Method for identifying bacterial wilt resistance through leaf injection inoculation in solanaceae crop seedling stage

Abstract

The invention discloses a method for identifying bacterial wilt resistance through leaf injection inoculation in a solanaceae crop seedling stage. The method specifically comprises the following steps: (1) preparing solanaceae crop seedlings; (2) preparing a ralstonia solanacearum suspension; (3) performing leaf injection inoculation; and (4) identifying the bacterial wilt resistance. According to the method, the ralstonia solanacearum suspension is injected into the leaves from the wounds of the leaves, the inoculation method is rapid, efficient, easy and convenient to operate, stable and reliable in result and capable of being implemented on various solanaceae crops, and a new means is provided for resistance identification of disease-resistant germplasm resources of the solanaceae crops on a large scale.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural planting, and more particularly to a method for identifying resistance to bacterial wilt by injection inoculation of leaves of solanaceous crops at the seedling stage. Background Art

[0002] Solanaceae crop bacterial wilt, caused by Ralstonia solanacearum, is the world's second largest bacterial disease, infecting more than 450 species of plants from 54 families. Bacterial wilt is characterized by rapid onset and severity, and its prevalence often causes a large reduction in crop yields or even crop failure, resulting in serious economic losses for growers. Currently, there is no effective chemical agent to control the disease. Therefore, cultivating and planting disease-resistant varieties to control bacterial wilt is currently the most effective, safest and most economical means of prevention and control.

[0003] In the process of breeding for bacterial wilt resistance, establishing a stable and accurate resistance identification system is a prerequisite for the screening of disease-resistant germplasm varieties and variety cultivation. At present, the main identification systems for bacterial wilt resistance include field disease nursery identification and inoculation identification. Among them, field disease nursery identification conducts variety identification and screening under natural disease conditions. The advantage is that it is more consistent with the actual production situation. However, there are many factors that affect the disease in the field, and the identification results in different years are unstable. In addition, the requirements for maintaining the disease nursery are high, and it takes up space and manpower. Therefore, it is more suitable for screening and verification in the later stage of breeding. In contrast, inoculation identification is less affected by space, manpower, and the environment, and is more suitable for the screening of large quantities of germplasm resources.

[0004] At present, in the evaluation of bacterial wilt resistance, root injury inoculation method, root immersion method and stem base injection method are often used in inoculation identification. Among them, the root injury inoculation method is widely used in the identification of resistance to R. solanacearum in tomatoes, peppers, tobacco, eggplants, potatoes and peanuts because it can reflect the infection ability of bacteria under natural conditions. However, this method has certain limitations. Its disease cycle is long, which takes about 14-21 days, and it is easily affected by the environment. There are often problems of unstable disease in different repetitions or batches. The root immersion method is more complicated to operate, and it needs to be transplanted after root immersion. The workload is large, and similar to the root injury method, it has the limitations of long disease cycle, susceptibility to environmental influences and instability. Although the stem base injection method is simple and fast, the operation of injecting the bacterial solution into the base of the plant stem is prone to uneven inoculation, and it is easy to cause excessive damage to the plant, resulting in plant growth and disease, resulting in inconsistent test results.

[0005] Therefore, how to establish a set of rapid, efficient, stable and suitable resistance identification technology for large quantities of materials is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for identifying resistance to bacterial wilt by injection inoculation of leaves of Solanaceae crops at the seedling stage, so as to overcome the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for identifying resistance to bacterial wilt by injection inoculation of leaves of Solanaceae crops at the seedling stage, specifically comprising the following steps:

[0009] (1) Preparing Solanaceae crop seedlings

[0010] Prepare and select uniformly grown, 5-6 leaf Solanaceae plant seedlings;

[0011] (2) Preparation of Ralstonia solanacearum suspension

[0012] (3) Leaf injection inoculation

[0013] Select two symmetrical lateral leaflets near the leaf axis in the middle of the Solanaceae crop plant, use a needle to make a small hole on the back of the leaf, and inject the solanacearum suspension into the leaf through the small hole in the leaf;

[0014] (4) Identification of resistance to bacterial wilt

[0015] After 7 days, the incidence of the disease was counted and the disease index was calculated to identify resistance to bacterial wilt.

[0016] Furthermore, in the above step (1), the Solanaceae crops include tomato, pepper, tobacco, nightshade, potato and eggplant.

[0017] Furthermore, the above step (1) is specifically as follows: the seeds of the Solanaceae crop are divided into 2 mL centrifuge tubes, an appropriate amount of double distilled water is added, the seeds are germinated in a constant temperature water bath at 55° C. for 15 min, and then dispersed on qualitative filter paper soaked in sterile water in a culture dish, gibberellin is added, and the seeds are placed in a culture box in dark light for cultivation. After 7 days, the seedlings with uniform germination and growth are selected and transplanted into potting soil, and the seeds are planted for about 5 weeks until they have 5-6 leaves.

[0018] Further, the above step (2) is specifically as follows: streak a small amount of Ralstonia solanacearum stock solution onto a TTC medium plate with a sterile inoculation loop, place the plate upside down in a 30°C incubator, culture for 2 days, pick a single colony in NB liquid culture medium, place the centrifuge tube in a 30°C shaking incubator, shake and culture at 180 rpm for 24 hours, then take out the centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, collect the bacteria, wash three times with sterile water, add an appropriate amount of sterile water to resuspend the bacteria, measure the OD600 value with an ultraviolet spectrophotometer, and dilute the Ralstonia solanacearum suspension to 1×10 8 CFU·mL -1 (OD600=0.1) was used for inoculation.

[0019] The further beneficial effects of the above are as follows. After injecting and inoculating the leaves of solanaceous crops with Ralstonia solanacearum, the resistance of plants can be stimulated. For example, the expression of disease-resistant genes such as SlPR1a, SlPR4, and SlPRb in resistant plants increased significantly 24 hours after inoculation. Based on the research result that Ralstonia solanacearum can rapidly infect plants through cotyledons at the seedling stage, the present invention established a method for efficiently identifying the resistance to bacterial wilt of solanaceous crops at the seedling stage based on leaf injection inoculation.

[0020] Further, step (3) above is specifically as follows: Select two symmetrically lateral small leaves near the leaf axis of the second and third compound leaves of solanaceous crop plants. Use the needle of a 1 mL syringe to make a small opening on the back of the leaf without piercing the leaf, and then use a syringe without a needle to suck the Ralstonia solanacearum suspension. Inject the Ralstonia solanacearum suspension into the leaf from the small opening of the leaf, and inject 0.5 mL of the Ralstonia solanacearum suspension into each leaf.

[0021] The further beneficial effects of the above are as follows. In the present invention, the Ralstonia solanacearum suspension is injected into the leaf from the wound of the leaf. This inoculation method is rapid, efficient, simple to operate, the results are stable and reliable, and it can be implemented on various solanaceous crops, providing a new means for the resistance identification of a large number of solanaceous crop disease-resistant germplasm resources.

[0022] Further, in step (4) above, the statistical method for the disease occurrence situation is as follows: On the premise that the plants without inoculation treatment grow normally, by observing the disease occurrence situation of the leaves and stems of solanaceous crop plants 7 days after inoculation, evaluate the disease occurrence level according to the disease transmission degree in the whole plant. Among them, 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.

[0023] Further, in step (4) above, the calculation formula for the disease index is: disease index = [∑(the number of diseased plants at each level × the corresponding disease level)] / (the highest disease level × the total number of inoculations).

[0024] Further, in step (4) above, the classification standard for the resistance to bacterial wilt is as follows: 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.

[0025] It can be seen from the above technical solutions that compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The method of the present invention is highly efficient and easy to operate, and can be used to quickly identify the bacterial wilt resistance of breeding materials of Solanaceae crops at the seedling stage under indoor conditions. Experimental results show that the disease resistance identification results can be obtained in only 7 days, and it is particularly suitable for the identification of bacterial wilt resistance of large-scale populations.

[0027] 2. The resistance identification results of the present invention are stable, reliable and easy to distinguish. The experimental results show that the leaf injection method of the present invention can achieve rapid and stable infection of Ralstonia solanacearum without damaging the main rhizomes of the plants, thereby minimizing the influence of human and environmental factors. In addition, its accuracy is higher than that of plants treated by the root injury inoculation method and the root immersion method, and can more truly reflect the resistance and sensitivity phenotype of the test plants.

[0028] 3. The method of the present invention can successfully and quickly induce plant resistance. Molecular experiments have shown that after leaf injection inoculation, the expression of disease resistance-related genes in resistant varieties is significantly higher than that in susceptible varieties. Therefore, it can also be further used for the study of the mechanism of disease resistance molecules in germplasm resources.

[0029] 4. The method of the present invention has wide applicability and can successfully infect different Solanaceae crops, providing a new candidate method for screening disease-resistant germplasm resources of Solanaceae crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Symptom differences (top) and plant mortality comparison (bottom) after leaf injection of eight resistant and susceptible tomato varieties.

[0031] Figure 2 Comparison of disease symptoms of AC and Guizhen No. 1 inoculated by leaf injection, root wound inoculation and root immersion respectively;

[0032] Figure 3 The difference in symptoms between AC and Guizhen No. 1 after inoculation with Ralstonia solanacearum; A: The difference in symptoms between AC and Guizhen No. 1 after leaf injection inoculation; B: The wilting rate of plants after leaf injection inoculation of AC and Guizhen No. 1, the red line represents AC, and the blue line represents Guizhen No. 1; C: The relative accumulation of Ralstonia solanacearum in the stems 1d, 3d, 5d and 7d after leaf injection inoculation of AC and Guizhen No. 1;

[0033] Figure 4 The expression analysis of tomato disease resistance-related genes SlPR1a, SlPR4 and SlPRb;

[0034] Figure 5 Phenotypes of leaves of different Solanaceae crops after injection with Ralstonia solanacearum. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 creative work are within the scope of protection of the present invention.

[0036] In the following examples and comparative examples:

[0037] 1. Test strains

[0038] Five strains of Ralstonia solanacearum that are representative of the region and have significant differences in virulence in Fujian were used as identification strains. The test strains were isolated and stored at the Institute of Plant Protection, Fujian Academy of Agricultural Sciences (-80°C). The details are shown in Table 1.

[0039] Table 1 Tested bacterial strains

[0040]

[0041] 2. Test tomatoes and tomato rootstocks

[0042] As shown in Table 2.

[0043] Table 2 Tomatoes and tomato rootstocks

[0044]

[0045]

[0046]

[0047] Among them, AC, Heinz1706, Tom Tomato, Z164, Bell Pepper, Ben's Tobacco, Yunyan 87 and Hangqie No. 1 were provided by the Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Minshu No. 2 was provided by the Crop Research Institute, Fujian Academy of Agricultural Sciences, and rootstock 1 to rootstock 10 were provided by the Crop Research Institute, Fujian Academy of Agricultural Sciences and Xiamen Ruyi Seedling Co., Ltd. Other materials such as Millennium and Guizhen No. 1 were purchased commercial varieties.

[0048] 3. Test culture medium

[0049] (1) NB liquid medium: 5.0 g / L glucose, 1.0 g / L casein hydrolysate, 10.0 g / L peptone. Adjust pH to 7.0 and sterilize at 121°C for 15 min.

[0050] (2) NA solid medium: Same as NB liquid medium, add 15.0 g / L agar powder before sterilization;

[0051] (3) TTC medium: Take the sterilized NA solid medium, wait until the temperature drops to about 50°C, add 2 mL of 0.5% TTC aqueous solution, mix and shake to obtain TTC medium.

[0052] Among them, 0.5% TTC formula: weigh 2,3,5-triphenyltetrazolium chloride (TTC) and dissolve it in sterile water to make the final concentration of 0.5%, filter and sterilize it with a 0.22 μm filter membrane, and store it in a 4°C refrigerator away from light.

[0053] 4. RT-qPCR amplification primers

[0054] As shown in Table 3.

[0055] Table 3 RT-qPCR primer sequences

[0056]

[0057] Example 1

[0058] The method for identifying resistance to bacterial wilt of Solanaceae crops by leaf injection inoculation at the seedling stage (leaf injection method) specifically comprises the following steps:

[0059] (1) Preparing Solanaceae crop seedlings

[0060] Pour full and disease-free tomato seeds into 2 mL centrifuge tubes, add double distilled water, germinate in a constant temperature water bath at 55°C for 15 min, and then spread on two layers of sterilized qualitative filter paper soaked in sterile water in a 9 cm culture dish. Add 2 mL of 100 mg·L -1 Gibberellic acid, then place the culture dish in a 25℃ light incubator for dark light culture. After 7 days, select the seedlings with consistent germination and growth and transplant them into potting soil. Plant them for 5 weeks until they have 5-6 leaves.

[0061] (2) Preparation of Ralstonia solanacearum suspension

[0062] The test strains RS210507F (Fuzhou City), RS240728F (Ningde City), RS220524F (Putian City), RS230928F (Sanming City) and RS240426F (Zhangzhou City) stored at -80°C were taken out, and the stock solution of Ralstonia solanacearum was streaked onto TTC medium plates with sterile inoculation loops. The plates were inverted in a 30°C incubator. After culturing for 2 days, single colonies were picked in NB liquid culture medium, and the centrifuge tubes were placed in a 30°C shaking incubator and shaken at 180 rpm for 24 h. The centrifuge tubes were then taken out and centrifuged at 6000 rpm for 5 min. The supernatant was discarded, the cells were collected, washed three times with sterile water, and the cells were resuspended in sterile water. The OD600 value was measured with an ultraviolet spectrophotometer, and the Ralstonia solanacearum suspension was diluted to 1×10 8 CFU·mL -1(OD600 = 0.1), mix in equal volumes to obtain a mixed Ralstonia solanacearum suspension for inoculation;

[0063] (3) Leaf injection inoculation

[0064] Select two symmetric lateral leaflets near the leaf axis of the second and third compound leaves of the tomato plant. Use a 1 mL syringe needle to make small openings on the back of the leaf without piercing the leaf. Then, use a syringe without the needle to suck up the mixed Ralstonia solanacearum suspension and inject the mixed Ralstonia solanacearum suspension into the leaf through the small opening on the leaf. Inject 0.5 mL of the mixed Ralstonia solanacearum suspension into each leaf;

[0065] (4) Identify the resistance to bacterial wilt

[0066] Seven days later, count the disease incidence and calculate the disease index to identify the resistance to bacterial wilt;

[0067] Among them, the statistical method for disease incidence is as follows: On the premise that the plants without inoculation treatment grow normally, observe the disease incidence of the leaves and stems of the tomato plants 7 days after inoculation, and evaluate the disease incidence level according to the disease transmission degree in the whole plant. Among them, level 0: no disease, no symptoms; level 1: the 4 lateral leaflets inoculated 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 whole plant wilt;

[0068] The calculation formula for the disease index is: Disease index = [∑(number of diseased plants at each level × corresponding disease level)] / (highest disease level × total number of inoculations);

[0069] The classification criteria for the resistance to bacterial wilt are as follows: 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.

[0070] Comparative example 1

[0071] The difference from Example 1 is only that step (3) is replaced by the root - wounding inoculation method: Before inoculation, use a scalpel to gently cut two knives around the roots of the tomato plant to artificially damage the root system of the plant, and then pour 10 mL of the mixed Ralstonia solanacearum suspension with OD600 = 0.1 around the root and stem of the plant.

[0072] Comparative example 2

[0073] The difference from Example 1 is only that step (3) is replaced by the root - dipping method: Immerse the roots of the tomato seedlings planted for 3 weeks in 20 mL of the mixed Ralstonia solanacearum suspension with OD600 = 0.1 for 15 minutes, and then replant the plants in the sterilized nutrient soil.

[0074] Performance Testing

[0075] 1. Feasibility of the leaf injection method of the present invention

[0076] First, eight tomato varieties with obvious differences in resistance and susceptibility to bacterial wilt were screened out by the root wound inoculation method in comparative example 1, among which AC, Qiangli, Tom, Qianxi and Heinz 1706 were susceptible, while Guizhen No. 1, Z164 and Wilsingen were resistant. Then, inoculation tests were carried out according to the leaf injection method in example 1, and the incidence was counted after 14 days, with three replicates. The results are shown in Figure 1. Figure 1 shown.

[0077] Depend on Figure 1 It can be seen that 14 days after leaf injection inoculation with Ralstonia solanacearum, the disease results of resistant and susceptible varieties were consistent with the screening results through root injury inoculation, and the difference between resistance and sensitivity was very obvious. 14 days after inoculation, the mortality rate of susceptible varieties inoculated by leaf injection method was slightly higher than that by root injury inoculation method, but there was no significant difference. In addition, it was observed that the symptoms of bacterial wilt were obvious enough after 7 days, the difference between replicates was small, the results were stable, reliable and easy to statistically analyze, and it can be applied to the rapid batch screening of germplasm resources resistant to bacterial wilt in the laboratory.

[0078] 2. Comparison of the leaf injection method of the present invention with the root injury inoculation method and the root immersion method

[0079] 1. Test tomatoes and tomato rootstocks: AC (susceptible) and Guizhen No. 1 (resistant)

[0080] 2. The three methods of inoculation of Ralstonia solanacearum are as follows:

[0081] Root injury inoculation method: as in comparative example 1, the incidence of disease was counted after 14 days and repeated 3 times.

[0082] Root immersion method: as in comparative example 2, the incidence of disease was counted after 10 days and repeated 3 times.

[0083] Leaf injection method: as in Example 1, the incidence was counted after 7 days, and repeated 3 times.

[0084] The results are as follows Figure 2 shown.

[0085] Depend on Figure 2 It can be seen that after 14 days of inoculation by wounded root inoculation, the incidence of AC was 50%, while Guizhen No. 1 was not sick; after 10 days of inoculation by root dipping method, some AC plants' leaves wilted, while Guizhen No. 1 was not sick; after 7 days of leaf injection inoculation, the incidence of AC reached 95%, while only the leaves of Guizhen No. 1 wilted at the inoculation site. The observation results show that compared with the wounded root inoculation method and the root dipping method, the advantages of the leaf injection method are simple operation, small inoculation amount, faster onset of bacterial wilt and clearer symptoms.

[0086] 3. Construction of the resistance identification technology system of the leaf injection method of the present invention

[0087] 1. Two varieties (AC and Guizhen No. 1) with obvious differences in resistance and susceptibility were selected, and the whole plant phenotypes were observed and compared for 7 days after leaf injection inoculation in Example 1. The results are as follows Figure 3 shown.

[0088] Depend on Figure 3 It can be seen that the leaves of AC and Guizhen No. 1 showed wilting symptoms at the inoculation site 1 day after injection; 3 days after injection, the leaves on the same leaf axis as the inoculated leaves of AC plants showed wilting symptoms, while Guizhen No. 1 did not show this symptom; 5 days after injection, except for the top leaves of AC plants, all other leaves wilted, while only the leaves at the inoculation site of Guizhen No. 1 wilted, and the plant still grew normally; 7 days after injection, the leaves of AC wilted, while only the leaves at the inoculation site of Guizhen No. 1 wilted, which did not affect its normal growth ( Figure 3 Meanwhile, the wilting rate of susceptible varieties AC reached 100% 6 days after inoculation ( Figure 3 (middle B).

[0089] Bacterial wilt is a vascular disease. In order to further clarify that leaf-injected Ralstonia can spread to the stems of plants through leaves, the stems of plants were cut at 1d, 3d, 5d and 7d after inoculation, and the relative accumulation of Ralstonia in the stems of plants was detected by RT-qPCR. The results showed that Ralstonia could be detected in the stems of susceptible variety AC and resistant variety Guizhen No. 1 3d after inoculation, and the content of Ralstonia in the stems of AC plants increased rapidly at 5d and 7d after inoculation compared with the relative accumulation of Ralstonia at 1d and 3d, but the content of Ralstonia in the stems of Guizhen No. 1 plants was lower at 3d, 5d and 7d after inoculation ( Figure 3 Middle C).

[0090] The above results show that the leaf injection method of the present invention can quickly and efficiently cause tomato plants to become diseased, and the symptoms are obvious and easy to distinguish, and is suitable for batch inoculation of tomato germplasm materials.

[0091] 2. The leaf injection method of Example 1 was used to inoculate 16 tomato materials for testing, and the identification results were compared with those of the root injury inoculation method of Comparative Example 1. The results are shown in Table 4.

[0092] Table 4 Phenotypes of tested tomato rootstocks in leaf injection and root wound inoculation

[0093]

[0094]

[0095] As shown in Table 4, the course of the disease of Ralstonia solanacearum inoculated by leaf injection method is clear, the difference between repetitions is smaller than that by root injury inoculation method, and the difference between resistant and susceptible varieties is more obvious, and the difference in the incidence of resistant and medium-resistant varieties can be clearly observed. The results of resistance identification showed that rootstock 6, rootstock 8 and rootstock 10 showed resistance; rootstock 1, rootstock 3, rootstock 4, rootstock 5, rootstock 7 and rootstock 9 showed medium resistance; tomato rootstock 6, tomato rootstock 405 and rootstock 2 showed susceptible. In summary, the leaf injection method can more truly reflect the resistance and susceptible phenotypes of the tested plants.

[0096] IV. Effect of the leaf injection method of the present invention on the expression of genes related to tomato resistance to bacterial wilt

[0097] The eight tomato varieties with obvious differences in resistance and susceptibility to bacterial wilt in "I. Feasibility of the leaf injection method of the present invention" were used for further analysis of the differential expression of disease resistance genes. Figure 4 shown.

[0098] Depend on Figure 4 It can be seen that 24 hours after leaf injection inoculation, the transcription levels of SlPR1a, SlPR4 and SlPRb genes in Guizhen No. 1, Anvil 4, Anvil 6, Anvil 7, Anvil 8 and Anvil 10 were significantly higher than those in AC, while the transcription levels of SlPR1a, SlPR4 and SlPRb genes in Anvil 2 were not significantly different from those in AC. This is consistent with the resistance results obtained by identification, further illustrating that the leaf injection method resistance identification system established by the present invention has certain reliability.

[0099] V. Applicability Analysis of the Leaf Injection Method of the Present Invention

[0100] In view of the need for identification of bacterial wilt resistance in other Solanaceae crops, the leaf injection method of Example 1 was applied to inoculate different Solanaceae crops such as Nicotiana benthamiana, Sanshengyan (Yunyan 87), pepper (sweet pepper), Solanum nigrum, potato (Minshu No. 2) and eggplant (Hangqie No. 1) to observe whether Ralstonia solanacearum can infect plants through leaf injection. The results are as follows Figure 5 shown.

[0101] Depend on Figure 5 It can be seen that 7 days after inoculation, all six tested Solanaceae crops showed varying degrees of wilt, which is consistent with the typical symptoms of bacterial wilt. The above results show that the leaf injection method of inoculation of Ralstonia solanacearum can successfully infect Solanaceae crops and cause rapid disease, providing a new candidate method for screening disease-resistant germplasm resources of Solanaceae crops.

[0102] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying resistance to bacterial wilt by injection inoculation of leaves of Solanaceae crops at the seedling stage, characterized in that: Specifically, it includes the following steps: (1) Prepare solanaceous crop seedlings Prepare and select seedlings of solanaceous crop plants with consistent growth and 5 - 6 leaves; (2) Prepare a suspension of Ralstonia solanacearum (3) Inoculate by leaf injection Select two symmetrically lateral small leaves near the leaf axis at the middle part of the solanaceous crop plant, make small openings on the back of the leaf with a needle, and inject the suspension of Ralstonia solanacearum into the leaf through the small openings on the leaf; (4) Identify the resistance to bacterial wilt After 7 days, count the disease incidence and calculate the disease index to identify the resistance to bacterial wilt.

2. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: In step (1), the solanaceous crops include tomato, pepper, tobacco, Solanum nigrum, potato and eggplant.

3. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: Step (1) is specifically as follows: Pour the seeds of solanaceous crops into 2 - mL centrifuge tubes respectively, add double - distilled water, incubate in a constant - temperature water bath at 55 °C for 15 min for germination, then disperse them on the qualitative filter paper moistened with sterile water in a petri dish, add gibberellin, and culture them in the dark in an incubator. After 7 days, select the seedlings with consistent germination growth and transplant them into potted soil, and plant for 5 weeks until they have 5 - 6 leaves.

4. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: The step (2) is specifically as follows: streak the stock solution of Ralstonia solanacearum onto a TTC medium plate with a sterile inoculation loop, place the plate upside down in a 30°C incubator, culture for 2 days, pick a single colony in NB liquid culture medium, place the centrifuge tube in a 30°C shaking incubator, shake and culture for 24 hours at 180 rpm, then take out the centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, collect the bacteria, wash three times with sterile water, add sterile water to resuspend the bacteria, measure the OD600 value with an ultraviolet spectrophotometer, and dilute the Ralstonia solanacearum suspension to 1×10 8 CFU·mL -1 Used for vaccination.

5. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: Step (3) is specifically as follows: Select two symmetrically lateral small leaves near the leaf axis of the 2nd and 3rd compound leaves of the solanaceous crop plant, make small openings on the back of the leaf without piercing the leaf with a 1 - mL syringe needle, then suck the suspension of Ralstonia solanacearum with a syringe without a needle, and inject the suspension of Ralstonia solanacearum into the leaf through the small openings on the leaf, and inject 0.5 mL of the suspension of Ralstonia solanacearum into each leaf.

6. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: In step (4), the statistical method for the disease incidence is as follows: On the premise of ensuring the normal growth of the non - inoculated plants, by observing the disease incidence of the leaves and stems of the solanaceous crop plants 7 days after inoculation, evaluate the disease incidence level according to the degree of disease spread in the whole plant. Among them, 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.

7. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: In step (4), the calculation formula for the disease index is: Disease index = [∑(number of diseased plants at each level × corresponding disease level)] / (highest disease level × total number of inoculations).

8. The method for identifying resistance to bacterial wilt of Solanaceae crops by injection inoculation of leaves at seedling stage according to claim 1, characterized in that: In step (4), the classification standard for the resistance to bacterial wilt is: Immune: DI = 0; Highly resistant: 0 < DI < 12.5; Resistant: 12.5 ≤ DI < 25; Moderately resistant: 25 ≤ DI < 50; Susceptible: 50 ≤ DI < 75; Highly susceptible: 75 ≤ DI ≤ 100.

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