Evaluation method for soft rot resistance level of aseptically cultured Zantedeschia aethiopica

Abstract

The invention relates to a method for evaluating the soft rot resistance level of aseptically cultured colored callalily. The method comprises the following steps: preparing in-vitro leaves of aseptically cultured colored callalily to be detected; placing the in-vitro leaves on a moisturizing device for moisturizing culture; preparing a bacterial liquid of the Erwinia solani, wherein the concentration of the bacterial liquid is lower than or equal to the quorum sensing threshold concentration of the Erwinia solani; inoculating a predetermined volume of the bacterial liquid at a petiole incision of the in-vitro leaf; at different time points after inoculation, the morbidity condition of the in-vitro leaves is observed and quantified; and evaluating the soft rot resistance level of the aseptically cultured colored callalily based on the quantitative result of the morbidity condition. On the basis of sterile and rapid propagation of the zantedeschia aethiopica, the inoculation method has the characteristics of simplicity, convenience, high efficiency and easiness in popularization, is of great significance to development of disease-resistant breeding of the zantedeschia aethiopica, and provides a new technology and method for disease-resistant breeding of other plants, plant-microorganism interaction and research in related fields of biotechnology.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically a method for assessing the resistance to soft rot in aseptically cultured colored calla lilies. Background Technology

[0002] The colored calla lily (Zantedeschia spp.), native to southern Africa, has been hailed as the "star of 21st-century flowers" due to its rich flower colors and beautiful plant shape after years of hybridization and selection. It has become a highly sought-after species in the global flower industry. However, bacterial soft rot is prevalent in this species, severely hindering its development. Breeding and creating new colored calla lily varieties resistant to soft rot is an effective way to address this pain point in the industry's development. Studies have reported that various pectinobacteria can cause soft rot in colored calla lilies, including *Pectobacterium aroidearum*, *P. zantedeschiae*, and *P. carotovorum* subsp. *carotovorum*. Furthermore, pathogens isolated from the soft rot tissues of other crops can also cause soft rot when inoculated into colored calla lilies.

[0003] Looking at the achievements of germplasm innovation in horticultural crops worldwide, aseptic culture technology has demonstrated significant advantages in shortening the breeding cycle and rapidly establishing large-scale populations. Li Yingzhang et al. (2000) used different sources of Erwinia amylovora (fire blight pathogen) and inoculated aseptically cultured apple tree stem segments, rooted seedlings, and greenhouse potted seedlings using the leaf-cutting method and the wick method. They found that tissue culture materials from different apple varieties showed varying sensitivities to the same strain; tissues of the same variety at different stages of in vitro culture showed no significant differences in sensitivity to the same pathogen; the sensitivity of aseptically cultured materials to pathogens decreased after rooting; disease-resistant varieties exhibited significant inhibitory effects on pathogen infection and colonization; and the results of in vitro and potted plant assessments of fire blight resistance were similar. Therefore, aseptic culture materials can be directly used for resistance selection. Using aseptic culture materials for disease resistance selection not only overcomes the limitations of the natural growing season but also enables rapid propagation to obtain genetically consistent large-scale populations for disease resistance assessment.

[0004] Accurately assessing the disease resistance level of different materials is crucial for the successful integration of aseptic culture technology and disease resistance breeding research. Inoculation with pathogens is the first step in resistance identification, and the effectiveness of inoculation determines the accuracy of the identification. Common inoculation methods for plant bacteria include spraying, needle pricking, and injection. Wang Xuejiao et al. (2018) found through comparison that spraying inoculation of calla lily leaves did not cause infection, needle pricking was difficult to control the initial bacterial load, while injection could control the inoculated bacterial load. Li Yingzhang et al. (2000), in studying the infection ability of *Phytophthora infestans* on apple seedlings in different states, used cotton thread as a medium, allowing the pathogen to penetrate from the container to the wound in the plant tissue, and then compared the differences in disease severity—this is known as the wick inoculation method. This method requires strict control of multiple factors, such as the height difference between the bacterial solution surface and the plant wound, the length of the cotton thread, and air humidity, to ensure good consistency between replicates.

[0005] The inoculation concentration of pathogens determines the outcome of plant-pathogen interactions and is one of the bases for assessing the resistance level of plant materials. Studies have shown that disease can only occur when the number of pathogens in a plant exceeds a certain concentration, which is called the quorum sensing (QS) threshold. Under natural conditions, the initial invasion concentration of pathogens will not exceed the QS threshold. Only after invasion, when pathogens absorb plant nutrients and reproduce, can they reach the threshold. The plant's disease resistance level and environmental factors determine whether pathogens can reproduce and their reproduction rate. Therefore, pathogens sometimes exist in a latent state within the plant, without causing disease. Only when the plant's immunity decreases or environmental factors change, causing the pathogen's reproduction rate to accelerate and its population exceeding the QS threshold, can disease be triggered. However, currently, when assessing plant resistance to soft rot, the inoculation concentration usually reaches 10. 9 CFU inoculation resulted in severe rotting of test samples 12-16 hours after inoculation, indicating rapid disease progression and failing to reflect the plant's inhibitory effect on pathogen invasion. Only when the inoculation concentration is close to the initial concentration of natural invasion can the arms race between plants and pathogens be reproduced, allowing for comparison of disease progression and accurate assessment of plant material resistance levels to pathogens.

[0006] The severity of disease varies depending on the inoculation site. Previous studies have shown that soft rot fungi are inoculated onto colored calla lilies at the following sites: bulbs, leaves, and petioles. Bulbs are often sliced ​​before inoculation (Gu Chunyan, 2009); petioles and leaves require puncture wounds before inoculation (Fan, 2020). Due to the complex vein network of leaves and the dense vascular bundles on the surface of petioles, it is difficult to ensure consistency in the tissue around the puncture wound and the path of lesion expansion between different replicates.

[0007] There is a lack of standardized research methods for identifying the disease resistance of aseptically cultured plant materials. To address this issue, the designers of this invention, based on the principles of pathogen-plant interaction, focused on evaluating the soft rot resistance of aseptically cultured colored calla lilies. The analysis covered multiple aspects, including the maintenance of viability of detached leaves, comparison of different inoculation sites, and selection of inoculation concentration. A water agar adhesion and moisturizing method for detached leaves of aseptically cultured colored calla lilies was established to prolong the viability of young tissues. Low concentrations (103) of several soft rot fungi with different pathogenicities were used. 6 CFU was inoculated at the petiole cut, delaying the onset of severe symptoms in the control group to 48 hours after inoculation, demonstrating the antagonistic interaction between the pathogen and the leaf tissue of colored calla lilies. However, according to existing methods, whether in pots or aseptically cultured plants, using high concentrations of CFU (10...)... 9 When CFU (Chemical Fumed Root Function) was inoculated onto detached leaves of colored calla lilies, disease began to appear within 12 hours of inoculation, with severe rot developing within 16 hours (Fan, 2020), failing to demonstrate the interaction between the plant and the pathogen. The method established in this invention approximates the natural disease progression of soft rot, showcasing the temporal dynamics of affinity between the plant material and the pathogen. At the pathogen-plant interaction level, it provides technical parameters for assessing the resistance level of aseptically cultured colored calla lilies to soft rot. Summary of the Invention

[0008] The purpose of this invention is to provide a method for assessing the resistance level of colored calla lilies to soft rot in aseptic culture. The method aims to utilize the principle of "pathogen-plant" interaction to demonstrate the disease progression of plant material lesions caused by pathogens under artificially controlled conditions, analyze the plant material's resistance to pathogen invasion, and thus assess its resistance level to the disease.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for evaluating the resistance level to soft rot in aseptically cultured colored calla lilies, comprising the following steps:

[0010] S1: Prepare sterile cultured detached leaves of colored calla lily to be tested;

[0011] S2: Place the detached leaf on a humidifying device for humidifying culture;

[0012] S3: Prepare a bacterial solution of soft rot pathogens, wherein the concentration of the bacterial solution is lower than or equal to the quorum sensing threshold concentration of the soft rot pathogens;

[0013] S4: Inoculate a predetermined volume of the bacterial solution at the petiole cut of the detached leaf;

[0014] S5: Observe and quantify the disease incidence of the detached leaves at different time points after inoculation;

[0015] S6: Based on the quantitative results of the disease occurrence, assess the resistance level of the aseptically cultured colored calla lilies to soft rot.

[0016] In step S2 of the present invention, the moisturizing device is a water agar plate, and the step of placing the detached leaf on the moisturizing device for moisturizing culture specifically involves placing the leaf face down on the surface of the water agar plate; the water agar plate contains agar, and the concentration of the agar in the water agar plate is 5.0 g / L-10.0 g / L;

[0017] In step S2, multiple detached blades are attached to the surface of the same water agar plate in a spaced-out manner;

[0018] In step S3, the concentration of the bacterial solution is 1×10⁻⁶. 6 CFU / mL - 1×10 7 CFU / mL;

[0019] In step S4, the petiole incision is obtained by horizontally cutting off the petiole end of the detached leaf to form an incision surface; and creating a wound on the incision surface.

[0020] In the aforementioned method, the wound is a hole formed by vertically piercing the central area of ​​the incision surface using a piercing tool, and the depth of the wound is 2-4 mm; in step S4, a predetermined volume of bacterial solution is injected into the petiole incision using a pipette; in step S5, quantifying the disease status of the detached leaf includes: measuring the total length of the main vein of the detached leaf, and measuring the length of the water-soaked lesion extending from the petiole incision along the main vein direction; calculating the disease severity value, whereby the disease severity value is the ratio of the length of the water-soaked lesion to the total length of the main vein.

[0021] In step S5, the disease severity is divided into multiple levels based on the severity value, and a disease index is calculated based on the levels; the disease index is calculated using the following formula:

[0022]

[0023] in, As a disease index, The onset of the disease is the first The number of blades in the grade, For the first The representative value corresponding to the level To observe the total number of leaves, This represents the highest incidence level.

[0024] The aforementioned disease progression is categorized into grades 0, 1, 2, 3, 4, and 5, with corresponding severity value ranges of 0, (0, 10], (10, 30], (30, 50], (50, 70], and (70, 100], respectively, and corresponding representative values. The numbers are 0, 1, 2, 3, 4, and 5 respectively.

[0025] According to the aforementioned disease index Assess disease resistance level: When When assessed as highly resistant; When, it is assessed as resistant; when At that time, it was assessed as moderately effective; when When, the assessment is as feeling; when At that time, the assessment was high sensitivity.

[0026] In step S3, the soft rot pathogen is selected from the genus Pectobacterium; preferably, the soft rot pathogen includes at least two strains with different pathogenicities; the at least two strains include a first strain and a second strain, wherein the first strain is more pathogenic than the second strain; preferably, the first strain is Pectobacterium aroidearum NJAU2 and the second strain is Pectobacterium aroidearum PccS1.

[0027] In step S1, the aseptically cultured colored calla lily detached leaves are taken from aseptic seedlings cultured on rooting medium for 35 to 55 days; in step S5, the different time points include 12 hours, 24 hours and 36 hours after inoculation. Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses aseptically cultured colored calla lily plants as experimental materials for the first time, representing field plants. It can get rid of seasonal restrictions, and the screening scale and working environment conditions are completely controllable. It has the characteristics of "more, faster, better and cheaper", and overcomes the shortcomings of the existing methods, such as large-scale cultivation, long time consumption, large amount of manual input and poor controllability. It provides technical support for accelerating the innovation of colored calla lily germplasm resistant to soft rot.

[0028] (2) In this invention, sterile cultured colored calla lily leaves are adhered to the surface of water agar, which has a moisturizing effect on the leaves but does not produce free water. The environmental humidity is stable and the leaf position is fixed. This not only prolongs the leaf vitality but also overcomes the shortcomings of the "absorbent paper + sterile water" moisturizing method: initially, when water is first added, the water flow when the container is moved will cause water to stick to the leaf surface and may even spread to the pathogen inoculation point, affecting the inoculation effect; later, when the delay time is long, water needs to be added to keep the leaves fresh, resulting in differences in environmental humidity before and after the test. (3) In this invention, vertical inoculation is performed at the petiole cut. Sterile toothpicks and other fixing equipment are used to create the wound. The surrounding plant tissue is a uniformly arranged microtubule tissue. Using this as the inoculation point has relatively good consistency and the inoculation solution penetration direction is fixed. This is different from the wound on the leaf or petiole surface, where the distribution of the surrounding vascular tissue is uncertain and the inoculation solution penetration direction cannot be controlled.

[0029] (4) This invention selects a relatively low concentration of bacterial solution for inoculation. The pathogen multiplies within the plant and reaches the quorum sensing (QS) threshold concentration, thereby causing disease. Measuring the morphological changes of plant leaves at different times can reflect the power struggle between the pathogen and the plant. If a bacterial solution concentration higher than the QS threshold is used for inoculation, severe symptoms will quickly appear in the rapidly progressing soft rot disease, making it difficult to capture the process of the struggle between the two and to evaluate the differences in the ability of different plants to inhibit the reproduction and spread of soft rot fungi (disease resistance).

[0030] (5) This invention is based on the sterile and rapid propagation of colored calla lilies. The inoculation method is simple, efficient and easy to promote. It is of great significance for carrying out disease-resistant breeding of colored calla lilies. At the same time, it provides new technologies and methods for disease-resistant breeding of other plants, plant-microbe interaction and biotechnology-related research. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of two methods for moisturizing detached leaves of colored calla lily plants in an embodiment of the present invention.

[0032] Part A demonstrates the water agar bonding method, while Part B demonstrates the filter paper and sterile water moisturizing method.

[0033] Figure 2 Comparative chart of disease incidence after inoculating different parts of colored calla lily leaves with soft rot fungus.

[0034] Figure 3 Comparative graph showing disease incidence after inoculating colored calla lily and Chinese cabbage leaves with soft rot pathogens from different sources.

[0035] Figure 4Disease index analysis at different time points after inoculating leaves of three colored calla lily strains with different concentrations of pectinobacterium.

[0036] Figure 5 This is a comparison of the disease progression after inoculation with low-concentration bacterial solution through petiole incision under two different moisturizing methods.

[0037] Part A consists of filter paper moistened with sterile water, while Part B consists of water agar adhesive for moisturizing. Detailed Implementation

[0038] The technical solution of this embodiment provides a method for evaluating the resistance level of colored calla lilies to soft rot in sterile culture. It includes the following: (1) Preparation of test plant materials: adventitious buds of colored calla lilies are tissue cultured, single plants are cut out, and after rooting culture with MS + 0.1% activated carbon for 45 days, complete leaves are taken, and the petiole length is kept at about 1.0 cm. They are placed in sterile water for later use.

[0039] (2) Maintaining the viability of plant materials: Take the prepared leaves out of the moist sterile water, with water adhering to the surface, and place them directly on the water agar plate with the leaf surface facing down, with different leaves spaced 0.5 cm apart and arranged neatly. (3) Types of pathogens inoculated: P. aroidearum PccS1 and P. aroidearum NJAU2 were isolated and identified from the soft rot tissue of colored calla lily in the field, and P. brasilense and P. solani were isolated and identified from the soft rot tissue of potato. Among them, Pectobacterium NJAU2 had the strongest pathogenicity, and P. solani had the weakest. (4) Selection of pathogen inoculation concentration: Compare different inoculation concentrations of bacterial solution and select the lowest inoculation concentration that can cause disease. (5) Selection of inoculation site location: Select inoculation sites with good repeatability of disease results and good consistency of pathogen diffusion in plant tissue. (6) Quantitative observation of the disease progression of soft rot: After inoculation with pathogens, the symptoms gradually worsen over time until severe cases appear. This process has a continuous period of time, and the differences in the severity of disease at different time points are accurately measured.

[0040] The following is a comparison of the methods used in different steps of the implementation process:

[0041] Preparation of aseptically cultured leaf samples of colored calla lilies. Adventitious buds from the propagated colored calla lily were cut into single plants, all intact leaves were removed, and they were vertically inoculated into rooting medium (MS + 1.0 g / L activated carbon + 30 g / L sucrose + 7.2 g / L agar). Ten plants were evenly inoculated into 9 cm diameter culture bottles and cultured for 45 days. Intact leaves with petioles of approximately 1.0 cm were then harvested and placed in sterile water. Each variety (strain) should be replicated in 3 groups, with no fewer than 5 plants in each group, and a total of no fewer than 15 leaves collected.

[0042] To maintain leaf viability, detached leaves with sterile water adhering to them are placed face down and smoothly affixed to water agar plates (water + agar 7.2 g / L sterilized at high temperature; poured into sterile square petri dishes before solidification, 3 mm thick, cooled, and ready for use). Adjacent leaves are spaced 0.5 cm apart. Figure 1 As shown in Figure A. The filter paper moisturizing method serves as a control; that is, a layer of sterile filter paper is placed in a flat-bottomed container, an appropriate amount of sterile water is added to moisten the filter paper, and the leaf is placed on the surface of the filter paper for later use. Figure 1 As shown in B.

[0043] Preparation of inoculum. Remove *Pectinobacterium* stored at -80℃ in glycerol, add 100 μL to LB medium, and incubate overnight at 28℃ with a shaker at 200 rpm for 12–16 h. Remove and transfer to fresh LB liquid medium at a 1:100 ratio, incubate at 28℃ with a shaker at 200 rpm for 4–5 h. Remove, centrifuge at 6000 rpm for 3 min, discard the supernatant, resuspend in sterile water, and adjust to OD200. 600 =1.0 (1×10 9 (CFU / mL), then diluted to 1×10⁻⁶. 8 1×10 7 and 1×10 6 CFU, for backup.

[0044] Colored calla lily leaves were inoculated with bacterial solution. The petiole exceeding 1 cm in length was horizontally removed with a scalpel, making a crescent-shaped incision. A 3 mm deep wound was made in the center of the crescent-shaped cut using a sterile toothpick. Simultaneously, using a sterile toothpick, a 3 mm long incision was made along the vein line at the base of the midrib or lateral veins in the middle of the colored calla lily leaf. 5 μL of the prepared bacterial solution was injected into the wound using a pipette. The bacterial solution concentration was 1 × 10⁻⁶. 8 1×10 7 and 1×10 6 CFU / mL. An equal volume of sterile water was inoculated as a control, with three replicates for each treatment. After inoculation, the petri dishes were sealed with plastic wrap and incubated at 28℃ under constant light (photoperiod L:D=16:8 h). At 12, 24, and 36 h post-inoculation, photographs were taken, and the total leaf length and the spread of water-soaked symptoms along the veins were measured. Disease severity was calculated as: spread length of water-soaked symptoms along the veins / vein length.

[0045] Disease severity is graded. Based on numerical values, the disease severity is divided into four levels: 0, I, II, III, IV, and V. The numerical ranges for severity are as follows: 0; greater than 0 and less than or equal to 10; greater than 10 and less than or equal to 30; greater than 30 and less than or equal to 50; greater than 50 and less than or equal to 70; and greater than 70 and less than or equal to 100. The representative values ​​for the six disease severity grades are 0, 1, 2, 3, 4, and 5, respectively.

[0046] Evaluation of soft rot resistance in colored calla lilies. Disease index (DI) was calculated based on the disease incidence rate of test samples under different treatments.

[0047]

[0048] in, Let be the number of samples for the i-th disease level. Let be the representative value for the i-th disease severity level. The total number of samples, The value represents the highest disease severity level; the resistance level of the plant material is determined based on the preset correspondence between the disease index range and the resistance level.

[0049] Then, based on the DI value distribution range of different samples, the resistance level to soft rot is determined:

[0050]

[0051] A comparison of different methods for each step in the implementation process.

[0052] (1) Method for maintaining the vitality (moisturizing) of detached colored calla lily leaves. Colored calla lily leaves are adhered to a water agar surface. The leaves can remain fresh for over 10 days without needing additional water. Humidity remains stable throughout the process, and the leaf position is fixed. Figure 1 As shown in Figure A. The filter paper is kept moist with sterile water. Initially, the sterile water may cause water to adhere to the leaf surface, and sometimes this water may spread to the inoculation site, such as... Figure 1 As shown in B, it interferes with inoculation; in the later stages (after 3 days), sterile water needs to be added, otherwise the leaves will wither.

[0053] (2) Comparison of pathogens inoculated at different locations on colored calla lily leaves. Microtubules are the main pathway for pathogens to spread within the plant. The soft-rot fungus *P. aroidearum* PccS1 (1×10⁻⁶) originating from colored calla lily was identified. 8 Inoculate wounds (CFU / mL) on different parts of the leaf (scratches on the midrib and lateral veins, petiole incisions), keep moist with absorbent sterile filter paper, and incubate for a certain period of time to observe the severity of disease. Symptoms indicate that inoculation along the midrib, such as... Figure 2 As shown in Figure A, the bacterial solution extends from the wound along the midrib to both ends, while simultaneously spreading to both sides of the midrib; inoculation occurs on the lateral veins of the leaf, as... Figure 2 As shown in B, the bacterial solution spreads irregularly from the wound along the lateral veins; inoculation is performed at the petiole cut, as... Figure 2As shown in Figure C, the bacterial solution spreads from the cut along the petiole to the midrib, and the water-soaked lesions extend orderly towards the leaf tip. The first two inoculation sites are currently the most commonly used. However, the midrib of tissue-cultured seedlings is relatively narrow, making it difficult to ensure the direction of the incision is perfectly aligned with the midrib, easily resulting in skewing. Furthermore, the midrib is ridge-shaped, making it easy for the inoculated bacterial solution to flow out. The lateral veins are even smaller and vary greatly in thickness, making it easy to tear the leaf when making the incision, inevitably causing bacterial solution loss. In both cases, the direction of bacterial solution diffusion after inoculation cannot be consistently maintained, resulting in lesion sizes that vary, affecting the accuracy of the test. Moreover, because inoculation via leaf vein wounds results in irregular lesions, it is usually necessary to measure the area to compare the differences in disease severity. In contrast, inoculation via petiole cuts allows the lesions to extend orderly along the midrib; measuring the length of the water-soaked lesions on the midrib is sufficient for comparison, making the operation simple and convenient.

[0054] (3) Comparison of pathogenicity of different strains of soft rot fungi. Assessing disease resistance requires comparing the degree of infection of the target species by multiple strains with varying pathogenicity. Therefore, five strains causing soft rot (1×10⁻⁶) were used. 8 CFU / mL was inoculated onto the cut surfaces of colored calla lily petioles adhered and kept moist on water agar. The strains were: *Pectobacterium solani* (isolated from potato), *P. aroidearum* PccS1 (isolated from colored calla lily), *P. aroidearum* NJAU2 (isolated from colored calla lily), *P. aroidearum* NJAU150 (isolated from konjac), and *P. brasilense* NJAU21 (isolated from clivia). Chinese cabbage petioles were used as a reference. The results showed that NJAU2 had the strongest pathogenicity; PccS1, NJAU150, and NJAU21 had similar pathogenicity, at an intermediate level; and *P. solani* had the weakest pathogenicity. Figure 3 As shown. Both NJAU2 and PccS1 were derived from field soft rot tissue of colored calla lilies, but from different collection locations: the former from Yunnan and the latter from Nanjing. Therefore, this invention selected NJAU2 and PccS1 as inoculation strains for evaluating the resistance of colored calla lilies to soft rot, and comprehensively evaluated the resistance of colored calla lily tissue culture seedlings to soft rot.

[0055] (4) Different concentrations (1×10⁻⁶) of Pectinobacterium aroidearum PccS1 8 1×10 7 and 1×10 6 CFU / mL was inoculated onto leaves of three different colored calla lily strains. Disease index analysis at different time points showed that inoculation concentrations greater than the QS threshold (1×10⁻⁶ CFU / mL) indicated disease indices at these concentrations. 7 The disease index of the three strains (CFU / mL) did not differ significantly at different time points after inoculation. Figure 4 B Figure 4 As shown in C, at low concentrations, the disease index of R2 and R3 did not differ significantly. However, 16-36 hours post-vaccination, the severity of disease in R1 was relatively mild compared to R2 and R3. Figure 4 As shown in Figure A.

[0056] (5) Two methods of humidification were used: filter paper with sterile water and leaf attachment to water agar plates. P. aroidearum PccS1 (10 μL) was injected into the petiole cut. 6 5 μL (CFU / mL) of sterile cultured colored calla lily leaves was applied. At 12 hours after inoculation, no symptoms were observed in the leaves, and the difference between the two humidification methods was minimal. As the disease progressed, the rotten area increased. Leaves kept moist with filter paper gradually showed yellowing, and as humidity decreased, adding sterile water caused the rotten tissue, containing pathogens, to spread outwards with the free water, affecting the normal disease process and making it difficult to measure the extent of the disease. Figure 5 As shown in Figure A. Using the water agar adhesion and moisturizing method, there is no liquid flow, the leaves remain viable, and inoculation is performed at the petiole cut. The bacterial suspension spreads stably along the midrib, clearly distinguishing between infected and healthy parts, allowing for accurate measurement. Figure 5 As shown in B.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. For those skilled in the art, within the scope of the present invention, variations and equivalent substitutions can be made based on the technical solutions and concepts of the above embodiments, according to specific requirements, species protection, and application objectives. All variations and equivalent substitutions still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the resistance to soft rot in aseptically cultured colored calla lilies, characterized in that: Includes the following steps: S1: Prepare sterile cultured detached leaves of colored calla lily to be tested; S2: Place the detached leaf on a humidifying device for humidifying culture; S3: Prepare a bacterial solution of soft rot pathogens, wherein the concentration of the bacterial solution is lower than or equal to the quorum sensing threshold concentration of the soft rot pathogens; S4: Inoculate a predetermined volume of the bacterial solution at the petiole cut of the detached leaf; S5: Observe and quantify the disease incidence of the detached leaves at different time points after inoculation; S6: Based on the quantitative results of the disease occurrence, assess the resistance level of the aseptically cultured colored calla lilies to soft rot.

2. The method according to claim 1, characterized in that: In step S2, the humidification device is a water agar plate, and the step of placing the detached leaf on the humidification device for humidification culture specifically involves placing the detached leaf with its leaf surface facing down on the surface of the water agar plate; the water agar plate contains agar, and the concentration of the agar in the water agar plate is 5.0 g / L-10.0 g / L; In step S2, multiple detached blades are attached to the surface of the same water agar plate in a spaced-out manner; In step S3, the concentration of the bacterial solution is 1×10⁻⁶. 6 CFU / mL - 1×10 7 CFU / mL; In step S4, the petiole incision is obtained by horizontally cutting off the petiole end of the detached leaf to form an incision surface; and creating a wound on the incision surface.

3. The method according to claim 2, characterized in that: The wound is a hole formed by vertically piercing the central area of ​​the incision surface using a piercing tool, and the depth of the wound is 2-4 mm.

4. The method according to claim 1, characterized in that: In step S4, the predetermined volume of bacterial solution is injected into the petiole incision using a pipette. In step S5, quantifying the disease status of the detached leaf includes: measuring the total length of the main vein of the detached leaf, and measuring the length of the water-soaked lesion extending from the petiole cut along the main vein direction; Calculate the severity value of the condition, which is the ratio of the length of the water-soaked lesion to the total length of the main vein.

5. The method according to claim 4, characterized in that: In step S5, the disease severity is divided into multiple levels based on the severity value, and a disease index is calculated based on the levels; the disease index is calculated using the following formula: in, As a disease index, The onset of the disease is the first Number of blades per grade For the first The representative value corresponding to the level To observe the total number of leaves, This represents the highest incidence level.

6. The method according to claim 11, characterized in that, The disease severity is classified into four levels: Level 0, Level 1, Level 2, Level 3, Level 4, and Level 5. The corresponding severity value ranges are 0, (0, 10], (10, 30], (30, 50], (50, 70], and (70, 100], respectively, with corresponding representative values. The numbers are 0, 1, 2, 3, 4, and 5 respectively. In step S6, based on the disease index Assess disease resistance level: When At that time, it was assessed as highly resistant; when When, it is assessed as resistant; when At that time, it was assessed as moderately effective; when When, the assessment is as feeling; when At that time, the assessment was high sensitivity.

7. The method according to claim 1, characterized in that: In step S3, the soft rot pathogen is selected from the genus Pectobacterium; preferably, the soft rot pathogen includes at least two strains with different pathogenicities.

8. The method according to claim 7, characterized in that: The at least two strains include a first strain and a second strain, wherein the first strain is more pathogenic than the second strain; preferably, the first strain is Pectobacterium aroidearum NJAU2 and the second strain is Pectobacterium aroidearum PccS1.

9. The method according to claim 1, characterized in that: In step S1, the aseptic cultured colored calla lily detached leaves are taken from aseptic seedlings cultured on rooting medium for 35 to 55 days.

10. The method according to claim 1, characterized in that: In step S5, the different time points include 12 hours, 24 hours and 36 hours after vaccination.

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