Method for identifying broad-spectrum disease resistance of tomato bacterial wilt

Abstract

The invention discloses a method for identifying broad-spectrum disease resistance of tomato bacterial wilt, which comprises the following steps: using seven mixed ralstonia solanacearum strains of different sequence variants as identification strains, transferring tomato seedlings with consistent pre-germination growth vigor to a culture medium inoculated with the identification strains for growth, observing and counting the growth condition and morbidity screening of the tomato seedlings, and identifying the broad-spectrum disease resistance of the tomato bacterial wilt. And verifying the screened broad-spectrum disease-resistant tomato variety by injecting an identification strain liquid into leaves in a seedling stage and performing disease resistance identification, and giving a disease resistance identification method comprising the steps of preparing a ralstonia solanacearum identification strain liquid, preparing solanaceae crop seedlings, screening the seedlings by a culture dish method and verifying the broad-spectrum disease resistance of the material by a leaf injection method. The method is convenient and efficient, results are stable and reliable, and a new means is provided for screening disease-resistant tomato germplasm resources on a large scale.

Description

Technical Field

[0001] The present invention relates to the field of agricultural identification of bacterial strains, and in particular to an identification method for broad-spectrum disease resistance of tomato. Background Art

[0002] Tomatoes (Solanum lycopersicum) are widely cultivated worldwide due to their adaptability and nutritious fruits. Tomato bacterial wilt, caused by the Solanaceae fungus Ralstonia solanacearum, is a soil-borne vascular bacterial disease with rich genetic diversity, infecting over 200 plant species. Research has shown that the strains that primarily infect Solanaceae vegetables in my country are strains of phylotype I / race 1 / biovar III and IV, with 11 sequence variants.

[0003] Multiple studies have shown that tomato bacterial wilt strains in Hubei belong to sequence variant 17, while those in Guangdong belong to sequence variants 1, 13, 14, 15, 17, 18, 34, 44, 45, 48, and 57. Tomato bacterial wilt strains in southern Jiangxi belong to sequence variants 13, 14, 15, 17, 18, 34, 44, and 48, and those in Fujian belong to sequence variants 14, 15, 16, 17, and 34. Bacillus solanacearum isolated from tomatoes, eggplants, and peppers can infect each other, making it difficult for a single resistant variety to exhibit strong resistance to all sequence variants.

[0004] Breeding varieties resistant to bacterial wilt can significantly reduce the damage caused by R. solanacearum to Solanaceae vegetables. Therefore, evaluating the resistance of germplasm resources using strains of different sequence variants as test strains is crucial for discovering germplasm resources with broad-spectrum resistance and developing scientific and effective bacterial wilt prevention and control measures.

[0005] Traditional root wound inoculation and root immersion methods suffer from long disease cycles, cumbersome procedures, and difficulty controlling the size of the artificial wound, resulting in poor inoculation results. The seedling dish method is simpler and more efficient than other inoculation methods. Under controlled conditions, it allows for rapid resistance testing of large batches of tomato materials, significantly reducing testing time and costs. Leaf injection, compared to other inoculation methods, offers advantages such as a smaller inoculum size, shorter testing cycles, easier statistical analysis of symptoms, and stable and reliable results.

[0006] Therefore, based on the demand for germplasm resources with broad-spectrum resistance to bacterial wilt in tomato breeding, it is a technical problem that needs to be solved urgently to establish a tomato broad-spectrum resistance identification technology based on a mixed identification strain of multiple bacterial wilt sequence variants, a seedling culture dish method and a leaf injection method. Summary of the Invention

[0007] In view of this, the present invention provides a method for identifying broad-spectrum disease resistance of tomato tomato bacterial wilt.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for identifying broad-spectrum resistance to tomato bacterial wilt disease is disclosed. The method uses a mixed strain of seven Ralstonia solanacearum strains with different sequence variants as identification strains, transplants pre-germinated tomato seedlings with consistent growth onto a culture medium inoculated with the identification strains for growth, observes and statistically analyzes the growth and morbidity of the tomato seedlings, and then verifies the selected broad-spectrum disease-resistant tomato varieties by injecting a bacterial solution of the identification strain into leaves at the seedling stage and performing disease resistance identification.

[0010] Preferably, the method further comprises preparing a bacterial solution of an identified strain of Ralstonia solanacearum, preparing Solanaceae crop seedlings, and performing disease resistance identification by seedling culture dish screening and leaf injection to verify the broad-spectrum disease resistance of the material.

[0011] Preferably, the preparation of the bacterial liquid of the Ralstonia solanacearum identification strain uses RS2107128F, RS210564F, RS211101F, RS210426F, GMI1000, RS210507F, and RS20240706SL strains identified as sequence variants 14, 15, 16, 17, 18, 34, and 48, respectively.

[0012] Preferably, the preparation of the bacterial solution of the identification strain of Ralstonia solanacearum comprises the following steps:

[0013] Seven strains were streaked onto TTC medium plates using a sterile inoculation loop. The plates were inverted and cultured in a 30°C biochemical incubator for 2 days. Single colonies were picked and placed in 50 mL centrifuge tubes containing NB medium. The centrifuge tubes were placed in a 30°C shaking incubator at 180 rpm for 24 hours, then centrifuged at 6000 rpm for 5 minutes. The supernatant was discarded to collect the bacteria. After washing three times with sterile water, the bacteria were resuspended in an appropriate amount of sterile water. The OD600 value was measured using a UV spectrophotometer. The bacterial suspensions of each strain were diluted to OD 600 =0.1,1×10 8 CFU·mL -1 , an equal volume of mixed bacterial suspension was used as the identification strain solution for inoculation.

[0014] Preferably, the preparation of the Solanaceae crop seedlings comprises the following steps:

[0015] 60 seeds of the tested germplasm resources were placed in 5 mL centrifuge tubes, and the surface was disinfected with an appropriate amount of 75% ethanol for 3 minutes and 30% sodium hypochlorite (containing 0.05% Silwet-L-77) for 10 minutes. The tubes were washed with sterile water for at least 5 times, and the centrifuge tubes were turned upside down during the process to ensure that the seeds were fully cleaned. Then, 3 mL of sterile water was added and the tubes were stored at 4 °C for 2 days to improve the germination rate and synchronization of the seeds. Then, the seeds were taken out of the 4 °C refrigerator and evenly placed in a 10 cm × 10 cm square culture dish containing 1 / 2 MS culture medium using sterilized tweezers. The square culture dish was placed in a 25 °C plant light incubator with a light intensity of 150 μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark, and the culture was carried out for 6 days.

[0016] Preferably, the seedling culture dish screening method comprises the following steps:

[0017] The mixed bacterial suspension was added to a 1 / 2MS plate, 3 mL / dish, and the culture dish was gently shaken to evenly distribute the bacterial suspension throughout the culture dish. The culture dish was allowed to stand at room temperature for 3-5 minutes. The excess bacterial suspension was removed with a pipette, and the culture dish was naturally air-dried for 15-20 minutes on a clean bench. Six-day-old tomato seedlings with uniform growth were transferred to a 1 / 2MS culture dish containing Ralstonia solanacearum. Twenty-one seedlings were placed in each culture dish, with 7 seedlings treated with each test germplasm resource, repeated in three groups, and clean water was used as the control group. The inoculated culture dishes were placed under the same growth conditions for 5 days, and the growth and incidence of the tomato seedlings were observed and counted, and the material resistance was determined according to NY / T1858.4-2010.

[0018] Preferably, the disease resistance identification of the material by the leaf injection method for verifying the broad-spectrum disease resistance comprises the following steps:

[0019] Disease-resistant materials were selected based on the results of resistance identification using the seedling culture dish method. The seedlings were transplanted into pots and grown for 5 weeks. Tomato plants with consistent growth were selected, and two symmetrical lateral leaflets near the axis of the second and third compound leaves were selected. A 1mL needle was used to gently poke a small hole on the back of the leaf. The bacterial suspension was then aspirated with a syringe without the needle and injected into the leaf through the wound. 0.5mL of bacterial suspension was injected into each leaf, and the control group was injected with clean water. The incidence of disease was counted after 7 days. Three replicates were performed, and 12 plants were inoculated in each replicate.

[0020] Preferably, the disease resistance identification of the material for verifying broad-spectrum disease resistance by the leaf injection method further comprises the following steps:

[0021] The criteria for evaluating the disease incidence level according to the degree of disease spread in the whole plant are as follows: Level 0: No disease, no symptoms; Level 1: The 4 lateral small leaves inoculated wilt and turn yellow; Level 3: The 2 compound leaves where the lateral small leaves inoculated wilt; Level 5: 3 - 4 compound leaves wilt; Level 7: All leaves except the top 1 - 2 leaves wilt; Level 9: All leaves of the whole plant wilt.

[0022] Preferably, it further includes calculating the disease index, where the calculation formula for the disease index is:

[0023]

[0024] Preferably, it further includes classifying the disease resistance of different variety populations. The criteria for classifying the disease resistance of different variety populations are as follows:

[0025] Immune I: DI = 0; Highly resistant HR: 0 < DI < 12.5; Resistant R: 12.5 ≤ DI < 25; Moderately resistant MR: 25 ≤ DI < 50; Susceptible S: 50 ≤ DI < 75; Highly susceptible HS: 75 ≤ DI ≤ 100.

[0026] The present invention has achieved the following technical effects compared with the prior art:

[0027] (1) The present invention has high applicability. The mixed discriminatory strains prepared by using multiple Ralstonia solanacearum sequence variants can screen disease - resistant tomato materials with broad - spectrum resistance.

[0028] (2) The present invention is efficient, convenient and fast. It can quickly identify the broad - spectrum resistance of breeding materials to Ralstonia solanacearum through the seedling petri - dish method under indoor conditions. The experimental results of Example 1 show that the disease - resistance identification results can be obtained in only 5 days, which is especially suitable for the disease - resistance identification of a large number of populations.

[0029] (3) The disease - resistance identification results of the present invention are stable and reliable. The leaf injection method of the present invention can achieve rapid and stable infection of Ralstonia solanacearum. The experimental results of Example 2 show that the resistant and susceptible phenotypes of the tested plants can be verified within only 7 days. Therefore, the method of combining the screening by the seedling petri - dish method and the verification by the leaf injection method can ensure the reliability and effectiveness of the identification results.

[0030] (4) The method of using leaf injection inoculation to verify the disease resistance to Ralstonia solanacearum adopted by the present invention can be further used for the mechanism research on the physiology and biochemistry related to the disease resistance of germplasm resources. Example 3 gives the mechanism research on the physiology and biochemistry related to the disease resistance of germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of symptom differences in the rapid identification of the broad - spectrum resistance of breeding materials to Ralstonia solanacearum by the seedling petri - dish method of the present invention. Among them,

[0032] (a) (b) Phenotypic differences between the broad-spectrum disease-resistant tomato variety Guizhen No. 1 and the susceptible tomato variety AC after 5 days of growth on 1 / 2 MS medium without inoculation or inoculation with the identification strain of Ralstonia solanacearum;

[0033] Figure 2 The phenotypic differences of the leaves of the six tomato materials of the present invention after injection and inoculation with a mixed suspension of Ralstonia solanacearum; among them, (a) is Guizhen No. 1, (b) is 204, (c) is 1440, (c) is 1520, (d) is AC, and (e) is Qianxi;

[0034] Figure 3 These are Fv / Fm fluorescence phenotype diagrams of leaves of the present invention before and after injection inoculation with Ralstonia solanacearum; wherein, (a1) is the Fv / Fm fluorescence phenotype of the broad-spectrum disease-resistant tomato variety Guizhen No. 1 plants before inoculation; (a2) is the Fv / Fm fluorescence phenotype of the broad-spectrum disease-resistant tomato variety Guizhen No. 1 plants after inoculation; (b1) is the Fv / Fm fluorescence phenotype of the susceptible tomato variety AC plants before inoculation; and (b2) is the Fv / Fm fluorescence phenotype of the susceptible tomato variety AC plants after inoculation. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] The invention discloses a method for identifying broad-spectrum disease resistance of tomatoes to bacterial wilt. The method uses a mixed strain of seven Ralstonia solanacearum strains with different sequence variants as identification strains, transplants pre-germinated tomato seedlings with consistent growth to a culture medium inoculated with the identification strains for growth, observes and statistics the growth of the tomato seedlings and the incidence rate for screening, and then injects the identification strain solution into the leaves at the seedling stage to verify the selected broad-spectrum disease-resistant tomato varieties and perform disease resistance identification.

[0037] The method also includes preparing bacterial liquid of the identification strain of Ralstonia solanacearum, preparing seedlings of Solanaceae crops, screening by seedling culture dish method, and verifying the disease resistance of the material by leaf injection method.

[0038] The bacterial suspensions of the identified Ralstonia solanacearum strains were prepared using strains RS2107128F, RS210564F, RS211101F, RS210426F, GMI1000, RS210507F, and RS20240706SL, which were identified as sequence variants 14, 15, 16, 17, 18, 34, and 48, respectively.

[0039] The preparation of the bacterial solution of the identification strain of Ralstonia solanacearum comprises the following steps:

[0040] Seven strains were streaked onto TTC medium plates using a sterile inoculation loop. The plates were inverted and cultured in a 30°C biochemical incubator for 2 days. Single colonies were picked and placed in 50 mL centrifuge tubes containing NB medium. The centrifuge tubes were placed in a 30°C shaking incubator at 180 rpm for 24 hours, then centrifuged at 6000 rpm for 5 minutes. The supernatant was discarded to collect the bacteria. After washing three times with sterile water, the bacteria were resuspended in an appropriate amount of sterile water. The OD600 value was measured using a UV spectrophotometer. The bacterial suspensions of each strain were diluted to OD 600 =0.1,1×10 8 CFU·mL -1 , an equal volume of mixed bacterial suspension was used as the identification strain solution for inoculation.

[0041] Preparation of Solanaceae seedlings includes the following steps:

[0042] 60 seeds of the tested germplasm resources were placed in 5 mL centrifuge tubes, and the surface was disinfected with an appropriate amount of 75% ethanol for 3 minutes and 30% sodium hypochlorite (containing 0.05% Silwet-L-77) for 10 minutes. The tubes were washed with sterile water for at least 5 times, and the centrifuge tubes were turned upside down during the process to ensure that the seeds were fully cleaned. Then, 3 mL of sterile water was added and the tubes were stored at 4 °C for 2 days to improve the germination rate and synchronization of the seeds. Then, the seeds were taken out of the 4 °C refrigerator and evenly placed in a 10 cm × 10 cm square culture dish containing 1 / 2 MS culture medium using sterilized tweezers. The square culture dish was placed in a 25 °C plant light incubator with a light intensity of 150 μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark, and the culture was carried out for 6 days.

[0043] The seedling dish screening method includes the following steps:

[0044] The mixed bacterial suspension was added to a 1 / 2MS plate, 3 mL / dish, and the culture dish was gently shaken to evenly distribute the bacterial suspension throughout the culture dish. The culture dish was allowed to stand at room temperature for 3-5 minutes. The excess bacterial suspension was removed with a pipette, and the culture dish was naturally air-dried for 15-20 minutes on a clean bench. Six-day-old tomato seedlings with uniform growth were transferred to a 1 / 2MS culture dish containing Ralstonia solanacearum. Twenty-one seedlings were placed in each culture dish, with 7 seedlings treated with each test germplasm resource, repeated in three groups, and clean water was used as the control group. The inoculated culture dishes were placed under the same growth conditions for 5 days, and the growth and incidence of the tomato seedlings were observed and counted, and the material resistance was determined according to NY / T1858.4-2010.

[0045] The disease resistance test for broad-spectrum disease resistance of materials using the leaf injection method includes the following steps:

[0046] Select disease-resistant materials according to the resistance identification results of the seedling culture dish method. Transplant the seedlings into pots and grow them for 5 weeks. Select tomato plants with consistent growth. Select two symmetrical lateral small leaves near the leaf axis of the second and third compound leaves. Use a 1 mL needle to gently make a small hole on the back of the leaf, and then use a syringe without a needle to suck the bacterial suspension and inject it into the leaf from the leaf wound; inject 0.5 mL of the bacterial suspension into each leaf. The control group is treated with injection of clear water. After 7 days, count the disease incidence; there are 3 replicates, and 12 plants are inoculated in each replicate.

[0047] The disease resistance identification for verifying the broad-spectrum disease resistance of the materials by the leaf injection method further includes the following steps:

[0048] The standard for evaluating the disease incidence level according to the disease spread degree in the whole plant is: level 0: no disease, no symptoms; level 1: the 4 lateral small leaves inoculated wilt and turn yellow; level 3: the 2 compound leaves where the inoculated lateral small leaves are located wilt; level 5: 3 - 4 compound leaves wilt; level 7: all leaves except the top 1 - 2 leaves wilt; level 9: all leaves of the whole plant wilt.

[0049] It further includes calculating the disease index. Among them, the calculation formula of the disease index is: ​​​​​​​​​​​​​​​​​​​​​​​​​​​, sucrose 30g·L -1 , agar powder 7g·L-1, pH adjusted to 5.7-5.9, sterilized at 121℃ for 15min;

[0059] 2) NB medium: glucose 5.0 g·L -1 , casein hydrolysate 1.0g·L -1 , peptone 10.0 g·L -1 Adjust the pH to 7.0 and sterilize at 121°C for 15 min.

[0060] 3) NA medium: Same as NB medium, add 15.0 g·L agar powder -1 , sterilize at 121℃ for 15min;

[0061] 4) TTC Medium: Take sterilized NA medium and, when the temperature drops to approximately 50°C, add 2 mL of 0.5% TTC aqueous solution. Mix and shake thoroughly to prepare TTC medium. (0.5% TTC formulation: Weigh 2,3,5-triphenyltetrazolium chloride (TTC) and dissolve it in sterile water to a final concentration of 0.5%. Sterilize the solution by filtering through a 0.22 μm filter and store in a refrigerator at 4°C away from light.)

[0062] The following example describes a method for detecting the relative accumulation of Ralstonia solanacearum in tomato plants after leaf inoculation:

[0063] Seven days after inoculation, a 1 cm section of stem was removed from the tomato plant stem 1 cm above the cotyledons using a sterilized single-sided razor blade (three samples were collected for each variety). The sample mass (0.1 g) was weighed and then surface-sterilized with 75% alcohol for 30 s. After removal, the sample was washed twice with sterile water, and excess surface water was absorbed. The sample was placed in a 1.5 mL centrifuge tube (containing two zirconium beads) and disrupted with a plant tissue disruptor. Total plant DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. DNA integrity was detected by 1% agarose gel electrophoresis. DNA concentration and A260 / A280 absorbance were determined using a Nanodrop 2000 / 2000c spectrophotometer. The sample was stored at -20°C until use.

[0064] The total plant DNA extracted by CTAB was diluted to 25 ng μL -1 The template used for qPCR was SlEF1a as the internal reference gene and 16sRNA as the target gene. As shown in Table 2, the relative accumulation of Ralstonia solanacearum was detected by qPCR.

[0065] The reaction system was as follows: 2×Taq Pro Universal SYBR qPCR masterMix 10 μL, DNA 2 μL, primer 0.8 μL, and RNase-free ddH2O added to 20 μL.

[0066] Reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing for 30s, 40 cycles. Add melting curve: 95℃ denaturation for 15s, 60℃ annealing for 60s, 95℃ denaturation for 15s. -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0067] Table 2: Primer sequences for the determination of Ralstonia solanacearum colonization

[0068]

[0069] The following examples describe methods for determining the activity of defense enzymes and the content of osmotic regulating substances:

[0070] The test materials were inoculated with Ralstonia solanacearum, and changes in defense enzyme activity and osmotic regulatory substance content were measured after inoculation. Healthy, uniformly growing tomato seedlings, five weeks old, were inoculated with leaf injection. When the bacterial wilt incidence rate reached 70% to 80%, 0.1 g of top leaves from the plants were weighed and sampled three times. Peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activity, and malondialdehyde (MDA) content were determined using corresponding kits (Sangon Biotech (Shanghai) Co., Ltd., China).

[0071] The plant multispectral fluorescence imaging analysis method in the following embodiments:

[0072] Combining the results of the seedling dish method and leaf injection screening and identification, 5-week-old tomato plants were selected for testing and inoculated by leaf injection. Water was also inoculated as a control. Three replicates were set up, with 12 plants in each replicate. Before and 48 hours after inoculation with Ralstonia solanacearum, the effects of stress on plants were screened using the Plant Explorer large-scale multispectral fluorescence imaging system from Beijing Huinuoread Technology Co., Ltd., based on chlorophyll a fluorescence imaging (ChlF). The photosynthetic characteristic Fv / Fm index of the tomato materials was measured to analyze the maximum light energy conversion efficiency or maximum photochemical efficiency. The change rate before and after biological stress was calculated as follows:

[0073]

[0074] The technical solution of the present invention is further described below in the form of embodiments in combination with the accompanying drawings and experimental data.

[0075] Example 1: Screening of disease-resistant tomato germplasm resources using the seedling culture dish method of the present invention

[0076] Preliminary resistance testing of 88 tomato accessions using the seedling plate method was performed. The results, shown in Table 3, indicate that among the tomato accessions inoculated with a suspension of a mixture of R. solanacearum strains of different sequence variants, four accessions were resistant, accounting for 4.54% of the total; 17 accessions were moderately resistant, accounting for 19.32% of the total; 19 accessions were moderately susceptible, accounting for 21.59% of the total; and 48 accessions were susceptible, accounting for 54.54% of the total. Among them, Guizhen No. 1, 204, 1440, and 1520 exhibited broad-spectrum resistance to the mixture of R. solanacearum strains of different sequence variants.

[0077] Table 3: Identification of resistance of tomato materials to bacterial wilt strains using the seedling culture dish method

[0078]

[0079]

[0080] Note: R: resistant; MR: moderately resistant; MS: moderately susceptible; S: susceptible.

[0081] Note: R: Diseaseresistance; MR: Moderateresistance; MS: Moderatesusceptible; S: susceptible.

[0082] Example 2: Verification of typical resistant tomato varieties by the leaf injection method of the present invention

[0083] Based on the results of the seedling culture dish method, the mixed bacterial suspension was inoculated into four disease-resistant materials, Guizhen No. 1, 204, 1440, and 1520, and two typical susceptible materials, AC and Qianxi, using the leaf injection method. At the same time, clear water was inoculated as a control, and detailed disease observation and data statistics were carried out. Figure 2 As shown in the figure, 7 days after inoculation, only the leaves at the inoculated site dried up in Guizhen No. 1, 204, 1440, and 1520, while all leaves except some top leaves of AC and Qianxi wilted. The relative accumulation of R. solanacearum in the stems was determined by qPCR 7 days after inoculation.

[0084] The results showed that R. solanacearum was detected in the stems of all six tomato accessions 7 days after inoculation. The relative accumulation of R. solanacearum in the stems of Guizhen 1, 204, 1440, and 1520 was lower, while that in AC and Qianxi was much higher than that in CK, as shown in Table 4. These results indicate that Guizhen 1, 204, 1440, and 1520 exhibited broad-spectrum resistance to a mixed suspension of R. solanacearum with different sequence variants, consistent with the resistance identification results obtained using the seedling Petri dish method.

[0085] Table 4: Relative accumulation of Ralstonia solanacearum in plant stems

[0086]

[0087] Note: Capital letters indicate extremely significant differences (P < 0.01); lowercase letters indicate significant differences (P < 0.05).

[0088] Note: Capitaletters represent extremely significant (P<0.01); Lowercase represent significant (P<0.05).

[0089] Example 3: Effects of the Inoculation of Ralstonia solanacearum on Defense Enzyme Activity, Osmotic Adjustment Substance Content, and Chlorophyll Fluorescence Phenotype

[0090] The activity levels of three defense enzymes and the content of one osmotic regulating substance in the six materials after inoculation with Ralstonia solanacearum were determined. The results showed that as shown in Table 5, the POD activity of Guizhen No. 1 (97.48 U / g), 204 (80.84 U / g), 1440 (156.93 U / g) and 1520 (164.06 U / g) was significantly lower than that of AC (347.14 U / g) and Qianxi (375.67 U / g); the POD activity of Guizhen No. 1 (550.04 U / g), 204 (586.26 U / g), 1440 (490.10 U / g) and 1520 (502.98 U / g) was significantly lower than that of AC (347.14 U / g) and Qianxi (375.67 U / g). The SOD activity of the six materials was significantly higher than that of AC (229.16 U / g) and Qianxi (219.30 U / g); the CAT activity levels in the six materials were in the range of 276.49-281.34 U / g, and the responses to the inoculation of Ralstonia solanacearum were consistent, with no significant differences; the MDA activities of Guizhen No. 1 (0.1278 μmol / g), 204 (0.1192 μmol / g), 1440 (0.1443 μmol / g) and 1520 (0.1491 μmol / g) were significantly lower than those of AC (0.2544 μmol / g) and Qianxi (0.2677 μmol / g).

[0091] The results showed that the lower the POD activity and MDA content after inoculation, the stronger the material's resistance to bacterial wilt; the higher the SOD activity after inoculation, the stronger the material's resistance to bacterial wilt.

[0092] In order to explore the effect of bacterial wilt inoculation on the chlorophyll fluorescence phenotype of tomato plants, tomato plants were placed in a plant multispectral imaging instrument before and after inoculation to measure the changes in their photosynthetic characteristics. The results showed that the chlorophyll fluorescence phenotype of the test tomato materials did not change much before and after inoculation with bacterial wilt, mainly green or yellow-green. The chlorophyll fluorescence phenotype of the susceptible materials changed significantly from green to purple. For example, Guizhen No. 1 and AC Figure 3Further calculations showed, as shown in Table 6, that significant differences existed between the resistant and susceptible materials before and after inoculation with Ralstonia solanacearum. Compared to pre-inoculation, the Fv / Fm ratios of the resistant materials Guizhen 1, 204, 1440, and 1520 changed by -3.49%, -8.50%, -6.32%, and -9.89%, respectively, while those of the susceptible materials AC and Qianxi changed by -31.50% and -29.63%, respectively. The results showed that the Fv / Fm ratio decreased significantly more in the susceptible materials than in the resistant materials 48 hours after inoculation.

[0093] Table 5: Effects of inoculation on defense enzyme activities and osmotic regulatory substance contents

[0094]

[0095] Note: Capital letters indicate extremely significant differences (P < 0.01); lowercase letters indicate significant differences (P < 0.05).

[0096] Note: Capital letters represent extreme significance (P<0.01); Lowercase represent significance (P<0.05).

[0097] Table 6: Changes in Fv / Fm of tomato plants before and after inoculation with Ralstonia solanacearum (%)

[0098]

[0099] Note: Capital letters indicate extremely significant differences (P < 0.01); lowercase letters indicate significant differences (P < 0.05).

[0100] Note: Capitaletters represent extremely significant (P<0.01); Lowercase represent significant (P<0.05).

[0101] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for identifying broad-spectrum resistance to tomato bacterial wilt, characterized in that: The method uses a mixed strain of seven Ralstonia solanacearum strains with different sequence variants as identification strains, transplants pre-germinated tomato seedlings with consistent growth onto a culture medium inoculated with the identification strains for growth, observes and statistically analyzes the growth of the tomato seedlings and the disease incidence, and then verifies the selected broad-spectrum disease-resistant tomato varieties by injecting the identification strain solution into the leaves at the seedling stage and performing disease resistance identification.

2. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 1, characterized in that: The method further comprises preparing bacterial solution of the identification strain of Ralstonia solanacearum, preparing seedlings of Solanaceae crops, screening by seedling culture dish method and verifying the broad-spectrum disease resistance of the materials by leaf injection method.

3. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, characterized in that: The bacterial liquid of the solanacearum identification strain is prepared using RS2107128F, RS210564F, RS211101F, RS210426F, GMI1000, RS210507F and RS20240706SL strains identified as sequence variants 14, 15, 16, 17, 18, 34 and 48, respectively.

4. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, wherein: The preparation of the bacterial solution of the identification strain of Ralstonia solanacearum comprises the following steps: Seven strains were streaked onto TTC medium plates using a sterile inoculation loop. The plates were inverted and cultured in a 30°C biochemical incubator for 2 days. Single colonies were picked and placed in 50 mL centrifuge tubes containing NB medium. The centrifuge tubes were placed in a 30°C shaking incubator at 180 rpm for 24 hours, then centrifuged at 6000 rpm for 5 minutes. The supernatant was discarded to collect the bacteria. After washing three times with sterile water, the bacteria were resuspended in an appropriate amount of sterile water. The OD600 value was measured using a UV spectrophotometer. The bacterial suspensions of each strain were diluted to OD 600 =0.1,1×10 8 CFU·mL -1 , an equal volume of mixed bacterial suspension was used as the identification strain solution for inoculation.

5. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, characterized in that: The preparation of the solanaceous crop seedlings comprises the following steps: 60 seeds of the tested germplasm resources were placed in 5 mL centrifuge tubes, and the surface was disinfected with an appropriate amount of 75% ethanol for 3 minutes and 30% sodium hypochlorite (containing 0.05% Silwet-L-77) for 10 minutes. The tubes were washed with sterile water for at least 5 times, and the centrifuge tubes were turned upside down during the process to ensure that the seeds were fully cleaned. Then, 3 mL of sterile water was added and the tubes were stored at 4 °C for 2 days to improve the germination rate and synchronization of the seeds. Then, the seeds were taken out of the 4 °C refrigerator and evenly placed in a 10 cm × 10 cm square culture dish containing 1 / 2 MS culture medium using sterilized tweezers. The square culture dish was placed in a 25 °C plant light incubator with a light intensity of 150 μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark, and the culture was carried out for 6 days.

6. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, characterized in that: The seedling culture dish method screening comprises the following steps: The mixed bacterial suspension was added to a 1 / 2MS plate, 3 mL / dish, and the culture dish was gently shaken to evenly distribute the bacterial suspension throughout the culture dish. The culture dish was allowed to stand at room temperature for 3-5 minutes. The excess bacterial suspension was removed with a pipette, and the culture dish was naturally air-dried for 15-20 minutes on a clean bench. Six-day-old tomato seedlings with uniform growth were transferred to a 1 / 2MS culture dish containing Ralstonia solanacearum. Twenty-one seedlings were placed in each culture dish, with 7 seedlings treated with each test germplasm resource, repeated in three groups, and clean water was used as the control group. The inoculated culture dishes were placed under the same growth conditions for 5 days, and the growth and incidence of the tomato seedlings were observed and counted, and the material resistance was determined according to NY / T1858.4-2010.

7. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, characterized in that: The disease resistance identification of the material for verifying broad-spectrum disease resistance by the leaf injection method comprises the following steps: Disease-resistant materials were selected based on the results of resistance identification using the seedling culture dish method. The seedlings were transplanted into pots and grown for 5 weeks. Tomato plants with consistent growth were selected, and two symmetrical lateral leaflets near the axis of the second and third compound leaves were selected. A 1mL needle was used to gently poke a small hole on the back of the leaf. The bacterial suspension was then aspirated with a syringe without the needle and injected into the leaf through the wound. 0.5mL of bacterial suspension was injected into each leaf, and the control group was injected with clean water. The incidence of disease was counted after 7 days. Three replicates were performed, and 12 plants were inoculated in each replicate.

8. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 2, characterized in that: The disease resistance identification of the material for verifying broad-spectrum disease resistance by the leaf injection method further comprises the following steps: The standard for evaluating the disease incidence level based on the degree of disease spread throughout the plant is level 0: no disease, no symptoms; level 1: the 4 inoculated lateral leaflets wilt and turn yellow; level 3: the 2 compound leaves where the inoculated lateral leaflets are located wilt; level 5: 3-4 compound leaves wilt; level 7: all leaves except the top 1-2 leaves wilt; level 9: all leaves of the entire plant wilt.

9. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 8, characterized in that: It also includes calculating the disease index, wherein the calculation formula of the disease index is:

10. The method for identifying broad-spectrum resistance to tomato bacterial wilt according to claim 8, characterized in that: It also includes the classification of disease resistance of different variety groups, among which the classification standards of disease resistance of different variety groups are: Immunity I: DI = 0; High resistance HR: 0 < DI < 12.5; Resistance R: 12.5 ≤ DI < 25; Moderate resistance MR: 25 ≤ DI < 50; Susceptible S: 50 ≤ DI < 75; Highly susceptible HS: 75 ≤ DI ≤ 100.

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