Chinese yam cross-regional introduction suitability detection and disease-resistant variety large-scale screening model based on leaf culture medium and application

Abstract

The invention discloses a Chinese yam cross-regional introduction suitability detection and disease-resistant variety large-scale screening model based on a leaf culture medium and application, and the model is characterized in that natural leaves of Chinese yam varieties to be introduced in different regions are used as raw materials to prepare a standardized leaf culture medium, and local dominant pathogenic bacteria of an introduction destination are inoculated; through the gradient inoculation and 28 DEG C incubator culture process, the resistance result of the variety to be introduced can be output within 6-7 days, and the detection accuracy and the operation feasibility are both considered. The model is especially suitable for grass-roots agricultural technology promotion departments, Chinese yam planting collaborations and agricultural product detection laboratories, can realize large-scale disease resistance screening of Chinese yam varieties to be introduced in different areas, and accurately judges the suitability of the varieties to be introduced to local pathogenic bacteria of introduction destinations. Introduction failure caused by mismatching of variety disease resistance and local pathogenic bacteria is avoided, and scientific support is provided for Chinese yam cross-region introduction decision making, industrial green prevention and control and high-quality disease-resistant variety popularization.

Description

Technical Field

[0001] This invention belongs to the technical field of plant cross-regional introduction adaptability detection and disease-resistant variety screening, specifically involving a model and application of cross-regional introduction adaptability detection and large-scale screening of disease-resistant varieties of yam based on leaf culture medium. Background Technology

[0002] As an important economic crop with both medicinal and edible uses in my country, the yam industry has seen continuous expansion in recent years, forming major production areas centered around Jiaozuo (iron stick yam) in Henan, Lixian (small white yam) in Hebei, and Heze (Chenji yam) in Shandong. Yam is not only a key pillar for increasing farmers' income in these production areas, but also demonstrates high added value potential in extended fields such as functional foods and traditional Chinese medicine processing. With the increasing demand for high-quality yams in the consumer market, the industry is gradually developing towards standardization and large-scale production.

[0003] With the expansion of yam cultivation across regions, the introduction of varieties from different areas is frequent. However, planting failures due to differences in pathogen adaptability are common during the introduction process. Currently, most introduction efforts focus only on the climatic adaptability of varieties (such as temperature and rainfall), neglecting the interaction between the introduced varieties and local yam pathogens. Some superior varieties from other regions, while exhibiting strong disease resistance in their native habitats, lack resistance to local dominant pathogens (such as a specific strain of anthracnose) after being introduced to new production areas, becoming highly susceptible varieties. This not only increases introduction costs but may also disrupt the local pathogen population balance, triggering new disease outbreaks. Existing disease resistance screening for yam varieties before cross-regional introduction relies heavily on field observations at the introduction site, which is insufficient to meet the practical need for "rapid and large-scale assessment of the resistance of different yam varieties to local pathogenic fungi" before introduction. There is an urgent need for a testing scheme that is close to field realities and easy to operate to fill this gap. Summary of the Invention

[0004] This invention overcomes the shortcomings of traditional disease-resistant yam variety screening and long introduction cycles, and provides a model and application for cross-regional introduction suitability testing and large-scale screening of disease-resistant varieties of yam based on leaf culture medium. This model is suitable for screening yam varieties resistant to leaf pathogenic fungi. The model needs to be operated in a controlled laboratory environment. Through standardized leaf culture medium preparation, pathogen inoculation and culture, accurate test results can be output within 6-7 days, taking into account both detection accuracy and operational feasibility. It is especially suitable for grassroots agricultural technology extension departments, yam planting cooperatives and agricultural product testing laboratories for pathogen suitability prediction before cross-regional introduction of yam, initial screening of disease resistance of new varieties, and secondary screening of disease resistance of main varieties promoted in major producing areas.

[0005] The yam cross-regional introduction suitability detection and disease-resistant variety large-scale screening model based on leaf culture medium provided by this invention has two advantages: First, it has high detection efficiency, which can significantly shorten the detection cycle compared with traditional detection methods, meet the requirements of rapid detection of resistance of different yam varieties to pathogenic fungi, and meet the timeliness requirements of introduction assessment. Second, it has a wide detection coverage, which can simultaneously detect the resistance of the yam to be introduced to a variety of pathogenic microorganisms (such as anthracnose fungus, black spot fungus, root rot fungus, etc.) common in the introduced production area, without the need to carry out separate detection procedures for a single pathogen, greatly improving the practicality and suitability of the detection.

[0006] To achieve the above objectives, this invention employs the following technical solution: a model for cross-regional introduction and adaptation testing of yam varieties and large-scale screening of disease-resistant varieties based on leaf culture medium. Natural leaves of yam varieties to be introduced from different regions are used as the core raw material for the culture medium, and yam leaf culture medium containing multiple leaf concentration gradients is prepared. The medium is then inoculated with native dominant pathogenic fungi from the target planting area and cultured at a constant temperature of 28℃. Within 6-7 days, the mycelial growth status of the pathogenic fungi and the phenotype of leaf lesions are observed to achieve large-scale disease resistance screening of yam varieties to be introduced from different regions, ultimately determining whether the introduced varieties are suitable for the target planting area.

[0007] Further specifying, the pathogenic fungi include Alternaria alternata (yam black spot pathogen), Colletotrichum gloeosporioides (yam anthracnose pathogen), and Fusarium oxysporum (yam root rot pathogen).

[0008] Furthermore, the model sets three leaf concentration gradients in the yam leaf culture medium preparation process: 1g / 100mL, 3g / 100mL, and 5g / 100mL. Through this multi-gradient dosage design, it can adapt to the growth of pathogenic microorganisms with different nutritional requirements, and at the same time cover the detection scenarios of yam varieties with high, medium, and low resistance levels, ensuring the universality and accuracy of the detection results.

[0009] Furthermore, the maximum concentration of the leaf application can reach 5g / 100mL. This concentration can provide sufficient natural nutrients for the growth of pathogenic fungi, ensure that the detection indicators such as pathogenic fungal colonies are clearly identifiable, and improve the reliability of the detection results.

[0010] The yam cross-regional introduction suitability detection and disease-resistant variety large-scale screening model based on leaf culture medium described in this invention has one of its core components as the standardized preparation of yam leaf culture medium. The specific process is as follows: First, leaves are collected and pretreated. According to the three leaf concentration gradients of 1g / 100mL, 3g / 100mL, and 5g / 100mL set by the model, leaves from healthy plants of the yam variety to be tested are selected. After collecting a sufficient amount of leaves for each gradient, they are immediately placed in an ice box at 4-6℃ (lined with sterile absorbent paper) and brought back to the laboratory within 1 hour. The required leaf weights for each gradient (1g, 3g, and 5g, respectively) are accurately weighed to ensure that the leaf quantity and target concentration are accurately matched, reducing subsequent concentration deviations. Next, clean the leaves by gently rinsing them 3-4 times with sterile water (changing the water each time to thoroughly remove surface dust and impurities). No additional disinfection is required. After rinsing, lay the leaves flat on sterile absorbent paper and let them air dry in a cool, ventilated place. Alternatively, gently absorb the moisture from the leaf surface with sterile absorbent paper to avoid residual moisture affecting the homogenization concentration and pathogen growth environment.Next, leaf homogenization was performed. The grinding operation was carried out outdoors, but all equipment, including grinding mortars, grinding rods, conical flasks, and sterile distilled water, were pre-sterilized at 121℃ for 20 minutes under high pressure to prevent exogenous microbial contamination. During the operation, the dried leaves were cut into 1-2cm square pieces and placed into sterile grinding mortars. Sterile distilled water (total 50mL) was added to each mortar in 3-4 batches. After each batch was added, the leaves were thoroughly ground with a sterile grinding rod until the leaf tissue formed a fine paste-like homogenate (without obvious tissue particles). This process of adding water in batches and thorough grinding was repeated. To minimize leaf residue in the grinding mortar and ensure complete dissolution of leaf nutrients and precise final homogenate concentration, after grinding, each gradient homogenate was completely transferred to its corresponding sterile conical flask (100mL), mixed thoroughly, and prepared leaf homogenate stock solutions of 1g / 50mL, 3g / 50mL, and 5g / 50mL. Subsequently, the leaf homogenate stock solutions were subjected to constant temperature treatment. Each gradient of leaf homogenate stock solution was placed in a 60℃ constant temperature water bath, and the water bath was maintained for 5 hours each time. This water bath operation was repeated every 12 hours, for a total of 4-5 times. Multiple constant temperature treatments promoted leaf nutrient absorption. The substances are fully released, while simultaneously killing the original microorganisms in the leaves. Finally, the culture medium is prepared and sterilized: First, prepare the "agar-water basal solution." Weigh out agar at a ratio of 20 g / L (add 1 g of agar to every 50 mL of sterile distilled water). Divide the prepared 50 mL agar-water solution into 100 mL Erlenmeyer flasks, seal the flasks, and autoclave at 121°C for 20 minutes. After sterilization, cool to 60-65°C in a sterile environment for later use (to avoid high temperatures damaging the nutrients in the leaves). This pre-dispensing and sterilization method avoids repeated batching in subsequent operations. To minimize the risk of contamination while ensuring precise dosage of each basal solution, and to facilitate rapid mixing with 50 mL of leaf homogenate stock solution, the agar-water basal solution at the same temperature is rapidly mixed with the temperature-controlled leaf homogenate stock solution in a clean bench (one 50 mL basal solution is mixed with one 50 mL homogenate at each gradient, ultimately preparing 100 mL / part culture medium with leaf concentrations of 1 g / 100 mL, 3 g / 100 mL, and 5 g / 100 mL). The mixture is immediately dispensed into sterile petri dishes and allowed to cool naturally to room temperature to solidify, yielding the yam leaf culture medium. This invention's model is applied to the suitability screening of yam varieties for cross-regional introduction and the identification of resistance to multiple pathogens.

[0011] This invention has the following advantages and beneficial effects: First, it has high detection efficiency and a short cycle. Compared with traditional methods, this invention, through standardized yam leaf culture medium preparation process and gradient concentration design, can significantly shorten the large-scale screening time for disease-resistant yam varieties. It is fast, low-cost, and especially suitable for disease resistance testing and screening before cross-regional introduction. Second, it is significantly cost-effective. Using the natural leaves of the yam variety to be tested as the core raw material of the culture medium, it eliminates the need for complex chemical composition preparation and expensive professional instruments, greatly reducing detection costs. It is particularly suitable for large-scale applications in grassroots agricultural technology extension departments, yam planting cooperatives, and other similar settings. Third, it has a wide detection coverage. Simultaneous detection of multiple pathogenic microorganisms eliminates the need for separate detection procedures for single pathogens, perfectly adapting to the actual situation of mixed infection by multiple pathogens in the field; fourth, its application scenarios are diverse and practical. It can be used for disease resistance adaptability testing of yam before its introduction to different regions. By simulating the local pathogen infection environment, it can predict the disease resistance adaptability of introduced varieties, avoiding cost waste and disease transmission risks caused by introduction failure. It can also assist in the screening of disease-resistant varieties. By comparing the leaf disease resistance phenotypes of different candidate varieties, it can quickly distinguish the differences in disease resistance between varieties, providing a scientific basis for yam disease resistance breeding and the selection of high-quality varieties, and effectively promoting the development of the yam industry towards high efficiency and high quality. Attached Figure Description

[0012] Figure 1 The study investigated the growth responses of three varieties (Huai Shan Yao No. 2, Yan Ye No. 1, and Shanxi Taigu) to three pathogens in three different concentrations of leaf culture medium. The study found that the nutrient content in the 1 g / 100 mL medium was too low, which limited the growth of the pathogens, while the pathogens could grow normally in the 3 g / 100 mL and 5 g / 100 mL media.

[0013] Figure 2 The test was conducted to assess the resistance of 12 varieties to three pathogens in a 5g / 100mL culture medium.

[0014] Figure 3 The study compared the leaf condition of the "control group (inoculated with sterile water)" and the "treatment group (inoculated with Alternaria alternata)" of two yam varieties (HL-1 and Tiegun), confirming the accuracy of the 5g / 100mL leaf culture medium in the test.

[0015] Figure 4 This is a PCA analysis of the metabolome of HL-1 and Dioscorea opposita leaves. Detailed Implementation

[0016] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0017] First, leaf collection and pretreatment were performed. Based on the model's three leaf concentration gradients (1g / 100mL, 3g / 100mL, and 5g / 100mL), leaves from healthy plants of the yam variety to be tested (between young and fully mature leaves) were selected. Sufficient leaves were collected for each gradient and immediately placed in a 4-6℃ ice box (lined with sterile absorbent paper) and brought back to the laboratory within one hour. The required leaf weights for each gradient (1g, 3g, and 5g, respectively) were accurately weighed to ensure precise matching of leaf quantity to the target concentration and reduce subsequent concentration deviations. Next, leaf cleaning was carried out. The weighed leaves were gently rinsed 3-4 times with sterile water (changing the sterile water each time to thoroughly remove surface dust and impurities), without additional disinfection. After rinsing, the leaves were laid flat on sterile absorbent paper and allowed to air dry naturally in a cool, ventilated place, or the surface moisture was gently blotted dry with sterile absorbent paper to avoid residual moisture affecting subsequent homogenization concentration and pathogen growth environment. Next, leaf homogenate preparation was carried out. The grinding operation was conducted outdoors, but all equipment, including grinding bowls, grinding rods, conical flasks, and sterile distilled water, were sterilized by high-pressure steam at 121℃ for 20 minutes in advance to prevent exogenous microbial contamination. During the operation, the dried leaves were cut into small square pieces of 1-2cm and placed into sterile grinding bowls. Sterile distilled water (total 50mL) was added to each grinding bowl in 3-4 batches. After each batch was added, the leaves were thoroughly ground with a sterile grinding rod until the leaf tissue was a fine paste-like homogenate (without obvious tissue particles). By adding water in batches and grinding thoroughly, the amount of leaf residue in the grinding bowl was reduced, ensuring that the nutrients in the leaves were fully dissolved and the final homogenate concentration was accurate. After grinding, the homogenates of each gradient were completely transferred to the corresponding sterile conical flasks (100mL), mixed, and leaf homogenate stock solutions of 1g / 50mL, 3g / 50mL, and 5g / 50mL were prepared. Subsequently, the leaf homogenate mother liquor was subjected to constant temperature treatment. The leaf homogenate mother liquors of different concentration gradients were placed in a 60℃ constant temperature water bath and kept at the temperature for 5 hours each time. The water bath operation was repeated every 12 hours for a total of 4-5 times. Through multiple constant temperature treatments, the nutrients in the leaves were fully released, while killing the original microorganisms in the leaves.Finally, the culture medium is prepared and sterilized: First, prepare the "agar-water basal solution". Weigh out agar at a ratio of 20 g / L (1 g of agar is added for every 50 mL of sterile distilled water). Dispense 50 mL of the prepared agar-water solution into 100 mL Erlenmeyer flasks, seal the flasks, and autoclave at 121°C for 20 minutes. After sterilization, cool to 60-65°C in a sterile environment for later use (to avoid high temperatures damaging the nutrients in the leaves). This pre-dispensing and sterilization method avoids the risk of contamination from repeated measurements in subsequent operations and ensures accurate dosage of each basal solution, facilitating rapid mixing with the 50 mL leaf homogenate stock solution. In a laminar flow hood, prepare the "agar-water basal solution" at the same temperature... The yam leaf culture medium was rapidly mixed with the homogenized leaf stock solution after constant temperature treatment (one 50 mL basal solution was mixed with one 50 mL homogenate for each concentration gradient, resulting in 100 mL / part culture medium with leaf concentrations of 1 g / 100 mL, 3 g / 100 mL, and 5 g / 100 mL). After mixing, the medium was immediately dispensed into sterile petri dishes and allowed to cool naturally to room temperature to solidify, thus obtaining the yam leaf culture medium. At the same time, a PDA culture medium was prepared as a control group (formula: 200 g of peeled and diced potatoes were boiled for 30 minutes, the filtrate was collected, 20 g of glucose and 20 g of agar were added, and distilled water was added to 1000 mL. The mixture was then sterilized at 121°C for 20 minutes). This medium was used simultaneously with the leaf culture medium for pathogen growth comparison detection to ensure the validity of the detection results.

[0018] Example 1

[0019] Resistance testing of different yam varieties based on this detection model:

[0020] Resistance testing of multiple yam varieties based on this detection model (including screening at a concentration of 5g / 100mL):

[0021] This embodiment aims to detect the resistance of different yam varieties to Alternaria alternata, Aspergillus niger, and Schizophyllum commune, and to verify the practicality of the model of the present invention. The experiment was repeated 3 times to control the error, and different treatments within the group were used as controls throughout the process.

[0022] Preparation of experimental materials: (1) Yam varieties: the initial screening was Huai yam No. 2, Yanye No. 1, and Shanxi Taigu (AC), and the secondary screening was Wenxian Taigu and 12 other varieties (DO and HL-1), which were free from pests and diseases; (2) Pathogens and fungal cakes: the three pathogens were activated by PDA medium and cultured at 28℃ for 7 days. The fungal cakes at the edge of the colony were taken with a 5mm punch that had been sterilized in advance for later use; (3) Equipment: in addition to grinding and sterilizing equipment, sterile tweezers and a 28℃ constant temperature incubator were prepared to ensure that the equipment in contact with the culture medium was sterile.

[0023] Step 1: Initial screening and concentration determination (1g / 100mL, 3g / 100mL, 5g / 100mL): Prepare three concentrations of leaf culture medium for AC according to the invention. Collect healthy leaves between young and mature stages, bring them back in an ice box at 4-6℃, add 50mL of sterile water in multiple batches, and grind until no particles remain. After the culture medium solidifies, punch a 5mm hole in the center. When inoculating, place the mycelium-side up mushroom cake into the hole, seal with sealing film, and incubate at 28℃ for 7 days. Use different concentration treatments within the group as controls to observe the uniformity of colony growth and differences in varietal resistance (see...). Figure 1 (Comparison of leaf culture medium results of three varieties, namely Huai Shan Yao No. 2, Yan Ye No. 1, and Shanxi Tai Gu, at three different concentrations) was conducted to determine that 5 g / 100 mL was the optimal concentration. At this concentration, the three pathogens grew stably, and the resistance differences among different varieties were clear, which could effectively eliminate nutritional interference.

[0024] The second step is to re-screen varieties (based on a concentration of 5g / 100mL): leaf culture medium with the same concentration of 5g / 100mL is prepared for 12 re-screened varieties, with 9 plates for each variety (3 plates for each of the 3 pathogens). After solidification, holes are punched, and the inoculation procedure is the same as the initial screening. The culture is carried out at 28℃ for 7 days, with different varieties within the group as controls.

[0025] Judgment of rescreening results (see) Figure 2 (Comparison of resistance differences in leaf culture media among 12 varieties for secondary screening): The criteria for judgment were high resistance (HR: no mycelium, no lesions, only mycelial cakes), moderate resistance (MR: a small amount of mycelium, no lesion spread), and susceptible (S: dense mycelium, lesion spread). Visual observation after 7 days: Using the differences between varieties within the group as a control, 11 varieties, including Wenxian Taigu, showed susceptibleness (dense mycelium on the culture medium surface, accompanied by lesion spread); only HL-1 showed high resistance. After inoculation with the three pathogens, no mycelium was produced or lesions were observed on the culture medium surface; only the initially inoculated mycelial cakes were visible. The differences in resistance among varieties were significant, and the verification results were reliable.

[0026] Step 3: Validation of the resistance stability of HL-1 high-concentration leaf culture medium: To further clarify the impact of increased leaf concentration on resistance screening results (adapting to potential differences in leaf nutrient environment during cross-regional introduction), and to investigate the interference of the physical properties of the culture medium at high concentrations on experimental procedures, two leaf concentration gradients of 10g / 100mL and 15g / 100mL were added. Common pathogens (Alternaria alternata, Fusarium oxysporum, and Colletotrichum gloeosporioides) were inoculated to conduct validation experiments. During the experiment, when preparing leaf culture media at each concentration according to the aforementioned standardized procedure, it was found that the leaf homogenates at concentrations of 10g / 100mL and 15g / 100mL, due to the increased proportion of leaf tissue, exhibited significantly increased viscosity after mixing with the agar-water basal solution. This resulted in slow flow rates and localized accumulation during pouring, making it difficult to spread evenly in the culture dishes. Therefore, leaf concentrations above 5g / 100mL are unsuitable as a concentration standard for cross-regional introduction screening.

[0027] Final conclusion: This model, through intra-group control with different treatments, determined that 5 g / 100 mL is the optimal concentration for rapid detection of disease resistance in yam leaf culture medium. Simultaneously, it screened HL-1, a variety highly resistant to *Alternaria alternata*, *Aspergillus niger*, and *Schizophyllum commune*, from 12 secondary-screening varieties. Further validation showed that leaf concentrations higher than 10 g / 100 mL are unsuitable for introduction screening. In summary, this model can provide precise concentration criteria for screening disease-resistant varieties during cross-regional introduction, and HL-1 is suitable for priority introduction and planting in areas with high incidence of the aforementioned three pathogens.

[0028] Example 2

[0029] HL-1 and resistance verification by live inoculation of Dioscorea opposita based on this detection model:

[0030] This embodiment uses HL-1 yam (screened highly resistant variety) and iron stick yam (susceptible control variety) as subjects. The reliability of the detection model of the present invention is verified by inoculating live leaves with Alternaria alternata spore suspension. The experiment is repeated 3 times, with the treatment group and the control group forming an intragroup control. Aseptic operation is carried out throughout the process.

[0031] For experimental materials, six healthy functional leaves from each of the two varieties (with petioles retained and consistent growth cycles) were selected, divided into a treatment group (3 leaves / variety) and a control group (6 leaves / variety). For spore suspension preparation, *Alternaria alternata* was cultured in PDA for 5-7 days, followed by washing of colonies with sterile PBS buffer. The collected solution was placed in a 10mL sterile EP tube containing steel beads, vortexed for 10 minutes to break the hyphae, filtered through three layers of sterile gauze, and the concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 6 The concentration was 1000 / mL, aliquoted, and stored at 4°C for later use. Regarding equipment and environment, all equipment that came into contact with the experiment (pipette tips, EP tubes, etc.) was sterilized by autoclaving at 121°C for 20 minutes. A 28°C constant temperature incubator, 75% ethanol, 3wt% sodium hypochlorite solution, and sterile distilled water were prepared. The entire experiment was conducted in a clean bench.

[0032] The experimental procedure consisted of three steps: First, leaf disinfection was performed. HL-1 and Chinese yam leaves were placed separately in sterile culture flasks and immersed sequentially in 75% ethanol for 30 seconds (gently shaking to ensure full contact between the front and back surfaces), followed by 3wt% sodium hypochlorite solution for 3-4 minutes. Then, the leaves were rinsed 3-4 times with sterile distilled water (replacing the water each time) until no disinfectant residue remained. Finally, the leaves were laid flat on sterile filter paper and allowed to air dry naturally in a laminar flow hood (until no obvious water stains were visible). Second, leaf treatment and inoculation were carried out using sterile... The dried leaves were picked up with tweezers, and a 5mm long wound (without penetrating the leaf mesophyll) was gently pierced on the right side of the center of the leaf surface (avoiding the veins) with a sterile knife. The leaves were then laid flat in a sterile petri dish lined with sterile water-moistened filter paper. For the treatment group, 20μL of spore suspension was added to the center of the wound using a sterile pipette, while for the control group, 20μL of sterile distilled water was added. Three replicate petri dishes were set up for each variety treatment group. Finally, the petri dishes were covered after inoculation and the edges were sealed with sterile sealing film. They were then placed in a 28℃ constant temperature incubator for seven days.

[0033] Results observation (see Figure 3 The comparison of the results of HL-1 and iron yam leaves inoculated with Alternaria alternata for 7 days showed that: in the control group (inoculated with sterile distilled water), the leaves of both HL-1 and iron yam remained fresh, with no lesions, mycelia or mold at the wounds, and the leaves were uniform in color without chlorosis or yellowing, with only slight healing marks on the wounds; in the treatment group, the leaves of iron yam showed obvious lesions, while the leaves of HL-1 yam in the treatment group showed no lesions, and the leaves maintained their original bright green color without chlorosis, mold or tissue rot, fully demonstrating high resistance characteristics, and the resistance difference between the two varieties in the treatment group was significant.

[0034] This experiment further verified the accuracy of the detection model of the present invention: the high resistance of HL-1 yam to Alternaria alternata is stable, which is in stark contrast to the susceptibility of iron yam. This shows that the detection results of the model can effectively predict the disease resistance of yam varieties in the actual environment, and provide a direct and reliable scientific basis for the promotion and planting of HL-1 yam in areas with high incidence of Alternaria alternata, as well as for the formulation of targeted disease control measures.

[0035] Example 3

[0036] Differences in metabolomics between HL-1 and Dioscorea opposita leaves:

[0037] Based on the aforementioned results of resistance differences between HL-1 yam (a highly resistant variety) and iron yam (a susceptible variety) in vivo inoculated with Alternaria alternata, in order to reveal the potential mechanism of HL-1's high resistance characteristics from a metabolic perspective, further metabolomics analysis was conducted on healthy leaves of the two varieties to clarify the key differences in metabolite composition and provide molecular metabolic basis for disease resistance research.

[0038] Leaf sample processing strictly followed uniform standards to ensure experimental repeatability and accuracy: healthy HL-1 and Dioscorea opposita leaves from the same batch and with the same growth status as those used in the live inoculation experiment were selected, with 6 biological replicates for each. Fresh leaves were freeze-dried to constant weight to avoid interference from moisture in metabolite extraction. The freeze-dried leaves were ground into fine powder using a pulverizer and passed through a 200-mesh sieve to remove coarse fiber impurities. 0.1g of leaf powder was accurately weighed and transferred to a 2mL centrifuge tube, 1mL of 70% methanol aqueous solution was added, and the mixture was vortexed and then ultrasonically extracted at room temperature for 30min to fully release intracellular metabolites. After extraction, the centrifuge tube was placed in a high-speed centrifuge and centrifuged at 12000rpm at 4℃ for 10min to separate the supernatant from the residue. Finally, the mixture was filtered through a 0.22μM organic phase microporous membrane to remove small particulate impurities, and the filtrate was transferred to a sample vial for analysis.

[0039] Metabolomics analysis was performed using ultra-high-resolution electrospray time-of-flight mass spectrometry (UHMS) to obtain qualitative and quantitative data on leaf metabolites from both varieties. Principal component analysis (PCA) was performed on the obtained metabolomics data, and the results showed that the sample points of HL-1 and Dioscorea opposita were significantly separated on the PCA plot (see [link to PCA analysis]). Figure 4 (PCA score diagram of leaf metabolome of HL-1 and Dioscorea opposita): The contribution rate of the first principal component (PC1) reached 41.1%, and the contribution rate of the second principal component (PC2) reached 21.5%, which indicates that there are significant differences between the two varieties at the leaf metabolome level.

[0040] Further screening criteria were used, with |log2(FC)|≥2 and P-value <0.05. To focus on key differentially expressed metabolites, metabolites with large fold differences were selected and compiled into a detailed table of differentially expressed metabolites (Table 1).

[0041] Table 1 Key Differential Metabolites Between HL-1 and Dioscorea opposita Leaves

[0042]

[0043] In summary, this metabolomics analysis not only confirmed significant differences in the leaf metabolomes of HL-1 and Dioscorea opposita (clear separation in PCA plots), but also screened out a number of key differentially expressed metabolites that may be involved in the HL-1 disease resistance process. The high expression of these metabolites is presumed to be an important metabolic basis for the stable and high resistance of HL-1 to Alternaria alternata. These results not only validate the resistance differences observed in the aforementioned in vivo inoculation experiments at the molecular metabolic level, but also provide a clear research direction and key clues for further in-depth analysis of the molecular mechanisms of HL-1 disease resistance and the discovery of disease resistance-related genes.

[0044] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium, characterized by: Using natural leaves of yam varieties from different regions as the core raw material for the culture medium, yam leaf culture medium containing multiple leaf concentration gradients was prepared. The medium was then inoculated with native dominant pathogenic fungi of the target planting area and cultured at a constant temperature of 28℃. Within 6-7 days, the mycelial growth status of the pathogenic fungi and the phenotype of leaf lesions were observed to achieve large-scale disease resistance screening of yam varieties from different regions and ultimately determine whether the varieties to be introduced are suitable for the target planting area.

2. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium as described in claim 1, characterized in that: The yam leaf culture medium preparation process uses three leaf concentration gradients: 1g / 100mL, 3g / 100mL, and 5g / 100mL. This multi-gradient dosage design can adapt to the growth of pathogenic microorganisms with different nutritional requirements, and at the same time cover the detection scenarios of yam varieties with high, medium, and low resistance levels, ensuring the universality and accuracy of the detection results.

3. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium according to claim 1, characterized in that: The pathogenic fungi include *Dioscorea black spot fungus*. Alternaria alternata Anthracnose of yam Colletotrichum gloeosporioides Root rot fungus of yam Fusarium oxysporum Pathogenic fungi, etc.

4. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium as described in claim 1, characterized in that... The preparation process of the yam leaf culture medium is as follows: (1) Leaf collection and pretreatment: Select leaves from healthy plants of yam varieties to be screened or introduced, store them in an ice box at 4-6℃ with sterile absorbent paper inside and bring them back to the laboratory. Accurately weigh 1g, 3g and 5g of leaves to correspond to three concentration gradients respectively; (2) Leaf cleaning treatment: Rinse the weighed leaves gently with sterile water 3-4 times, changing the sterile water each time to remove surface dust and impurities. After rinsing, spread them flat on sterile absorbent paper to air dry naturally, or use sterile absorbent paper to absorb the surface moisture; (3) Preparation of leaf homogenate: Cut the dried leaves into 1-2cm square pieces, put them into a grinding mortar sterilized by high pressure steam at 121℃ for 20min, add 50mL of sterile distilled water in multiple batches, grind thoroughly until a fine paste homogenate without obvious tissue particles is formed, and transfer (3) Prepare leaf homogenate stock solutions with concentrations of 1g / 50mL, 3g / 50mL, and 5g / 50mL in sterile conical flasks; (4) Homogenization and constant temperature treatment: Place the homogenate stock solutions of each concentration gradient in a 60℃ constant temperature water bath, keep warm for 5 hours each time, repeat the water bath operation every 12 hours, and perform it 4-5 times in total to achieve sterilization and promote the release of substances in the leaves; (5) Final preparation and sterilization of culture medium: Prepare agar-water base solution at a ratio of 20g / L, sterilize it with high pressure steam at 121℃ for 20 minutes, and then cool it to 60-65℃. Mix it with leaf homogenate stock solutions of the same temperature at a ratio of 1:1 in a clean bench to prepare 100mL / part of yam leaf culture medium with concentrations of 1g / 100mL, 3g / 100mL, and 5g / 100mL. Dispense the medium into sterile culture dishes and let it cool and solidify naturally.

5. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium as described in claim 1, characterized in that: The optimal concentration of the yam leaf culture medium is 5 g / 100 mL. At this concentration, it is possible to clearly distinguish the differences in resistance to specific pathogenic fungi among the yam varieties to be introduced, meeting the precise requirements for variety resistance grading during large-scale screening. It can also simulate the natural nutritional environment of the leaves in the field of the introduction destination, so that the test results truly reflect the actual resistance level of the varieties to local pathogenic fungi, providing a reliable basis for determining whether the varieties are suitable for the target planting area.

6. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium according to claim 1, characterized in that: The criteria for large-scale screening of disease-resistant yam varieties are as follows: high-resistant varieties show no mycelial growth or lesions on the surface of the culture medium, with only the initial inoculation cake visible; moderately resistant varieties show a small amount of mycelial growth and no lesion spread; susceptible varieties show dense mycelia and lesions spreading along the inoculation point. For cross-regional introduction compatibility testing of yam, the dominant pathogen strain of the destination should be selected for inoculation, and the criteria are as follows: suitable varieties show high or moderate resistance; unsuitable varieties show susceptibility.

7. The model for cross-regional introduction and adaptability testing of yam and large-scale screening of disease-resistant varieties based on leaf culture medium according to any one of claims 1-6, characterized in that: The model is applicable to grassroots agricultural technology extension departments, yam planting cooperatives, and agricultural product testing laboratories. It is used for the initial screening of disease resistance in new yam varieties, the secondary screening of disease resistance in the main producing areas, and the prediction of disease resistance in yam varieties before cross-regional introduction.

8. The application of the model according to any one of claims 1-6 in the large-scale screening of disease-resistant yam varieties, characterized in that: For multiple candidate yam varieties, the disease resistance levels of each variety are tested and compared using a model to quickly screen out varieties with high resistance to the target pathogen, providing a basis for disease-resistant yam breeding and the promotion of high-quality varieties.

9. The application of the model according to any one of claims 1-6 in the detection of the adaptability of yam varieties, characterized in that: Collect dominant pathogen strains at the destination of introduction, and use models to detect the resistance of the introduced varieties to the strains. Predict the risk of disease occurrence of the introduced varieties in the local field, and avoid introduction failure and the emergence of new diseases caused by the interaction between foreign varieties and local pathogens.

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