Pinellia tuber soft rot fungus infection dynamic detection method

Abstract

The invention discloses a pinellia tuber soft rot fungus infection dynamic detection method, which comprises: applying a pH indicator to a to-be-detected surface of a plant tissue sample, and carrying out timing observation after the application; the color change range of the pH indicator at least covers pH 5.0-pH 7.0. The invention provides a dynamic monitoring system combining chromogenic reaction and pH value change, the method can be used for dynamic detection of soft rot fungus infection of pinellia tuber tubers, the method is simple and convenient in detection process operation, does not need complex equipment, is suitable for laboratory environment and can also be used for rapid field detection, the detection efficiency is improved, and the detection scene is expanded.

Description

Technical Field

[0001] This invention belongs to the field of plant pathology detection technology, specifically relating to a method for dynamic detection of soft rot pathogen infection in Pinellia tuber. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Pinellia ternata ( Pinellia ternata Pinellia ternata is an important traditional Chinese medicinal herb, but it is often severely affected by soft rot during cultivation, leading to tuber decay and reduced yield. Soft rot is mainly caused by pathogens such as Pectinobacterium, which cause softening, discoloration, and rotting of the tuber tissue after infection. Currently, there is no systematic detection method for the physiological and pathological changes during the infection process of Pinellia ternata tubers by soft rot pathogens.

[0004] Existing methods for early diagnosis of plant soft rot mostly rely on molecular detection or tissue isolation and culture, which are cumbersome, time-consuming, and require specialized equipment, making it difficult to achieve rapid and intuitive judgment in the field or on-site.

[0005] Therefore, developing a simple, intuitive, and reproducible method for detecting infection dynamics is of great significance for early warning of diseases in Pinellia ternata, breeding of disease-resistant varieties, and integrated prevention and control. Summary of the Invention

[0006] Currently, the detection of soft rot in Pinellia ternata relies heavily on field observation and traditional isolation and culture methods, which are time-consuming, have low sensitivity, and are difficult to dynamically monitor the infection process. To address these shortcomings, the present invention aims to provide a method for the dynamic detection of soft rot pathogens in Pinellia ternata tubers. This method is a rapid and visual detection method for soft rot in Pinellia ternata tubers based on changes in tissue pH.

[0007] The core of this invention lies in the discovery and utilization of a key physiological response: during the infection of plant tissues by soft rot fungi, a regular increase (alkalization) in the local microenvironment of the tissue occurs. By applying a specific pH indicator to the surface of the infected tissue and utilizing the correlation between its color change and pH value, a rapid and visual assessment of the disease's development can be achieved.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for dynamic detection of soft rot pathogen infection in Pinellia tuber, comprising the following steps: The pH indicator was applied to the surface of the plant tissue sample to be tested, and the results were observed after timing. The pH indicator has a color change range that covers at least pH 5.0 to pH 7.0, and more preferably pH 6.0 to pH 7.0.

[0009] In some embodiments of the present invention, the pH indicator is selected from bromocresol purple or bromothymol blue.

[0010] More preferably, the pH indicator is bromocresol purple, which is yellow when pH < 5.2 and purple when pH > 6.8, with the color changing in between.

[0011] In some embodiments of the present invention, the pH indicator is a bromocresol purple solution or a bromothymol blue solution, and the concentration of the pH indicator solution is 0.1%~2% w / v.

[0012] In some embodiments of the present invention, the pH indicator is in the form of test paper or spray, wherein the test paper is prepared by impregnating paper with bromocresol purple or bromothymol blue solution and then drying it.

[0013] Furthermore, the dynamic monitoring method also includes determining the degree of infection based on the color change of a pH indicator.

[0014] Furthermore, when the color is yellow or remains yellow, it indicates that the pH is below 6.0, the tissue has not undergone significant alkalization, and may be healthy or in the very early stages of infection.

[0015] A pale purple or purplish-red color indicates a pH value between approximately 6.0 and 6.5, suggesting that the tissue has begun to alkalize and that the soft rot bacteria have started to become active, indicating that the infection is in its early to middle stages.

[0016] A rapid change to deep purple indicates a pH level above approximately 6.5, significant alkalization of the tissue, and suggests active soft rot pathogens, indicating the disease has progressed to the middle to late stages. For enhanced contrast, photographs can be taken against white and black backgrounds.

[0017] Furthermore, the dynamic monitoring method also includes the correlation of pH value of the test tissue. The method involves: immediately after staining with the indicator, taking another tissue sample treated with the same method, grinding and centrifuging it, collecting the supernatant, and measuring its actual pH value using a precision pH meter. Through statistical analysis, a correlation model between "color grade - staining time - measured pH value" is established. The color change of the test surface after applying the indicator is observed, and the pH state of the tissue is determined based on the correlation between color and pH value; wherein, an increase in tissue pH value is positively correlated with the degree of soft rot infection.

[0018] In some embodiments of the present invention, the plant tissue sample is selected from healthy tissue or tissue suspected of being infected by soft rot fungi.

[0019] In some embodiments of the invention, the plant tissue sample is selected from intact tissue or processed sections or wounds. Preferably, it is cut plant tissue. It can be longitudinally or transversely cut.

[0020] The plant tissue in question is the tuber of Pinellia ternata.

[0021] Preferably, observation and recording are performed within 1-5 minutes after the indicator is applied.

[0022] Secondly, the present invention provides the following applications of the method for dynamic detection of soft rot pathogen infection in Pinellia tuber: (1) Disease research on artificially inoculated samples under laboratory conditions; (2) Initial screening of suspected disease samples in the field; (3) Preliminary evaluation of disease resistance screening of germplasm resources; (4) Non-destructive testing of diseases in stored tubers, fruits and vegetables.

[0023] The biological principle upon which this invention is based is as follows: When soft rot pathogens such as *Pectinobacterium* infect plant tissues, they secrete cell wall-degrading enzymes such as pectinase, which decompose the pectin in the plant cell walls to produce products such as galacturonic acid. Further metabolism leads to the accumulation of ammonia, causing a significant increase (alkalization) in the pH value of the microenvironment surrounding the infection point. This change is an early and key characteristic of disease development. After *Pinellia ternata* is infected by soft rot pathogens such as *Pectinobacterium*, the pH value of the tissue gradually changes over time. In their research, the inventors surprisingly discovered a fixed pattern between the tissue pH value and the infection time. Based on this change, the infection time and extent can be inferred. A pH indicator that is sensitive to color changes within this pH range can accurately and conveniently detect soft rot in *Pinellia ternata*.

[0024] The technical solution of this invention is further characterized by the fact that it does not require tissue culture and is not limited to laboratory research. Experiments can be conducted directly on field-grown Pinellia ternata tubers to detect disease incidence. Moreover, the reference indicators are not limited to color changes but also include pH prediction. This invention also uses a pH meter to detect pH and determine the pattern of these changes, thereby verifying the feasibility of using the pH indicator specified in this invention to detect disease incidence in Pinellia ternata tubers.

[0025] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: This invention provides a dynamic monitoring system combining a colorimetric reaction with a specific pH indicator and pH changes, which can be used for the dynamic detection of soft rot pathogen infection in Pinellia ternata tubers. This method has the following characteristics: 1. The testing process is simple to operate and requires no complicated equipment. It is suitable for both laboratory environments and rapid field testing, improving testing efficiency and expanding testing scenarios. 2. Based on the method of this invention, combined with colorimetric reaction and pH measurement, dynamic visualization and quantification of the infection process are achieved; 3. The detection method provided by this invention has good repeatability and stable results, and can provide technical support for disease-resistant breeding and disease control of Pinellia ternata. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 This is the process for preparing a suspension of Bacillus soft rot; Figure 2 This is a schematic diagram of the color change in the bromocresol purple color reaction; Figure 3 This is a colorimetric image of the supernatant after centrifugation of homogenized infected tissue using bromocresol purple solution. Figure 4 It is a curve showing the pH value change during the infection process. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] The core of the rapid visual detection method for plant soft rot provided by this invention lies in using a color indicator that is sensitive to alkaline pH to capture and visualize this physiological change.

[0030] In some embodiments of the present invention, a method for dynamic detection of soft rot pathogen infection in Pinellia ternata tubers is provided, comprising the following steps: (I) Selection and preparation of pH indicators The pH indicator used in this invention should cover a color change range from healthy plant tissue (typically acidic, pH approximately 5.5-6.0) to tissue severely alkalized by soft rot pathogens (pH can reach above 6.5). Because the pH change range is relatively small, only rising from about 6 to 6.8, ordinary pH test strips cannot clearly distinguish this range. Preferred indicators for this invention may be bromocresol purple (color change point pH 6.3, yellow to purple) or bromothymol blue (color change point pH 7.0, yellow to blue), etc.

[0031] The concentration of the indicator solution is typically 0.1% to 2% (w / v), and the solvent is water or low-concentration ethanol. Too low a concentration may result in indistinct color development, while too high a concentration may cause an excessively dark background or waste of resources. A typical preparation method is as follows: Weigh 0.1-1.0 g of bromocresol purple powder, dissolve it in 100 mL of distilled or deionized water, and stir until completely dissolved to obtain the working solution.

[0032] (II) Preparation of plant tissue samples The sample to be tested can be intact tissue or a processed section or wound. For clearer observation, it is generally recommended to cut the tissue to create a fresh, flat section to expose more of the internal tissue and the indicator reaction. For tuberous and root vegetables (such as Pinellia ternata and potato), longitudinal or transverse sections can be made. The sample surface should be clean and free from excessive external moisture.

[0033] (III) Testing Operation and Result Interpretation Using a dropper, pipette, or sprayer, apply a small amount (e.g., 5-50 µL) of the indicator working solution evenly to the surface of the tissue to be tested. Start timing and observation immediately after application.

[0034] Observe the color change over 1 to 10 minutes, especially 1 to 5 minutes. Color interpretation should be performed using a standard colorimetric chart or calibrated with a buffer solution of known pH. For example, when using bromocresol purple: Yellow or remaining yellow: indicates a pH of approximately 6.0, with no significant alkalization of the tissue, possibly indicating health or very early stages of infection.

[0035] A pale purple or purplish-red color indicates a pH value between approximately 6.0 and 6.5, suggesting that the tissue has begun to alkalize and that the soft rot bacteria have started to become active, indicating that the infection is in its early to middle stages.

[0036] A rapid change to deep purple indicates a pH level above approximately 6.5, significant alkalization of the tissue, and suggests active soft rot pathogens, indicating the disease has progressed to the middle to late stages. For enhanced contrast, photographs can be taken against white and black backgrounds.

[0037] (iv) Verification using combinations with other methods To establish more accurate quantitative or semi-quantitative standards, this method can be correlated with traditional pH meter measurements. Specifically, this could involve immediately after indicator staining, taking another tissue sample treated with the same method, grinding and centrifuging it, collecting the supernatant, and measuring its actual pH value using a precision pH meter. Through statistical analysis, a correlation model between "color grade - staining time - measured pH value" can be established, thereby making the results of this visualization method more objective and comparable.

[0038] (v) Application Scenarios Expansion The method of this invention is not only applicable to disease research on artificially inoculated samples under laboratory conditions, but after simple adaptation, it can also be used for: preliminary screening of suspected disease samples in the field, preliminary evaluation of disease resistance screening of germplasm resources, and non-destructive detection of diseases in stored tubers, fruits and vegetables.

[0039] When used in the field, the indicator can be formulated into portable test strips or sprays.

[0040] It should be noted that the above embodiments are merely illustrative examples of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. For those skilled in the art, any adjustments or substitutions made to the type and concentration of the indicator, the sample processing method, the observation time point, etc., without departing from the concept of the present invention, should be covered within the scope of protection of the present invention.

[0041] The following specific embodiment further illustrates the implementation process and technical effects of the present invention. This embodiment uses Pinellia ternata tubers as material, Pectinobacterium as the pathogen, and bromocresol purple as an indicator.

[0042] Example 1 A method and validation experiment for dynamic detection of soft rot pathogen infection in Pinellia ternata tubers includes the following steps: (1) Preparation of Pectinobacterium The preparation process of pectinobacterium is as follows Figure 1 As shown, pathogenic pectinobacteria ( ) were taken from storage at -80℃. Pectobacterium sp. The bacterial strain (Xu Jiawei. Identification and Control Research of Soft Rot Pathogen in Pinellia Producing Areas of my country [D]. Hubei University of Traditional Chinese Medicine, 2023. DOI:10.27134 / d.cnki.ghbzc.2023.000652.) was inoculated under aseptic conditions. A small amount of bacterial growth was picked up with an inoculation loop and inoculated into a 10 mL sterile centrifuge tube containing 4 mL of LB liquid medium. The inoculated centrifuge tube was placed in a constant temperature shaker at 28℃ and cultured at 180 rpm for 12 h (overnight) to allow the bacterial culture to reach the late logarithmic growth stage.

[0043] After culturing, centrifuge the bacterial suspension at 5000 rpm for 5 min and discard the supernatant. Add an appropriate amount of sterile ddH2O to the bacterial pellet and gently resuspend by pipetting. Measure the absorbance (OD) of the bacterial suspension at 600 nm using a UV spectrophotometer. 600 Continue to adjust the bacterial suspension concentration with sterile ddH2O until the OD value is reached. 600 The value is 0.8±0.05, which is the final concentration of the bacterial suspension for infection. Store it at 4℃ for later use (it is recommended to use it within 2 hours).

[0044] (2) Preparation of tuber samples Select healthy, uniformly sized Pinellia ternata ( Pinellia ternata Fifty fresh tubers were repeatedly rinsed with deionized water to remove surface dirt and sand, and then blotted dry with sterile filter paper. Each tuber was longitudinally sectioned along its long axis to ensure a smooth cut. All samples were placed in an inoculation tray lined with sterile, moistened filter paper, kept moist, and ready for use.

[0045] (3) Inoculation and infection Use a pipette to aspirate the OD prepared in step (1). 600 10 µL of a 0.8% bacterial suspension was evenly dropped onto the entire cross-section of the longitudinally sectioned tuber or applied to surface wounds. The inoculated tubers were then placed in a moistened tray and allowed to incubate at room temperature (25±1℃). Samples (including longitudinally sectioned and wound samples) were collected at five time points: 0 h, 3 h, 6 h, 9 h, and 12 h post-inoculation, with three biological replicates at each time point.

[0046] (4) Bromocresol purple staining Preparation of 1% bromocresol purple solution: Accurately weigh 0.2g of bromocresol purple powder, dissolve in 20mL of ultrapure water, mix until completely dissolved, the solution is orange-yellow. Immerse longitudinal sections of infected tubers (or longitudinal sections of wound samples) taken at various time points into this staining solution, and observe and photograph the color changes at 0min, 1min, 3min, and 5min after immersion. The staining process is photographed against both white and black backgrounds to enhance color contrast. Results are as follows. Figure 2 As shown, after 0 hours of infection with the soft rot fungus, healthy Pinellia ternata tubers appear yellow to pink after staining with bromocresol purple. After 3 hours of infection, the tubers gradually turn light purple. From 6 to 12 hours of infection, the color of the tubers gradually deepens to dark purple. With increasing infection severity, the bromocresol purple staining of the Pinellia ternata tubers deepens from yellow to dark purple.

[0047] (5) pH value test (for verification) Take 1 g of infected tuber tissue, add 5 mL of ultrapure water, and homogenize thoroughly under ice bath conditions. Centrifuge the homogenate at 4℃ and 5000 rpm for 5 min, collect 4 mL of the supernatant, and measure its pH value using a calibrated pH meter. Each sample was tested in triplicate, and the average value was taken as the pH result at that time point.

[0048] (6) Results and Data Analysis Observations revealed that 0-hour samples immediately turned yellow after staining and remained stable for 5 minutes. 3-hour samples began to change from yellow to light purple after 1 minute of staining, and became a more pronounced purple after 3 minutes. 6, 9, and 12-hour samples rapidly turned dark purple within 30 seconds of contact with the staining solution. Figure 3 As shown.

[0049] pH meter measurement results are as follows: Figure 4 As shown, the average pH values ​​of the tissue homogenates at 0 h, 3 h, 6 h, 9 h, and 12 h of infection were 6.09±0.025, 6.26±0.015, 6.4±0.02, 6.52±0.015, and 6.58±0.015, respectively. Analysis of variance indicated significant differences in pH values ​​at each time point (P<0.05).

[0050] Conclusion: The color change of bromocresol purple (yellow → light purple → dark purple) perfectly matched the upward trend of the measured pH value in the tissue, and the color change was clearly identifiable to the naked eye 3 hours after infection, much earlier than the appearance of visible rot symptoms (usually after 12 hours). This example successfully verified the effectiveness and sensitivity of this method for rapid and visual detection of the infection process of Pinellia ternata tuber soft rot.

[0051] Comparative Example 1 Phenol red was used as the pH indicator to replace bromocresol purple in Example 1 for dynamic detection of soft rot pathogen infection in Pinellia tuber; other operating methods were the same.

[0052] As a result, the soft rot pathogen infection was detected at 0 h, meaning that the healthy Pinellia tuber turned yellow after phenol red staining; at 3 h to 12 h, the Pinellia tuber still turned yellow after staining, making it impossible to detect the infection time in a timely manner. Phenol red was not sensitive enough as a pH indicator for the dynamic detection of soft rot pathogen infection in Pinellia tuber.

[0053] Comparative Example 2 Dynamic detection of soft rot pathogen infection in Pinellia ternata tubers was performed using commonly used pH test strips. One g of infected tuber tissue at five time points (0h, 3h, 6h, 9h, and 12h) was added to 5 mL of ultrapure water, thoroughly homogenized under ice bath conditions, centrifuged, and the supernatant was dropped onto pH test strips. After approximately 3 seconds, the result was compared with a colorimetric card. The results showed that the pH value at all five time points was 5 on the colorimetric card, making it impossible to distinguish pH changes at different time points.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for dynamic detection of soft rot pathogen infection in Pinellia ternata tubers, characterized in that, Includes the following steps: The pH indicator was applied to the surface of the plant tissue sample to be tested, and the results were observed after timing. The pH indicator has a color change range that covers at least pH 5.0 to pH 7.

0.

2. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber as described in claim 1, characterized in that, The pH indicator is selected from bromocresol purple or bromothymol blue, and the solution concentration of the pH indicator is 0.1%~2% w / v.

3. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber according to claim 1, characterized in that, The pH indicator is in the form of test paper or spray, and the test paper is prepared by soaking paper in bromocresol purple or bromothymol blue solution and then drying it.

4. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber according to claim 1, characterized in that, The dynamic monitoring method also includes determining the degree of infection based on the color change of a pH indicator.

5. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber as described in claim 1, characterized in that, When the color is yellow or remains yellow, it indicates that the pH is below 6.0, the tissue has not undergone significant alkalization, and may be healthy or in the very early stage of infection; A pale purple or purplish-red color indicates a pH value between approximately 6.0 and 6.5, suggesting that the tissue has begun to alkalize and that the soft rot bacteria have started to become active, indicating that the infection is in its early to middle stages. A rapid change to dark purple indicates a pH value higher than approximately 6.5, significant alkalization of the tissue, and suggests active soft rot pathogens, indicating that the disease has progressed to the middle or late stages.

6. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber according to claim 1, characterized in that, The dynamic monitoring method also includes the correlation of pH value of the tissue to be tested. The method is as follows: after staining with indicator, another tissue sample with the same treatment is taken immediately, ground, centrifuged and the supernatant is taken, and its actual pH value is measured with a precision pH meter; through statistical analysis, a correspondence model between color grade, infection time and measured pH value is established.

7. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber as described in claim 1, characterized in that, The plant tissue samples were selected from healthy tissue or tissue suspected of being infected by soft rot fungi.

8. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber as described in claim 1, characterized in that, The plant tissue sample is selected from intact tissue or processed sections or wounds; preferably, it is cut plant tissue; it is longitudinal or transverse.

9. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber according to claim 1, characterized in that, The plant tissue is the tuber of Pinellia ternata, and the soft rot pathogen is Bacillus pectinophilus.

10. The method for dynamic detection of soft rot pathogen infection in Pinellia tuber as described in any one of claims 1 to 9 is characterized in that, Including the following aspects: (1) Disease research on artificially inoculated samples under laboratory conditions; (2) Initial screening of suspected disease samples in the field; (3) Preliminary evaluation of disease resistance screening of germplasm resources; (4) Non-destructive testing of diseases in stored tubers, fruits and vegetables.

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