Accurate evaluation method for tomato rootstock resistance to elephantopus scaber root-knot nematode

Abstract

The invention belongs to the field of tomato seedling cultivation, and particularly relates to a method for accurately evaluating the level of tomato rootstock resistance to elephantopus scaber root-knot nematode. On the basis of testing indexes such as a disease index, a root knot index, an egg index, a propagation coefficient and a soil microorganism species number of tomato rootstocks, a rootstock growth index is combined, an index (Simpson index) algorithm of species diversity in a soil microorganism community is creatively utilized, evaluation parameters are optimized, and the evaluation accuracy is improved. The measured indexes are adopted to calculate membership functions and soil microbial diversity indexes to evaluate the resistance of the tomato rootstock to be detected to the root-knot nematode, so that the rootstock variety for resisting the tomato rootstock to the root-knot nematode can be better and comprehensively clustered and analyzed, and the resistance of the tomato rootstock to the tomato rootstock to the root-knot nematode can be accurately evaluated; and a novel method is provided for screening the tomato rootstock for resisting the rhus chinensis root-knot nematode.

Description

Technical Field

[0001] This invention belongs to the field of tomato seedling cultivation, specifically involving a method for accurately evaluating the resistance level of tomato rootstock to weevils and root-knot nematodes. Background Technology

[0002] Meloidogyne enterolobii, a newly discovered tropical root-knot nematode, has attracted widespread attention in recent years. It is distributed across tropical and subtropical regions of Asia, Africa, Europe, and North America. This nematode has an extremely wide host range, including various vegetables in the legume, solanaceae, and cucurbitaceae families, as well as important food and economic crops such as soybeans, corn, cotton, tobacco, guava, and lychee. Unlike major root-knot nematode species such as *M. incognita* and *M. javanica*, *M. enterolobii* can overcome resistance mediated by multiple resistance genes, including Mh (potato), Mi-1 (tomato), N (pepper), and Mir1 (soybean), and parasitize and reproduce on resistant tomatoes, peppers, and cowpeas, causing yield losses of over 65%. Therefore, it is listed internationally as one of the most harmful plant pathogenic nematodes, and is included in the A2 alert list by European and Mediterranean plant protection organizations.

[0003] In response to the damage caused by the bean root-knot nematode, research has been conducted on various aspects, including breeding disease-resistant varieties and chemical control. Grafting is one of the most economical, effective, and convenient measures. Therefore, screening for resistant rootstock varieties is crucial. Based on the characteristics of the bean root-knot nematode (which can overcome resistance mediated by multiple resistance genes such as Mh (potato), Mi-1 (tomato), N (pepper), and Mir1 (soybean) and parasitize and reproduce on resistant tomatoes, peppers, and cowpeas), more parameters should be considered when screening and applying bean root-knot nematode-resistant rootstocks to accurately reflect the resistance level of the rootstock itself.

[0004] Whether grafted tomato seedlings in production can control root-knot nematodes depends primarily on the resistance level of the tomato rootstock. However, current methods that classify resistance solely using disease resistance indicators or root knot indices are prone to bias due to differing classification standards and cannot objectively evaluate resistance levels. Therefore, developing a precise evaluation method for *Heliotropium indicum* root-knot nematodes is of great significance for screening rootstocks resistant to *Heliotropium indicum*. Summary of the Invention

[0005] This invention provides a method for accurately evaluating the resistance level of tomato rootstock to weevils and root-knot nematodes.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for accurately evaluating the resistance level of tomato rootstock to weevils and root-knot nematodes includes the following steps:

[0008] (1) Cultivate tomato rootstock seedlings to be tested. When the seedlings have 5 true leaves, select 10 seedlings of uniform growth for each rootstock variety and inoculate them with Elephant Ear Root-knot Nematode. Each rootstock is set as a control treatment without nematode.

[0009] (2) Fifty days after inoculation, the growth indicators, disease resistance indicators, reproduction coefficient and soil microbial species of each plant were measured.

[0010] (3) The resistance of the rootstock of the test rootstock to the weevils root-knot nematode was evaluated using the measured indicators.

[0011] Furthermore, in step (2), the growth indicators include: plant height, stem diameter, above-ground fresh weight and root fresh weight; the disease resistance indicators include: root knot index, egg index, disease index and reproduction coefficient.

[0012] Furthermore, in step (3), the membership function and / or soil microbial diversity index are calculated using the measured indicators; the larger the membership function value, the stronger the resistance of the tomato rootstock to be tested to the root-knot nematode; the smaller the soil microbial diversity index value, the stronger the resistance of the tomato rootstock to be tested to the root-knot nematode.

[0013] Furthermore, the membership function calculation formula is: X(μ)=1-(X-Xmin) / (Xmax-Xmin); where X is the measured value of a certain index of the grafted rootstock after 50 days of inoculation, Xmax is the maximum value of a certain index of all rootstocks, and Xmin is the minimum value of a certain index of all rootstocks.

[0014] Furthermore, the soil microbial diversity index mentioned is the Simpson index, calculated using the formula: D = 1 - ∑(ni / N) 2 D represents the Simpson index, ni represents the number of individuals of the i-th species, and N represents the sum of the number of individuals of all species. The smaller the Simpson index, the higher the species diversity of the sample, especially the richness and diversity of soil-specific dominant bacteria, which will inhibit the soil environment suitable for the growth of soil-borne diseases (root-knot nematodes), thereby improving the plant's resistance to soil-borne diseases.

[0015] Furthermore, the method for determining the soil microbial diversity index is as follows: Root soil is collected and stored in an ultra-low temperature freezer at -80℃ for later use. Metagenomics and high-throughput sequencing methods are used to determine the sequence of the 16S rRNA gene of soil microorganisms, and the number of soil microbial species is counted through gene alignment analysis. Preferably, the soil microorganisms are bacteria or fungi. More preferably, the soil microorganisms are bacteria.

[0016] Furthermore, the methods for determining the growth indicators and disease resistance indicators are as follows: the height of the rootstock, stem diameter, fresh weight of the above-ground parts and fresh weight of the roots are measured using a ruler, vernier caliper and electronic scale respectively; the roots are collected, washed, dried and weighed; and the root knot index, egg index, disease index and propagation coefficient are calculated.

[0017] The root knots of each individual plant are counted, and the root knot index (GI) = number of root knots per plant / fresh weight of roots per plant;

[0018] The number of eggs in each individual plant was counted, and the egg index (EI) was calculated as: number of eggs per plant / fresh weight of roots per plant.

[0019] The disease index (DI) for each variety is calculated based on the disease incidence of individual plants. DI = ∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level) × 100;

[0020] Soak the chopped roots in a 1.2% sodium hypochlorite solution at 200 r·min. -1 Shake for 10 minutes, count the eggs under a microscope, and calculate the reproduction number (RF) as the number of eggs per plant / the initial inoculation amount per plant.

[0021] A method for screening tomato rootstocks resistant to weevils and root-knot nematodes includes the following steps:

[0022] (1) Using the precise evaluation method described above, calculate the membership function and / or soil microbial diversity index to evaluate the resistance level of the tomato rootstock to the weevils root-knot nematode;

[0023] (2) Screen the resistance of the tomato rootstock to weevils to the root-knot nematode based on the calculated membership function and / or soil microbial diversity index.

[0024] Furthermore, in step (2), the larger the membership function value, the stronger the resistance of the tomato rootstock to be tested to the root-knot nematode; the smaller the soil microbial diversity index value, the stronger the resistance of the tomato rootstock to be tested to the root-knot nematode.

[0025] Furthermore, the soil microorganisms are bacteria or fungi. Preferably, the soil microorganisms are bacteria.

[0026] The beneficial effects of this invention are:

[0027] This invention, based on tests of tomato rootstocks for disease index, root knot index, egg index, and reproduction coefficient, combines rootstock growth indicators with a creative algorithm utilizing the Simpson index (a species diversity index in soil microbial communities) to optimize evaluation parameters. It uses the measured indicators to calculate membership functions and soil microbial diversity indices to evaluate the resistance of test rootstocks to *Heliotropium indicum* (a type of bean nematode). This allows for better comprehensive cluster analysis to identify rootstock varieties resistant to *Heliotropium indicum*, and provides a precise evaluation of tomato rootstock resistance to *Heliotropium indicum*, offering a new method for screening tomato rootstocks resistant to *Heliotropium indicum*. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Cluster analysis for rootstock resistance.

[0030] Figure 2 Simpson's index for each rootstock. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] I. Experimental Methods

[0034] Once the seedlings have developed 5 true leaves, 10 uniformly growing seedlings from each rootstock variety are selected for inoculation. Mature root-knot nematode eggs are collected from the roots of infected water spinach, placed in clean water to hatch into second-instar larvae (J2), and then inoculated onto the roots of the seedlings at a rate of 2,000 larvae per seedling. A control treatment without nematodes is included for each rootstock.

[0035] II. Project Measurement

[0036] Fifty days after inoculation, root soil samples were collected from the plants to determine the species diversity of the soil microbial community. The roots were collected, washed, dried, and weighed for various index measurements. Root knot index, egg cluster index, disease index, and reproduction coefficient were calculated.

[0037] (1) Growth indicators

[0038] Fifty days after nematode inoculation, six nematode-inoculated rootstocks were randomly selected. Plant height, stem diameter, above-ground fresh weight, and root fresh weight were measured using a ruler, calipers, and electronic scale. For each grafted rootstock variety, the average value of two plants was used as one replicate plot. The relative growth of rootstock seedlings was calculated as follows: Relative growth of rootstock seedlings (%) = (Measurement value in the root-knot nematode-inoculated plot / Measurement value in the control plot) × 100%. (The relative growth of rootstock seedlings refers to the change in growth rate and amount of rootstocks treated with and without nematode inoculation over a certain period, providing a scientific and objective evaluation of the impact of root-knot nematodes on rootstock growth.)

[0039] (2) Disease resistance indicators

[0040] While measuring the growth indicators of the grafted rootstock, its disease resistance indicators were also measured simultaneously.

[0041] The root knots of each individual plant are counted, and the root knot index (GI) is calculated as: number of root knots per plant / fresh weight of roots per plant.

[0042] The number of eggs in each individual plant is counted, and the egg index (EI) is calculated as: number of eggs per plant / fresh root weight per plant.

[0043] The proportion of root knots on the root system of each individual plant was estimated to classify the disease status of the plants. The classification criteria were: Grade 0, no root knots on any root system, no infection; Grade 1, 1%–20% of the root system had root knots; Grade 2, 21%–40% of the root system had root knots; Grade 3, 41%–60% of the root system had root knots; Grade 4, 61%–80% of the root system had root knots; Grade 5, 81%–100% of the root system had root knots. The Disease Index (DI) for each variety was calculated based on the disease status of individual plants. DI = ∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × highest level) × 100. The DI value can be used to measure the disease resistance of each variety, with the specific standards as follows: DI = 0, immune; 0 < DI ≤ 20, highly resistant; 20 < DI ≤ 40, moderately resistant; 40 < DI ≤ 60, resistant; 60 < DI ≤ 80, moderately susceptible; DI > 80, highly susceptible.

[0044] Soak the chopped roots in a 1.2% sodium hypochlorite solution at 200 r·min. -1 Shake for 10 min, then count the eggs under a microscope. Reproduction factor (RF) = number of eggs per plant / initial inoculation amount per plant. Rootstocks with RF < 1 are considered resistant to root-knot nematodes.

[0045] The higher the values ​​of the four indicators GI, EI, DI, and RF, the weaker the resistance of the rootstock to the root-knot nematode of the bean shrub.

[0046] (3) Membership function calculation

[0047] Membership function values ​​of growth index and disease resistance index of grafted rootstock 50 days after inoculation with root-knot nematodes.

[0048] Calculation formula: X(μ)=1-(X—Xmin) / (Xmax-Xmin).

[0049] In the formula, X represents the measured value of a certain indicator of the grafted rootstock 50 days after inoculation, Xmax represents the maximum value of the same indicator across all rootstocks, and Xmin represents the minimum value of the same indicator across all rootstocks. A larger membership function value indicates stronger resistance in a particular variety.

[0050] (4) Soil microbial diversity index

[0051] Soil microbial community alpha diversity analysis primarily assesses the richness and diversity of microbial communities in environmental samples using multiple diversity indices, and explores differences in diversity indices between control and treatment groups through inter-group difference tests. Commonly used metrics include Chao1 (richness index), Shannon (diversity index), and Simpson (diversity index). By observing various index values, information such as species diversity can be obtained, and significant differences in index values ​​between samples can be detected. Soil microbial community diversity measurement method: Root soil was collected and stored at -80℃ for later use. Metagenomics and high-throughput sequencing methods were used to determine the sequences of 16S rRNA genes of soil microorganisms. Gene alignment analysis was conducted to count the number of soil microbial species.

[0052] The Chao 1 index is an index that measures species richness. The value indicates the level of species richness; the larger the index, the higher the species richness. The Shannon index is used to measure species diversity, and the Simpson index is also used to measure species diversity. The larger the Shannon index and the smaller the Simpson index, the higher the species diversity of the sample.

[0053] Chao1 = Sobs + n1(n1-1) / 2(n2+1), where Sobs is the number of observed OTUs, n1 is the number of OTUs with only 1 sequence, and n2 is the number of OTUs with only 2 sequences.

[0054] The Shannon index is calculated as follows: H'=-Σ(pi×ln(pi)), where pi represents the richness of the i-th species relative to all species (i.e., number of individuals / total number of individuals), and ln represents the natural logarithm.

[0055] The Simpson index is calculated as follows: D = 1 - ∑(ni / N) 2Where D represents the Simpson index, ni represents the number of individuals of the i-th species, and N represents the sum of the number of individuals of all species.

[0056] III. Results Analysis

[0057] 3.1 Traditional Identification and Evaluation of Root-Knot Nematode Resistance

[0058] Significant differences existed in disease resistance indicators among different tomato rootstocks infected with the root-knot nematode *Heliotropium indicum*. Regarding the root knot index, the modified *Torubam* rootstock had the lowest index, indicating the strongest resistance, followed by *Totosga*, *Huimei* rootstock, and *Beck*, while *Millennium* showed the weakest resistance. In terms of egg count, the modified *Torubam* and *Huimei* rootstocks had the lowest egg counts, with no difference between them, indicating the strongest resistance. *Beck* and *Totosga* were next, while *Millennium* had the highest egg count, indicating the weakest resistance. Regarding disease index, the modified *Torubam* rootstock had the lowest disease index at 18.33, indicating high resistance, followed by *Huimei* rootstock, *Totosga*, and *Beck*, with disease indices of 22.59 / 33.24 and 34.53 respectively, indicating moderate resistance. *Millennium* had the highest disease index at 84.67, indicating susceptibility.

[0059] Resistance was evaluated based on the reproduction coefficient; rootstocks with RF < 1 were considered resistant to root-knot nematodes. Only the modified Torubam rootstock was resistant; the others were susceptible.

[0060] Table 1. Effects of *Heliotropium indices* on disease resistance in different tomato rootstocks.

[0061]

[0062] 3.2 The present invention optimizes parameters and comprehensively evaluates resistance.

[0063] (1) Effects of inoculation with *Heliotropium indicum* root-knot nematode on growth indicators of different tomato rootstock seedlings

[0064] Fifty days after inoculation with *Heliotropium indicum* root-knot nematode, seedlings from different tomato rootstocks showed decreased plant height, stem diameter, aboveground fresh weight, and root fresh weight compared to the control, indicating that infection with *Heliotropium indicum* root-knot nematode affected seedling growth. Regarding plant height, the modified *Torubam* and *Huimei* rootstocks were the tallest, indicating that nematode infection had a relatively small impact on plant height. *Beck* and *Totosga* rootstocks followed, while *Millennium* rootstock was significantly affected. Regarding stem diameter, there was no significant difference among the modified *Torubam*, *Huimei* rootstock, and *Totosga* rootstocks, indicating a relatively small impact on stem diameter. *Millennium* rootstock was significantly affected. Regarding aboveground fresh weight, the modified *Torubam* and *Huimei* rootstocks were the heaviest, followed by *Totosga*, while *Millennium* rootstock had the lightest. Regarding root fresh weight, the modified *Torubam* and *Huimei* rootstocks had the highest root fresh weight, followed by *Beck* and *Totosga*, while *Millennium* rootstock had the lowest. Calculations of the coefficients of variation for different growth indicators show that plant height has the largest coefficient of variation at 5.31%, indicating that root-knot nematode infection has a significant impact on seedling height, followed by aboveground fresh weight and root fresh weight. Therefore, plant height, aboveground fresh weight, and root fresh weight can all be used as reference indicators for evaluating rootstock resistance to root-knot nematodes in bean plants.

[0065] Table 2. Effects of *Heliotropium indicum* root-knot nematode on growth indicators of seedlings from different tomato rootstocks.

[0066]

[0067] (2) Membership function values ​​of relevant indicators of tomato rootstock seedlings after inoculation with *Heliotropium indicum* root-knot nematode

[0068] Table 3 shows that, by calculating the total membership function values ​​of growth and disease resistance indicators of tomato grafted rootstocks 50 days after inoculation with *Hymenochaeus edulis*, the highest total membership function value was found for the tomato grafted rootstock 'Improved Torubam' (5.36), followed by 'Huimei Zanba' and 'Totosga'. 'Millennium' had the lowest total membership function value (3.88), indicating that 'Improved Torubam' exhibited the strongest resistance to *Hymenochaeus edulis*, followed by 'Huimei Zanba' and 'Totosga', while 'Millennium' showed the weakest resistance. Therefore, evaluating rootstock resistance to nematodes using the comprehensive membership function values ​​of trait indicators provides a true and objective assessment. Good rootstocks not only need strong disease resistance but also good growth conditions to achieve both disease resistance and high yield.

[0069] Table 3. Effects of the root-knot nematode of *Heliotropium indicum* on the membership function values ​​of relevant indicators in watermelon rootstock seedlings.

[0070]

[0071] (3) Cluster analysis of resistance to root-knot nematode in tomato rootstock seedlings

[0072] Cluster analysis was performed using plant height (PH), stem diameter (SD), aboveground fresh weight (SFM), root fresh weight (RFM), root knot index (GI), egg index (EI), disease index (DI), and reproduction coefficient (RF) as indicators. Figure 1 The six rootstocks can be categorized into three groups: resistant to root-knot nematode in tomatoes (modified Torubam, Huimei Rootstock, and Totosga); moderately resistant to root-knot nematode in tomatoes (Beck and Qiangli); and susceptible to the disease (Millennium). The cluster analysis results are consistent with the membership function values, indicating that the membership function can accurately evaluate the comprehensive characteristics of rootstocks and can serve as a screening index for tomato rootstocks resistant to root-knot nematode in tomatoes.

[0073] (4) Determination of soil microbial diversity index for different resistant rootstocks

[0074] Soil microbial community alpha diversity analysis is an important tool for measuring and comparing the complexity and change of biological communities. Alpha diversity mainly analyzes the degree of species diversity, analyzing both species abundance and evenness of individual distribution within a community for one or more samples.

[0075] Soil microorganisms are hailed as the "soul" of soil. Soil microorganisms mainly refer to the bacteria, fungi, actinomycetes, and algae living in the soil; they are an indispensable and important component of soil. These tiny microorganisms, invisible to the naked eye, are the core of soil biofertility and important regulators of soil life. Crop growth is inseparable from soil; roots grow in the soil, and different crops exhibit varying degrees of adaptability to different soil types. Crop roots play different roles, enriching or attracting beneficial microorganisms for utilization, promoting healthier crop growth and improving disease resistance. By analyzing soil microbial community diversity (Chao index, Shannon index, Simpson index), combined with disease resistance indicators, we can identify root soil microbial community diversity closely related to disease resistance, guiding more scientific and rational selection of root-knot nematode-resistant rootstock materials.

[0076] By analyzing the diversity of bacterial and fungal microbial communities in the root soil of different rootstocks infected with *Eriocaulon buergerianum*, it was found that the Simpson index showed significant differences in both fungal and bacterial microbial community diversity among different rootstocks. Other indices, such as chao, shannon, and ace, did not show significant differences among different rootstocks. In terms of root soil bacterial and microbial community diversity (Table 4), the modified Torubam rootstock had the lowest Simpson index value of 0.0176, which was significantly different from other rootstocks. The next highest value was that of the Huimei rootstock, with a Simpson index value of 0.0294, which was also significantly different from other rootstocks. The Millennium rootstock had the highest Simpson index value. Correlation analysis with disease index revealed that a lower Simpson index value for soil (bacterial) microbial diversity corresponds to a lower disease index, and vice versa. For example, for improved Torubam, the Simpson index for soil (bacterial) microbial diversity was 0.0176, and the disease index was 18.33; for Huimei Anvil, the Simpson index was 0.0294, and the disease index was 22.59. This indicates a positive correlation between the Simpson index and the disease index, and a negative correlation with disease resistance.

[0077] Table 4. Effects of *Heliotropium indicum* root-knot nematode on the diversity index of root soil microbial communities (bacteria) of different tomato rootstocks.

[0078]

[0079] Regarding the diversity of root soil fungal microbial communities (Table 5), the Simpson indices of improved Torubam, Huimei Rootstock, and Totosca were 0.24, 0.24, and 0.29, respectively, with no significant differences among the three. However, they showed significant differences compared to Beck, Strong, and Millennium. Beck and Strong were next, with Simpson indices of 0.36 and 0.37, respectively, showing significant differences compared to Millennium. Millennium had the highest Simpson index value of 0.42. Correlation analysis with disease index revealed that a lower Simpson index value for soil (fungal) microbial diversity corresponds to a lower disease index. For example, the fungal Simpson indices for improved Torubam, Huimei Anvil, and Totosca were 0.24, 0.24, and 0.29, respectively, with no significant difference among them. Their disease indices were 18.33, 22.59, and 33.24, respectively. However, compared with bacterial Simpson and disease indices, the distinction in resistance was not yet clear.

[0080] Table 5. Effects of *Heliotropium indicum* root-knot nematode on the diversity index of root soil microbial communities (fungi) of different tomato rootstocks.

[0081]

[0082] By comparing the Simpson index of fungal and bacterial microbial community diversity, significant differences were found in different rootstocks. However, when compared with the disease index, the Simpson index of bacterial microbial community diversity showed a stronger positive correlation with the disease index and a more obvious distinction in resistance. Therefore, when screening index parameters for rootstocks resistant to weevils and root-knot nematodes, the Simpson index of bacterial microbial community diversity should be given priority as the parameter.

[0083] A lower Simpson index value for soil (bacterial) microbial diversity indicates a higher enrichment and attraction of species around the rootstock's root system, particularly a high abundance and diversity of dominant soil-specific bacteria. This suppresses the soil environment conducive to the growth of soil-borne diseases (root-knot nematodes), thereby increasing the plant's resistance to these diseases. Therefore, the Simpson index for soil (bacterial) microbial diversity can be added as an evaluation parameter in the screening system for rootstocks resistant to weevils and root-knot nematodes.

[0084] Traditional methods for evaluating rootstocks resistant to root-knot nematodes tend to focus on indicators such as disease index, root knot index, egg index, and reproduction coefficient. However, during our experiments, we found that some materials, even with the same number of infected root knots after inoculation with root-knot nematodes, exhibited different aboveground growth patterns. In some cases, there were no significant differences in plant height and aboveground fresh weight compared to uninoculated treatments. This suggests that although these materials were infected by nematodes, the impact on their growth was relatively small, indicating a degree of resistance. Therefore, if evaluated solely based on disease index, root knot index, egg index, and reproduction coefficient, the material might be identified as a susceptible variety. Thus, the aforementioned evaluation methods cannot accurately assess rootstock resistance.

[0085] This invention collects and measures the diversity of soil microbial communities in the root systems of different rootstocks, and analyzes the alpha diversity index of soil microbial communities. The results show that the Simpson index has significant differences among different rootstocks, while other indices, such as Chao1 and Shannon, do not show significant differences among different rootstocks. The smaller the Simpson index value of soil microbial diversity, the higher the diversity of species enriched and attracted around the root system of the rootstock. In particular, the high abundance and diversity of soil-specific dominant bacteria will inhibit the soil environment suitable for the growth of soil-borne diseases (root-knot nematodes), convert more nutrients through the root system to supply the aboveground growth, and thus improve the plant's resistance to soil-borne diseases (root-knot nematodes).

[0086] This invention, based on resistance indicators, comprehensively considers aboveground growth conditions (plant height, stem diameter, aboveground fresh weight, etc.) and introduces the Simpson index of root system soil microbial diversity to evaluate the enrichment, attraction, and utilization of soil microorganisms by different rootstocks. More soil microorganisms enriched in the root system will inhibit the soil environment suitable for root-knot nematode growth, and the microorganisms will convert more soil nutrients to supply aboveground growth through the root system, reducing the impact of root-knot nematodes on the plant. This is also a way for rootstocks to comprehensively utilize environmental factors to achieve disease resistance, and it can provide a more accurate and comprehensive evaluation of the overall resistance of tomato rootstocks to weevils and root-knot nematodes.

[0087] Therefore, the present invention uses the Simpson index of root soil microbial (preferably bacterial) diversity as an indicator to accurately evaluate the resistance of rootstocks to weevils root-knot nematodes.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for horizontally precise evaluation of tomato rootstock resistance to Meloidogyne exigua, characterized by, It comprises the following steps: (1) cultivating the tomato rootstock seedlings to be tested, and selecting uniform seedlings for inoculating with Meloidogyne javanica when the seedlings grow 5 true leaves; each rootstock is set as a control without inoculation with the nematode; (2) 50 days after inoculation, the growth index, disease resistance index, reproduction coefficient and soil microbial species number of each plant are determined; (3) the resistance of the tested rootstock to M. javanica is evaluated by using the determined indexes.

2. The evaluation method according to claim 1, characterized by, In the step (2), the growth index includes plant height, stem diameter, aboveground fresh weight and root fresh weight; the disease resistance index includes root knot index, egg mass index, disease index and reproduction coefficient.

3. The evaluation method according to claim 2, characterized by, The determination method of the growth index and the disease resistance index is as follows: the plant height, stem diameter, aboveground fresh weight and root fresh weight of the rootstock are determined respectively, the root system is collected, washed, dried and weighed, and the root knot index, egg mass index, disease index and reproduction coefficient are calculated; The root knots of each single plant are counted, and the root knot index (GI) = single plant root knot number / single plant root fresh weight; The egg masses of each single plant are counted, and the egg mass index (EI) = single plant egg mass number / single plant root fresh weight; The disease index (DI) of each variety is calculated according to the disease condition of single plant; DI = ∑(each level of diseased plant number × corresponding level) / (total number of investigated plants × highest level) × 100; The cut roots were soaked in 1.2% sodium hypochlorite solution, 200 r·min -1 Shake for 10 min, count the eggs under a microscope, and the reproductive factor (RF) = the number of eggs per plant / the initial inoculation amount per plant.

4. The evaluation method according to claim 1, wherein In the step (3), the membership function and / or the soil microbial diversity index are calculated by using the determined indexes; the greater the value of the membership function, the stronger the resistance of the tested tomato rootstock to M. javanica; the smaller the value of the soil microbial diversity index, the stronger the resistance of the tested tomato rootstock to M. javanica.

5. The evaluation method according to claim 3, wherein The calculation formula of the membership function is X(μ) = 1-(X-Xmin) / (Xmax-Xmin); in the formula, X: the determined value of a certain index of the grafted rootstock 50 days after inoculation, Xmax: the maximum value of a certain index of all rootstocks, and Xmin: the minimum value of a certain index of all rootstocks.

6. The evaluation method according to claim 3, wherein The soil microbial diversity index is Simpson index, and the calculation formula is: D=1-∑(ni / N) 2 , wherein D represents Simpson index, ni represents the number of individuals of the i th species, and N represents the sum of the number of individuals of all species.

7. The evaluation method according to any one of claims 4 to 6, characterized in that, The determination method of the soil microbial diversity is as follows: the root system soil is collected and stored in a-80℃ ultra-low temperature refrigerator for standby; the sequence of the 16S rRNA gene of the soil microorganism is determined by using the metagenome and high-throughput sequencing method, the number of soil microbial species is counted by comparing and analyzing the genes; preferably, the soil microorganism is bacteria or fungi; more preferably, the soil microorganism is bacteria.

8. A method for screening tomato rootstocks resistant to M. javanica, comprising the following steps: (1) the resistance of the tested tomato rootstock to M. javanica is evaluated by using the membership function and / or the soil microbial diversity index calculated by using the above-mentioned precise evaluation method; (2) the level of the tested tomato rootstock resistant to M. javanica is screened according to the calculated membership function and / or soil microbial diversity index.

9. The method of claim 8, wherein, In the step (2), the greater the value of the membership function, the stronger the resistance of the tested tomato rootstock to M. javanica; the smaller the value of the soil microbial diversity index, the stronger the resistance of the tested tomato rootstock to M. javanica.

10. The method of claim 9, wherein, The soil microorganism is bacteria or fungi; preferably, the soil microorganism is bacteria.

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