A method for rapidly evaluating effect of tobacco biocontrol agent based on root box phenotype system

Abstract

The application discloses a kind of tobacco biocontrol agent effect rapid evaluation method based on root box phenotype system, adopt the cultivation tobacco of slanting transparent root box, make root system stick wall growth;After bacteria are applied, nondestructive imaging is carried out to root system at multiple preset time points, and phenotype parameters such as total root length are extracted;Further, through root system growth rate (RGR), relative growth index (RPI) Cumulative Effect Value (CEV) Quantitative model is constructed, combined with disease specificity weight coefficient and grading standard, the objective determination of biocontrol effect grade is realized.Compared with prior art, the beneficial effects of the present application are: 7~21 days are completed in evaluation, without washing root, grading and other cumbersome operation, save 7~14 days in single experiment;Nondestructive imaging avoids the loss of root system caused by traditional destructive sampling, can accurately capture subtle differences;Extract multiple morphological parameters, realize the time sequence tracking of root system development process, provide basis for action mechanism analysis;Unify the sampling process of fungicide application, improve comparability.

Description

Technical Field

[0001] This invention relates to the field of tobacco plant protection and microbial technology, specifically to a rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems. Background Technology

[0002] Tobacco (Nicotiana acum L.) is an important economic crop in my country. Root-knot nematode disease, bacterial wilt (Ralstonia solanacearum), and black shank (Phytophthora parasitica) are the three major diseases of tobacco production, causing serious damage and huge economic losses. At present, biological control has become an important development direction for tobacco disease control due to its environmental friendliness and strong sustainability. In the research and development and application of biocontrol agents, effect evaluation is a key link in screening superior strains, optimizing compound schemes, and guiding field application. However, the existing biocontrol agent effect evaluation methods have the following shortcomings: (1) The evaluation index is single and lacks dynamic observation. At present, the effect evaluation of tobacco biocontrol agents mainly relies on traditional agronomic trait measurement (plant height, stem diameter, number of leaves) or destructive sampling to measure root parameters (root length, root dry weight), and manual calculation of disease index. The former is difficult to reflect the underground growth status, while the latter has a long experimental cycle, is cumbersome to operate, time-consuming and laborious, and cannot conduct continuous dynamic observation of the same plant. (2) Existing phenotypic monitoring devices are mainly designed for breeding and have not been specifically optimized for biocontrol screening and evaluation. In recent years, plant phenotypic monitoring devices based on root boxes have been publicly reported. For example, Chinese invention patent CN117433449B discloses a method for joint identification of aboveground and underground plant phenotypic characteristics based on root boxes, which achieves real-time non-destructive imaging of crop roots through an RGB camera and a darkroom environment. However, this device provides a general plant phenotypic identification platform and has not established standardized inoculant application schemes, sampling time windows, and effect quantification evaluation models for the specific application scenario of tobacco biocontrol agent effect evaluation, making it difficult to directly use for standardized effect evaluation of biocontrol agents. (3) Effect judgment is highly subjective and lacks a standardized scoring system. Existing biocontrol effect evaluations mostly rely on the percentage of control efficacy or empirical visual inspection, lacking objective evaluation standards based on root system quantitative data.

[0003] In summary, there is an urgent need to develop a method that can fully utilize non-destructive root phenotypic monitoring technology, be specifically optimized for tobacco biocontrol agents, and achieve standardized quantitative evaluation. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for rapid evaluation of the efficacy of tobacco biocontrol agents based on root box phenotype systems, comprising the following steps:

[0006] S1: Tobacco plants are cultivated using a root box device, which is set at an angle and has a transparent wall surface for root growth and imaging.

[0007] S2: Apply the tested biocontrol agent to tobacco plants and set up a blank control group without applying the agent;

[0008] S3: At multiple preset time points before and after the application of the fungicide, non-destructive image acquisition of tobacco roots at the transparent wall of the root box is performed using an imaging device to obtain a root image dataset at each time point.

[0009] S4: Extract root phenotypic parameters from images at each time point using image processing algorithms. The root phenotypic parameters include at least the total root length.

[0010] S5: Calculate the root growth rate (RGR) using the following formula:

[0011]

[0012] Where P is the root phenotypic parameter, and Δt is the time interval between two adjacent samplings;

[0013] S6: Calculate the relative root growth index (RPI) at each time point using the following formula:

[0014]

[0015] S7: Calculate the cumulative effect value (CEV) using the following formula:

[0016]

[0017] Where Wi is the weighting coefficient at each time point, and ;

[0018] S8: Based on the CEV value and in accordance with the preset grading standard, determine the biocontrol effect level of the biocontrol agent on tobacco.

[0019] Preferably, the biocontrol agent in S2 is applied by root irrigation, with a concentration of 1×107~1×108 CFU / mL and a dosage of 500mL per plant.

[0020] Preferably, the biocontrol agent in S2 is applied by root irrigation on the 3rd day after tobacco transplanting.

[0021] Preferably, the preset time point in S3 varies depending on the type of disease being tested, specifically as follows:

[0022] For bacterial wilt: the day after pathogen inoculation is designated as T0, the 3rd day as T1, the 7th day as T2, and the 14th day as T3;

[0023] For black shank disease: the day after pathogen inoculation is designated as T0, the 3rd day as T1, the 5th day as T2, and the 7th day as T3;

[0024] For root-knot nematode disease: the day after pathogen inoculation is designated as T0, the 7th day as T1, the 14th day as T2, and the 21st day as T3.

[0025] Preferably, the weighting coefficients in S7 are set as follows: W1=0.2, W2=0.35, W3=0.45.

[0026] Preferably, the preset grading standard in S8 is:

[0027] For bacterial wilt and black shank: CEV ≥ 50% is considered highly effective, 30% ≤ CEV < 50% is considered moderately effective, and CEV < 30% is considered ineffective.

[0028] For root-knot nematode disease: CEV≥40% is considered highly effective, 20%≤CEV<40% is considered moderately effective, and CEV<20% is considered ineffective.

[0029] Preferably, the root phenotypic parameters in S4 also include one or more of the following: root projected area, number of root tips, and number of root branches.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1. Rapid Screening of Biocontrol Strains. Based on the obtained time-series tracking images and detailed root morphology analysis, the effectiveness of biocontrol strains can be evaluated within 7-21 days, assisting in the screening of the most effective strains from a large number of candidate strains. Compared with existing potted plant screening, a single experiment saves 7-14 days of experimental cycle, greatly improving the efficiency of biocontrol agent screening. This method indirectly assesses the damage and control effect of biocontrol strains on bacterial wilt, black shank, and root-knot nematode diseases by observing root growth, saving time spent on disease index classification and calculation. In addition, this method eliminates the need for root washing, drying, and weighing of potted plants during growth data acquisition, reducing the time required for sample processing.

[0032] 2. Improve the accuracy of biocontrol strain evaluation. Currently, widely used pot experiments can only assess biocontrol strains through macroscopic indicators such as plant fresh weight and dry weight. This method relies solely on destructive sampling to collect data after the experiment. Because tobacco plants have numerous fine and weak fibrous roots, significant root loss often occurs during soil separation, affecting the accuracy of the results. This method eliminates the need for root separation from the soil, extracting various morphological parameters from root images to accurately capture subtle differences in biocontrol bacteria and reduce operational errors.

[0033] 3. Multidimensional Analysis of Root Growth. This method can extract various morphological parameters from images, such as total root length, root projected area, number of root tips, and number of root branches, which helps to evaluate root growth from multiple dimensions. Furthermore, by taking multiple root photographs at different time points, this method allows for the tracking of tobacco root development across different time dimensions, thus helping to discover the mechanism of action of biocontrol bacteria and providing guidance for the efficient application of subsequent biocontrol strains and the development of compound inoculants.

[0034] 4. Standardized operating procedures and evaluation system. Standardized application protocols for microbial agents (timing, concentration, dosage, and application location) and time sampling protocols (T0, T1, T2, T3) have been established, which solves the problem of inconsistent operation of existing evaluation methods and improves the comparability and repeatability of evaluation results.

[0035] 5. Quantitative Evaluation System. A quantitative evaluation model (RGR, RPI, CEV) based on root phenotypic time series data was proposed, and a standardized criterion for judging the effect level (high efficiency / medium efficiency / low efficiency) was established, overcoming the shortcomings of traditional methods that rely on subjective experience judgment. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the root box device and imaging system in this invention;

[0037] Figure 2 These are morphological images of tobacco roots in the treatment and control groups in this embodiment of the invention. AC: Root morphology; DF: Root skeleton after image segmentation and extraction. A, D: Inoculated with Bacillus amyloliquefaciens; B, E: Control; C, F: Inoculated with Brevibacillus brevis.

[0038] Figure 3 This is a graph showing the change of root growth rate (RGR) over time in an embodiment of the present invention.

[0039] In the picture: 1. Flat root box; 2. Angle steel frame; 3. Imaging camera. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Please see Figure 1-3 Figure 1 shows that the present invention provides a technical solution: a method for rapid evaluation of the efficacy of tobacco biocontrol agents based on root box phenotype systems, comprising the following steps.

[0042] S1: Root box cultivation of tobacco plants

[0043] The root box phenotypic monitoring device disclosed in Chinese invention patent CN117433449B is used. Tobacco plants are cultivated using the root box device, which is tilted and has a transparent wall for root adhesion and imaging. The root box device includes: a tilted flat root box 1, a support 2 for supporting the root box, and a screen, an imaging camera 3, and a light source system positioned around the root box.

[0044] S2: Standardized application of biocontrol agents

[0045] The tested biocontrol agent was applied to tobacco plants, and a blank control group was set up without the application of the agent.

[0046] The application concentration of the biocontrol agent is 1×10⁻⁶. 7 ~1×10 8 The concentration of CFU / mL was 500mL per plant. Application was by root drenching, where the fermentation liquid was poured over the base of the stem within a 5cm radius. The biocontrol agent was applied by root drenching on the 3rd day after tobacco transplanting. The control group received an equal volume of sterile water. Five replicates were set up for both the control and treatment groups, and pathogen inoculation was performed according to method S3.

[0047] S3: Pathogen inoculation

[0048] Bacterial wilt, black shank, and root-knot nematode are important diseases of tobacco, and these three pathogens are mainly used as testing targets for biocontrol strains in production. Inoculation is performed on the same day as the application of the biocontrol strain. The inoculation methods for these three pathogens are as follows:

[0049] Inoculation with *Ralstonia solanacearum*: A non-invasive root drenching method was used. The *Ralstonia solanacearum* pathogen was incubated on NA medium at 28°C for 48 hours, and then adjusted to 10⁻¹⁰ with sterile water. 8 CFU / mL. Apply to the soil at the base of the stem at a 10% water-to-soil ratio.

[0050] Inoculation with *Tobacco Black Shank* fungus: The stem base injection method is used. After conidia are produced, the zoospore count is adjusted to 10. 5To obtain 10–20 μL of zoospore suspension, insert a syringe into the cortex at the base of the tobacco stem.

[0051] Tobacco root-knot nematode inoculation: Freshly collected root-knot nematode eggs are incubated indoors, and second-instar larvae are collected. 500–1000 second-instar larvae are inoculated into the soil at the base of each tobacco seedling.

[0052] S4: Temporal acquisition of root phenotypic images

[0053] Non-destructive image acquisition of tobacco roots at the transparent wall of the root box was performed using imaging equipment to obtain a dataset of root images at various time points.

[0054] The preset time points vary depending on the type of disease being tested. For bacterial wilt, the test times are: day 1 (T0), day 3 (T1), day 7 (T2), and day 14 (T3) after pathogen inoculation. For black shank, the test times are: day 1 (T0), day 3 (T1), day 5 (T2), and day 7 (T3) after pathogen inoculation. For root-knot nematode disease, the test times are: day 1 (T0), day 7 (T1), day 14 (T2), and day 21 (T3) after pathogen inoculation.

[0055] S5: Extraction of root phenotypic parameters

[0056] The root phenotypic parameters P of the images at each time point are extracted using image processing algorithms. The parameters include total root length, root projected area, number of root tips, and number of root branches.

[0057] Image processing algorithms include image segmentation, skeletonization, and topology analysis algorithms.

[0058] S6: Calculation of Root Growth Rate (RGR)

[0059] Based on the root phenotypic parameters at each time point, the root growth rate (RGR) is calculated using the following formula:

[0060]

[0061] Where P is the selected root phenotypic parameter (such as total root length), and Δt is the time interval (days) between two adjacent samplings.

[0062] S7: Calculation of the Relative Root Growth Index (RPI)

[0063] Calculate the relative root growth index (RPI) at each time point using the following formula:

[0064]

[0065] Where P processes, T i The root phenotypic parameter values ​​of the treatment group at time Ti, P control, T iThe values ​​represent the root phenotypic parameters of the control group at time point Ti.

[0066] S8: Calculation of the Cumulative Root Vigor Effect Value (CEV)

[0067] Calculate the cumulative effect value (CEV) using the following formula:

[0068]

[0069] Where Wi is the weighting coefficient at each time point.

[0070] Furthermore, the weighting coefficients are W1=0.2, W2=0.35, and W3=0.45.

[0071] S9: Effect Level Determination

[0072] Based on the CEV value and in accordance with the preset grading standards, the level of the biocontrol agent's disease prevention and growth promotion effect on tobacco is determined.

[0073] The grading criteria are as follows: bacterial wilt and black shank disease with a CEV ≥ 50% are considered highly effective, 30% ≤ CEV < 50% are considered moderately effective, and CEV < 30% are considered ineffective.

[0074] After root-knot nematodes develop the disease, the root system will swell, increasing the total root length during analysis. This offsets some of the increase in root system caused by the growth promotion. Therefore, when evaluating root-knot nematodes, CEV≥40% is considered highly effective, 20%≤CEV<40% is considered moderately effective, and CEV<20% is considered ineffective.

[0075] Example 1

[0076] This embodiment provides a specific implementation method for a rapid evaluation method of the efficacy of tobacco biocontrol agents based on root box phenotype systems.

[0077] 1. Preparation of the root box device

[0078] This invention employs the root box phenotypic monitoring device disclosed in Chinese invention patent CN117433449B. The device includes: a frame (a frame structure, measuring 210cm × 190cm × 90cm, with black light-blocking cloth covering all four sides to form a monitoring chamber); a root box (90cm × 50cm × 1.5cm, made of acrylic sheet, installed at a 45° angle within the monitoring chamber); two RGB cameras (resolution 5496 × 3672), respectively located on the upper and lower levels of the monitoring chamber, for capturing images of the underground (root system) and aboveground parts; a light source system (5 visual lights) for providing uniform illumination; and a PC connected to the RGB cameras via MVS software for real-time display of crop images. This invention only uses root system image acquisition and analysis, and does not involve the aboveground parts.

[0079] 2. Cultivation of Tobacco Plants

[0080] Fill the root boxes with potting soil and supply water to the soil through the top of the boxes. Sow tobacco seeds in the root boxes, ensuring adequate water and nutrient supply. After germination, provide adequate light using fluorescent lamps. Once the above-ground parts and roots of the tobacco have grown to a suitable stage, prepare for inoculum treatment and phenotypic data collection.

[0081] 3. Preparation and application of microbial agents

[0082] The activated biocontrol bacterial strains were inoculated into liquid culture media and fermented under optimal fermentation conditions to obtain fermentation broths. Both fermentation broths were then diluted with sterile water to a final concentration of 1×10⁻⁶. 7 ~1×10 8 CFU / mL, for later use. The first root drenching application was performed on the 3rd day after tobacco transplanting, with 500 mL of bacterial suspension per plant. The control group received an equal volume of sterile water.

[0083] 4. Time-series acquisition of root phenotypic images

[0084] The preset time points vary depending on the type of disease being tested. For bacterial wilt, the test times are the day after inoculation (T0), day 3 (T1), day 7 (T2), and day 14 (T3). For black shank, the test times are the day after inoculation (T0), day 3 (T1), day 5 (T2), and day 7 (T3). For root-knot nematode disease, the test times are the day after inoculation (T0), day 7 (T1), day 14 (T2), and day 21 (T3). The following procedures are performed for each test:

[0085] (1) Place the root box in the monitoring room, turn on the light source and adjust it to a suitable brightness, close the light-shielding sliding door and eliminate external light source interference.

[0086] (2) Connect the RGB camera to the PC using MVS software, acquire the root system image and store it.

[0087] (3) Repeatedly scan the same plant at each time point to ensure consistent imaging conditions.

[0088] 5. Image Processing and Parameter Extraction

[0089] (1) The obtained root system image is labeled and cropped, and the root system segmentation is performed by training the Segformer neural network model to obtain the root system segmentation map.

[0090] (2) Use Python to process the segmentation graph into a skeleton, extract the root skeleton based on the 8-connected mode, and retain the root length information.

[0091] (3) Extract root phenotypic parameters at each time point: total root length, root projected area, number of root tips, and number of root branches.

[0092] The root length is calculated using the following formula:

[0093]

[0094] In the formula, l represents the root system length, K is the conversion factor between pixels and actual size, N1 is the number of pixels connected diagonally, N2 is the number of pixels connected horizontally and vertically, and r1 and r2 are the compensation lengths.

[0095] The root surface area is calculated using the following formula:

[0096]

[0097] In the formula, S_r is the root system surface area, and P_r is the total number of pixels in the root system.

[0098] 6. Calculation of Effect Evaluation

[0099] (1) Calculate the root growth rate (RGR):

[0100] Taking the total root length in the bacterial wilt biocontrol agent experiment as an example,

[0101] (sky),

[0102] ,

[0103] (sky).

[0104] (2) Calculate the relative root growth index (RPI):

[0105]

[0106] Similarly, calculate RPI_T2 and RPI_T3.

[0107] (3) Calculate the cumulative effect value of root activity (CEV):

[0108]

[0109] (4) Evaluation of efficacy level: In the evaluation of bacterial wilt and black shank, CEV≥50% is considered highly effective, 30%≤CEV<50% is considered moderately effective, and CEV<30% is considered poorly effective. In the evaluation of root-knot nematodes, CEV≥40% is considered highly effective, 20%≤CEV<40% is considered moderately effective, and CEV<20% is considered poorly effective.

[0110] Example 2

[0111] This example demonstrates a practical evaluation of the effectiveness of Bacillus brevis in controlling tobacco root-knot nematodes using this method.

[0112] 1. Experimental Design

[0113] Three treatment groups were set up: (1) a water control group (CK); (2) Bacillus shortness of breath inoculation, 1×10 7 (CFU / mL). Simultaneously, 500 southern root-knot nematodes were inoculated, with 5 biological replicates for each treatment.

[0114] 2. Experimental Procedure

[0115] Tobacco cultivation, microbial agent application, root system image time-series acquisition, and parameter extraction were performed according to the method in Example 1. Total root length data were recorded at each time point from T0 to T3.

[0116] 3. Experimental Results

[0117]

[0118] 4. Effect Evaluation

[0119]

[0120] The compound microbial agent was determined to be highly effective in promoting tobacco growth.

[0121] Comparative Example 1: Traditional Destructive Sampling Method for Potted Plants

[0122] Methods: Tobacco plants were treated with the same concentration of Bacillus shortness of breath. On day 21 after nematode inoculation (the endpoint only), the entire plant was pulled up, the roots were washed, the root length was measured, and the control efficacy was calculated.

[0123] Disadvantages: It cannot capture the dynamic growth rate (RGR) at the T1 and T2 stages; single sampling is prone to errors due to individual differences; damage to the root system makes it impossible to continuously track the same plant.

[0124] Comparative Example 2: Static Evaluation Method Based on a Single Indicator

[0125] Method: The control effect is calculated using only the total root length at time T3, without introducing weighting coefficients and cumulative effect value (CEV).

[0126] Defects: The key colonization and resistance-inducing functions of biocontrol bacteria in the early stages of disease occurrence (T1, T2) are ignored, resulting in a one-sided evaluation result that cannot truly reflect the sustained protective ability of the inoculant.

[0127] This method applies tilted root box non-destructive imaging technology to evaluate the efficacy of tobacco biocontrol agents, solving the problem that traditional methods require plant destruction for root observation. Different sampling time points are set for the different pathological processes of bacterial wilt, black shank, and root-knot nematode, making the evaluation more closely aligned with disease occurrence patterns. The introduction of time-weighted coefficients (W1=0.2, W2=0.35, W3=0.45) highlights the contribution of the agent's effect in the mid-to-late stages of disease progression, reflecting the agent's sustained protective ability better than simple averages. Different CEV thresholds are set for different types of diseases, improving the specificity and accuracy of the evaluation results. In summary, this method utilizes a tilted root box device to sequentially achieve non-destructive temporal imaging of the root system, extract multi-dimensional root phenotypic parameters, construct a cumulative effect value (CEV) evaluation system with time weighting, and achieve graded determination of different disease targets. Compared with traditional destructive sampling and single-endpoint evaluation, this method has significant advantages in being dynamic, non-destructive, high-throughput, and quantitative.

[0128] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems, characterized in that: Includes the following steps: S1: Tobacco plants are cultivated using a root box device, which is set at an angle and has a transparent wall surface for root growth and imaging. S2: Apply the tested biocontrol agent to tobacco plants and set up a blank control group without applying the agent; S3: At multiple preset time points before and after the application of the fungicide, non-destructive image acquisition of tobacco roots at the transparent wall of the root box is performed using an imaging device to obtain a root image dataset at each time point. S4: Extract root phenotypic parameters from images at each time point using image processing algorithms. The root phenotypic parameters include at least the total root length. S5: Calculate the root growth rate (RGR) using the following formula: Where P is the root phenotypic parameter, and Δt is the time interval between two adjacent samplings; S6: Calculate the relative root growth index (RPI) at each time point using the following formula: S7: Calculate the cumulative effect value (CEV) using the following formula: Where Wi is the weighting coefficient at each time point, and ; S8: Based on the CEV value and in accordance with the preset grading standard, determine the biocontrol effect level of the biocontrol agent on tobacco.

2. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The biocontrol agent in S2 is applied by root irrigation at a concentration of 1×10⁷~1×10⁸ CFU / mL, with a dosage of 500 mL per plant.

3. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The timing for applying the biocontrol agent in S2 is as follows: root irrigation on the 3rd day after tobacco transplanting.

4. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The preset time points in S3 vary depending on the type of disease being tested, specifically: For bacterial wilt: the day after pathogen inoculation is designated as T0, the 3rd day as T1, the 7th day as T2, and the 14th day as T3; For black shank disease: the day after pathogen inoculation is designated as T0, the 3rd day as T1, the 5th day as T2, and the 7th day as T3; For root-knot nematode disease: the day after pathogen inoculation is designated as T0, the 7th day as T1, the 14th day as T2, and the 21st day as T3.

5. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The weighting coefficients in S7 are set as follows: W1=0.2, W2=0.35, W3=0.

45.

6. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The preset grading standard in S8 is as follows: For bacterial wilt and black shank: CEV ≥ 50% is considered highly effective, 30% ≤ CEV < 50% is considered moderately effective, and CEV < 30% is considered ineffective. For root-knot nematode disease: CEV≥40% is considered highly effective, 20%≤CEV<40% is considered moderately effective, and CEV<20% is considered ineffective.

7. The rapid evaluation method for the efficacy of tobacco biocontrol agents based on root box phenotype systems according to claim 1, characterized in that: The root phenotypic parameters in S4 also include one or more of the following: root projected area, number of root tips, and number of root branches.

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