight leaf discs were placed in 10 ml centrifuge tubes at different treatment stages. 8 ml of deionized water was added, and the tubes were evacuated by a vacuum pump for 6 min. The tubes were then placed in a 28℃ constant temperature shaker and shaken for 4 h. The initial conductivity R1 was measured. The centrifuge tubes were then placed in a boiling water bath for 20 min, removed, and shaken in a shaker for 30 min. After cooling to room temperature, the tubes were shaken well and the conductivity R2 was measured. REC = R1 / R2×100%.

[0083] (3) SOD and CAT activity assay

[0084] SOD activity was determined by the hydroxylamine method and CAT activity was determined by the ammonium molybdate method. The assay kits were purchased from Nanjing Jiancheng Biological Institute, and the experimental data were measured using an enzyme-linked immunosorbent assay (ELISA) reader according to the kit instructions.

[0085] 1. Changes in the size of the lesions

[0086] 4 μL of 1×10⁵ lily varieties were used for 5 different varieties. 6 After inoculation with CFU / mL spore suspension, leaf lesions were recorded every 24 hours. The results are shown in Figure 5. Figure 6 'Praffindo', 'Competition', and 'Palazzo' showed subtle infection phenotypes on leaf discs as early as 2 days after inoculation, indicating rapid disease progression. 'Palazzo' showed the fastest growth rate in lesion diameter with increasing treatment time, reaching 90% incidence on the entire leaf disc by 6 days after inoculation, demonstrating high susceptibility to gray mold. 'Robinna' showed the mildest disease, with no obvious lesions appearing by 7 days after inoculation, exhibiting the strongest resistance to gray mold. The lesion area of ​​all varieties stabilized 5 days after inoculation, consistent with the stabilization time of the incidence rate observed in previous assessments, indicating that using the incidence rate 5 days after inoculation as an indicator of disease resistance has a certain degree of reliability.

[0087] 2. Changes in REC

[0088] During infection, pathogens produce a large amount of toxins and corresponding enzymes that damage tissue cells. In this process, a large amount of electrolytes leak from the cells, and the REC value directly reflects the degree of damage to plant cells. Figure 7 shows the REC changes of five lily varieties at different treatment stages. The REC changes after inoculation varied among the varieties. Throughout the treatment process, the REC changes of 'Robinna', 'Praffindo', and 'Sorbonne' were relatively small, all less than 5%; 'Competition' showed a slight upward trend, reaching 3.68% at 1 dpi and increasing to 10.56% at 7 dpi. With the extension of treatment time, the REC of 'Parazor' showed a significant upward trend, reaching 31.43% on day 7, an increase of 27.38%, indicating that Botrytis cinerea inoculation caused greater damage to the leaves of 'Parazor'.

[0089] 3. Changes in SOD and CAT activity

[0090] When plants encounter adverse conditions, they produce SOD and CAT to scavenge reactive oxygen species. SOD then converts oxygen from the plant into reactive oxygen species through a dismutation reaction. 2 -CAT can convert H2O2 into H2O and O2, preventing the excessive accumulation of reactive oxygen species that lead to lipid peroxidation of plant cell membranes and slowing down cell death. The two work synergistically to remove excess reactive oxygen species from cells (Acosta-Motos et al., 2015). When plants experience stress, the higher the activity of their antioxidant enzymes, the stronger their ability to scavenge reactive oxygen species, and the greater their adaptability to stress.

[0091] SOD and CAT activities in leaf discs were measured on days 0, 1, 2, 3, 4, and 5 after inoculation. As shown in Figure 8, enzyme activities of different lily varieties increased to varying degrees after inoculation, showing a trend of first increasing and then decreasing. The SOD activities of 'Robinna' and 'Praffindo' increased rapidly after inoculation, reaching their maximum values ​​after 3 days, increasing by 147 and 114 enzyme activity units respectively compared to before inoculation, indicating that they responded quickly to the infection stress of Botrytis cinerea. The enzyme activity levels of 'Sorbonne', 'Competitive', and 'Parazor' reached their maximum relatively later. The SOD activity of 'Competitive' increased by 105 enzyme activity units, and the enzyme activities decreased to varying degrees on days 4-5 after inoculation.

[0092] Depend on Figure 9 It can be seen that the CAT activity after inoculation showed an overall trend of first increasing and then decreasing, consistent with the trend of SOD. Before inoculation, 'Robina' and 'Praffindo' had higher CAT activity, reaching their maximum values ​​on the 3rd day after inoculation, reaching 36.76 and 32.04 enzyme activity units respectively. Both could quickly mobilize enzyme activity to resist adversity when subjected to gray mold infection. 'Sorbonne', 'Competitive', and 'Parazor' reached their maximum enzyme activity 3-4 days after inoculation, with relatively lower CAT activity. As the stress time increased, the CAT activity of all lily varieties decreased. Five days after inoculation, the enzyme activity in the leaf discs was lower than before treatment, with 'Robina' showing the smallest decrease.

[0093] 4. Overall Evaluation

[0094] The membership function method was used to evaluate the resistance of different lily germplasm resources to gray mold. The formula is as follows: u(Xi) = (Xi - Xmin) / (Xmax - Xmin) ① u(Xi) = 1 - (Xi - Xmin) / (Xmax - Xmin) ②

[0095] Indicators positively correlated with resistance to gray mold in lilies were calculated using formula ①, while those negatively correlated were calculated using formula ②. u(Xi) is the membership function value of the i-th indicator for each lily variety, u(Xi)∈[0,1]; Xi represents the measured value of the i-th indicator for each lily variety; Xmax and Xmin are the maximum and minimum values ​​of the i-th indicator for all tested varieties.

[0096] To accurately reflect the resistance of the five lily species to gray mold, the changes in lesion size, REC, SOD, and CAT activity were combined with membership function formulas for comprehensive evaluation. Lesion size and REC were calculated using formula ②, while SOD and CAT activity were calculated using formula ①. The average value of each membership function value was accumulated and ranked. The larger the membership function value, the stronger the disease resistance. The results of the disease resistance of the five lily species are shown in Table 3. The order from strongest to weakest is: 'Robinna', 'Praffindo', 'Sorbonne', 'Competition', and 'Parazor', which is consistent with the previous results of the incidence rate identification.

[0097] Table 3. Comprehensive evaluation of gray mold resistance in five lily varieties

[0098]

[0099] In summary, the results of the three examples above show that the optimal concentration of the inoculated spore suspension is 1×10⁻⁶. 6 The optimal observation time is 5 days after inoculation of detached leaf discs with spore suspension, when the disease condition is stable. The disease incidence rate at 5 dpi is used as the final incidence rate result. This invention provides a method for identifying resistance to gray mold in lilies.

Claims

1. A method for identifying resistance to gray mold in lilies, the method comprising: The method comprises the following steps: (1) disinfecting the lily leaves to be identified, and making leaf discs from the leaves using a puncher, wounding the center of the leaf discs, placing the leaf discs on 0.4% water agar, inoculating a spore suspension of a Botrytis strain, using PDB medium as a control, and then placing in a 23°C light incubator, dark treatment for 0-48 hpi, and then a light cycle of 12 h light per day; (2) calculating the incidence rate after the incidence is stable according to the recorded incidence, and averaging the values obtained by repeating multiple times, i.e. the incidence rate of each variety; Incidence rate = (number of diseased leaf discs - number of diseased leaf discs in the control group) / number of inoculated discs x 100% (3) obtaining the membership function value of the corresponding index of each lily variety according to the incidence rate obtained in step (2), and ranking the resistance of lily Botrytis according to the membership function value, grade I highly resistant: 0.80≤u(Xi)≤1; grade II low resistant: 0.50≤u(Xi)<0.80; grade III low susceptible: 0.30≤u(Xi)<0.50; grade IV highly susceptible: 0≤u(Xi)<0.

30.

2. The method of claim 1, wherein, The concentration of the spore suspension of the said Botrytis strain is 1 x 10 6 cfu / ml.

3. The method according to claim 1 or 2, characterized in that, The inoculation amount of the spore suspension of the Botrytis strain is 4 μl.

4. The method of claim 2, wherein, The preparation process of the spore suspension of the Botrytis strain is: (a) inoculating the Botrytis strain on a solid culture medium, and culturing for 2 weeks at 22°C in the dark to produce conidia; (b) Prepare liquid medium, take 10-30 ml and pour into the solid medium prepared in step (a), scrape the spores and mycelium on the surface of the medium, filter the mycelium, obtain the spore solution, and dilute with liquid medium to obtain a spore suspension with a spore concentration of 1 x 10 6 cfu / ml. (b) Prepare liquid medium, take 10-30 ml and pour into the solid medium prepared in step (a), scrape the spores and mycelium on the surface of the medium, filter the mycelium, obtain the spore solution, and dilute with liquid medium to obtain a spore suspension with a spore concentration of 1 x 10 6 cfu / ml.

5. The method of claim 4, wherein, The solid culture medium is 1 / 2 PDA; and the liquid culture medium is PDB.

6. The method of claim 1, wherein, In step (2), the incidence is stable at 5 d after inoculation of the spore suspension on the detached leaf discs, and the incidence at 5 dpi is taken as the final incidence rate result.

7. The method of claim 1, wherein, At the lily bud stage, mature functional leaves are selected for identification.

8. The method of claim 1, wherein, The leaves are made into 1 cm diameter leaf discs using a puncher.

9. The method of any one of claims 1-4, wherein, The Botrytis strain is Botrytis cinerea.

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