Watermelon breeding method for improving disease resistance

Abstract

The invention relates to a watermelon breeding method for improving disease resistance, which comprises the following steps: S1, establishing a plurality of groups of parallel evolution systems, each group comprising a donor region and a verification region; s2, inoculating the ith virus to the ith group of donor region plants (i = 1, 2... N); s3, after inoculation, an in-vitro leaf identification method is adopted to eliminate plants; s4, extracting dead plant viruses; s5, respectively inoculating the extracted strain and the original strain to the plants in the verification area, if the evolutionary virus disease is more serious, replacing the original virus with the evolutionary virus, inoculating the evolutionary virus to the donor area, and skipping to execute S3; s6, selfing the same group of plants to cultivate seedlings; and S7, hybridizing the watermelon plants in different groups in pairs, planting watermelon seedlings cultivated by hybridizing the two groups in a new donor area and a verification area, inoculating watermelon viruses inoculated by donors of previous generations to the watermelon plants, and skipping to execute the step S3 until the last group is left. Watermelon seedlings with stable antibodies of various protoviruses and evolutionary viruses can be cultivated in a short time.

Description

Technical Field

[0001] The invention belongs to the technical field of test equipment, and in particular relates to a watermelon breeding method with improved disease resistance. Background Art

[0002] Watermelon, one of the most popular summer fruits, has long suffered from severe damage to its yield and quality due to viral diseases. According to statistics, viral diseases such as cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and squash leaf curl virus (SqMV) reduce global watermelon production by 40%-60% annually, with incidence rates exceeding 60% in protected cultivation and continuous cropping areas. These viruses cause leaf mosaics, deformities, fruit shrinkage, and even plant death, directly leading to a decrease in marketable fruit yield.

[0003] Currently, watermelon cultivation relies primarily on chemical pesticides for pest control, but these methods have significant limitations. Traditional control methods primarily rely on spraying antibiotics (such as Ningnanmycin and Virus A). While these drugs can inhibit viral proliferation, long-term use can easily lead to drug resistance in the virus. Studies have shown that after two growing seasons of continuous use of the same pesticide, the frequency of drug-resistant genes in the virus population can rise to over 60%, resulting in a 40%-50% decrease in control effectiveness. Furthermore, drug residues can potentially harm human health through the food chain. For example, Ningnanmycin has a half-life of 7-10 days in watermelon fruit. If used within 7 days before harvest, its residues may exceed the EU limit of 0.01 mg / kg.

[0004] Based on the above, there is an urgent need to cultivate a watermelon seedling with stable antibodies to multiple original viruses and evolved viruses to reduce the harm of viruses to watermelon plants and improve the safety of watermelon consumption. Summary of the Invention

[0005] The present invention provides a watermelon breeding method for improving disease resistance, which can cultivate watermelon seedlings with stable antibodies to multiple original viruses and evolved viruses in a short period of time, thereby reducing the impact of viruses on watermelon plants and laying a foundation for green food cultivation.

[0006] The technical solution adopted by the present invention is:

[0007] A watermelon breeding method for improving disease resistance comprises the following steps:

[0008] S1. Obtain N types of watermelon viruses and establish multiple parallel evolution systems, each containing a donor region and a validation region;

[0009] S2, inoculating the i-th virus into the i-th group of donor area plants (i=1, 2...N);

[0010] S3, after inoculation, the plants were eliminated using the detached leaf identification method;

[0011] S4. Applying immune inducers to the plants that have not been eliminated and extracting viruses from the dead plants;

[0012] S5. Inoculate the extracted virus strain and the original virus strain into the watermelon plants in the verification area respectively. If the symptoms of the evolved virus are more severe, define the extracted virus as the evolved virus, replace the original virus with the evolved virus and inoculate it into the donor area, and jump to S3.

[0013] S6. Self-pollination of surviving plants in the donor area to cultivate watermelon seedlings;

[0014] S7. Hybridize watermelon plants of different groups in pairs, with one group hybridized only once. Plant the watermelon seedlings cultivated from the two hybridization groups in the new donor area and verification area of ​​the same group. Inoculate the watermelon plants with the watermelon virus that has been inoculated by their previous donors. Jump to S3 and execute until the last group remains.

[0015] Furthermore, the detached leaf identification method is performed as follows:

[0016] (a) Take the top young leaves of the donor area, disinfect their surface, and place them on MS medium containing 2% sucrose;

[0017] (b) The back of the leaf was inoculated with the same virus as the donor most recently inoculated;

[0018] (c) culturing at 25°C for 48 h under light;

[0019] (d) Number of lesions ≥ 4 / cm 2 Or if the lesion area is ≥25%, it will be eliminated.

[0020] Furthermore, the immune inducer is a 0.1 mM salicylic acid + 50 μg / mL chitosan oligosaccharide compound solution, and the spraying amount is 5 mL / plant.

[0021] Furthermore, in step S5, when the number of evolved virus spots on a single leaf increases by more than 15%, the extracted virus is defined as an evolved virus.

[0022] Furthermore, watermelon viruses include CMV, WMV, PRSV, and SqMV;

[0023] After step S7, step S8 is further performed to detect the resistance gene sites SNP_CMV-R, SNP_WMV-R, SNP_PRSV-R, and SNP_SqMV-R, and retain the individual plants containing all four sites.

[0024] Furthermore, in step S7, before hybridizing the two donor areas, the male flowers of the watermelon plants in one donor area are removed, and the female flowers of the watermelon plants in the other donor area are removed, and artificial directional pollination is performed and the plants are isolated by bagging.

[0025] Furthermore, the volume ratio of the viruses applied in the previous generations in step S7 is calculated according to the following formula:

[0026]

[0027] P k : Volume ratio coefficient of the kth generation virus;

[0028] m: number of viruses;

[0029] k: current virus generation number (k=1, 2, ..., m).

[0030] Furthermore, the inoculation concentration of the latest generation is 10 4 CFU / mL±10%; inoculation volume is 2 μL / strain.

[0031] Furthermore, in step S7, after the last group of watermelons bears fruit, watermelon seedlings are cultivated using watermelon seeds.

[0032] Furthermore, S8, watermelon seeds with a single watermelon weight not less than the average weight of the watermelon variety and a center sugar content not less than the average center sweetness of the watermelon variety are selected for seed preservation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Achieving directed evolution of broad-spectrum disease resistance

[0035] Through multiple parallel evolutionary systems and iterative viral evolution mechanisms, the virus is forced to evolve into a more virulent strain under the pressure of immune inducers. The resistance genes of surviving plants undergo extreme screening by the "evolved virus," gaining dual resistance to the original strain and potential variants, breaking through the bottleneck of traditional breeding, which often results in single-strength resistance and is easily ineffective.

[0036] 2. Efficient aggregation of multiple resistance genes

[0037] Through pairwise hybridization and mixed virus attack, the hybrid offspring are required to simultaneously resist all previous generations of viruses of the parental group (including evolved strains). Through forced hybridization aggregation and cross-generational virus stress testing, the synergistic superposition of N types of virus resistance genes is quickly achieved, cultivating germplasm resources with a wider range of resistance coverage.

[0038] 3. Establish a proactive disease resistance defense system

[0039] Using the "Evolved Virus Validation Zone" to simulate the natural evolutionary path of viruses, we reverse-screen plant resistance using more virulent strains. The resulting varieties possess advanced adaptability, significantly reducing the risk of resistance failure due to future viral mutations.

[0040] 4. Breakthrough in the efficiency bottleneck of disease-resistant breeding

[0041] The in vitro leaf identification method is used to achieve high-throughput early screening, and a parallel system is used to simultaneously process multiple pathogens; immune inducers are used to activate potential resistance, and the watermelon seedlings cultivated in a single system are hybridized in pairs and then eliminated, so that the hybridized seedlings inherit the antibodies of the two donors. The watermelon seedlings are then inoculated with all the virus antibodies that have been inoculated by their previous donors. After the watermelon seedlings are cultivated, the two new systems are hybridized again until the last group remains. This not only greatly shortens the cultivation time, but also ensures that the finally cultivated watermelon seedlings have multiple stable virus antibodies. DETAILED DESCRIPTION

[0042] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0043] Example

[0044] The present invention provides a watermelon breeding method for improving disease resistance, comprising the following steps:

[0045] S1. Obtain N types of watermelon viruses and establish multiple parallel evolution systems, each containing a donor region and a validation region;

[0046] S2, inoculating the i-th virus into the i-th group of donor area plants (i=1, 2...N);

[0047] S3, after inoculation, the plants were eliminated using the detached leaf identification method;

[0048] S4. Applying immune inducers to the plants that have not been eliminated and extracting viruses from the dead plants;

[0049] S5. Inoculate the extracted virus strain and the original virus strain into the watermelon plants in the verification area respectively. If the symptoms of the evolved virus are more severe, define the extracted virus as the evolved virus, replace the original virus with the evolved virus and inoculate it into the donor area, and jump to S3.

[0050] S6. Self-pollination of surviving plants in the donor area to cultivate watermelon seedlings;

[0051] S7. Hybridize watermelon plants of different groups in pairs, with one group hybridized only once. Plant the watermelon seedlings cultivated from the two hybridization groups in the new donor area and verification area of ​​the same group. Inoculate the watermelon plants with the watermelon virus that has been inoculated by their previous donors. Jump to S3 and execute until the last group remains.

[0052] Before cultivating seedlings, the types of watermelon viruses that are easily infected can be selected according to the geographical location where the watermelon seedlings need to be planted and the geographical characteristics of the planting area. In this embodiment, four types are set. Four parallel evolution systems are set according to the four virus types. Each group includes a donor area and a verification area. Watermelon seedlings are planted in the donor area and the verification area. The four viruses are inoculated into all watermelon plants in the donor area of ​​the four evolution systems respectively. The in vitro leaf identification method can be used to quickly detect watermelon plants with low antibodies that need to be eliminated. Then, immune inducers are applied to the watermelon plants so that some watermelon plants may produce more virulent evolved viruses. Based on the vast majority of evolved viruses The evolved virus is more potent and will cause the watermelon plant to die. Of course, the cause of watermelon death may also be caused by individual defects or the cultivation environment. Therefore, the virus of the dead plant is extracted, and the extracted strain and the original strain are respectively inoculated into the watermelon plants in the verification area. If it is judged that the symptoms of the evolved virus are more serious, the evolved virus replaces the original virus and is inoculated into the donor area and the process jumps to S3, so that the watermelon plants have antibodies to both the original virus and the evolved virus. This can shorten the time for judging and extracting the evolved virus. However, if the symptoms of the evolved virus are not more serious than those of the original virus, it means that the death is caused by the original virus and individual reasons. All surviving watermelon plants in the donor area are self-pollinated to cultivate watermelon seedlings.

[0053] Based on the above, four groups of seedlings with both original and evolved viral antibodies were cultivated. Then, two of these four groups of seedlings were paired and hybridized, forming two groups for systematic cultivation. The process then skips to step S3 until the last group remains. This method not only significantly shortens the cultivation time of watermelon seedlings, but also ensures that the final watermelon seedlings possess multiple, stable viral antibodies.

[0054] Preferably, the detached leaf identification method is performed as follows:

[0055] (a) Take the top young leaves of the donor area, disinfect their surface, and place them on MS medium containing 2% sucrose;

[0056] (b) The back of the leaf was inoculated with the same virus as the donor most recently inoculated;

[0057] (c) culturing at 25°C for 48 h under light;

[0058] (d) Number of lesions ≥ 4 / cm 2 Or if the lesion area is ≥25%, it will be eliminated.

[0059] In the process of cultivating watermelon seedlings, first, the top young leaves of the donor area plants are taken and the surface is disinfected, and then placed in MS medium containing 2% sucrose to obtain sterile explants and provide nutritional support, thereby creating a suitable in vitro culture environment. This step not only improves the accuracy of the experiment, but also promotes the growth of leaves. Next, the same virus as the donor area is rubbed and inoculated on the back of the leaves to verify the stability of the resistance of the plants in the donor area and screen out plants that are resistant to the virus. At the same time, this step also provides experimental materials for studying the interaction between viruses and plants. Subsequently, the inoculated leaves are cultured under light conditions of 25°C for 48 hours to provide a suitable growth environment and observe the virus infection process, thereby accelerating the virus infection and facilitating subsequent observation and recording. According to the threshold value of the number and area of ​​lesions (the number of lesions ≥4 / cm 2 or lesion area ≥ 25%), and timely eliminate plants that are sensitive to viruses to ensure that the varieties finally cultivated have higher virus resistance and stability.

[0060] Preferably, the immune inducer is a mixture of 0.1 mM salicylic acid and 50 μg / mL chitosan oligosaccharide, with a spraying amount of 5 mL / plant.

[0061] Spraying an immune inducer composed of 0.1mM salicylic acid and 50μg / mL chitosan oligosaccharides can induce stronger resistance in plants. This combination may also produce a synergistic effect, significantly improving plant resistance to viruses and reducing the occurrence and harm of viral diseases. Furthermore, this inducer is environmentally safe and residue-free, helping to increase watermelon yield and quality, meeting the requirements of sustainable development in modern agriculture. Furthermore, the pressure from increased plant resistance increases the likelihood of induced viral evolution.

[0062] Preferably, in step S5, when the number of evolved virus spots on a single leaf increases by more than 15%, the extracted virus is defined as evolved virus.

[0063] The evolved virus and the original virus were inoculated into the validation area by friction inoculation for 72 hours, and the increase of virus spots was observed.

[0064] Preferably, watermelon viruses include CMV, WMV, PRSV and SqMV;

[0065] After step S7, step S8 is further performed to detect the resistance gene sites SNP_CMV-R, SNP_WMV-R, SNP_PRSV-R, and SNP_SqMV-R, and retain the individual plants containing all four sites.

[0066] After cultivating disease-resistant watermelon seedlings, the team focused on four viruses that pose significant threats to the watermelon industry: CMV, WMV, PRSV, and SqMV. Using molecular marker-assisted selection (MAM-assisted selection), they detected four SNPs closely linked to resistance genes: SNP_CMV-R, SNP_WMV-R, SNP_PRSV-R, and SNP_SqMV-R. This enabled efficient screening of individual plants carrying all four resistance genes. By retaining individual plants harboring all four loci, the team achieved the aggregation of multiple resistance genes, significantly enhancing the broad-spectrum resistance of watermelons to a variety of viruses.

[0067] Preferably, in step S7, before hybridizing the two donor areas, the male flowers of the watermelon plants in one donor area are removed, and the female flowers of the watermelon plants in the other donor area are removed, and artificial directional pollination and bagging are carried out for isolation, which can avoid the loss of virus resistance due to self-pollination of individual plants.

[0068] Preferably, the volume ratio of the viruses applied in the previous generations in step S7 is calculated according to the following formula:

[0069]

[0070] P k : Volume ratio coefficient of the kth generation virus;

[0071] m: number of viruses;

[0072] k: Current virus generation number, which can also be expressed as the serial number of the current virus inoculation (k=1, 2, ..., m).

[0073] When there are four viruses, the first generation virus = 0.4, the second generation virus = 0.3, the third generation virus = 0.2, and the fourth generation virus = 0.1.

[0074] By calculating the proportion of viral volume applied over generations, we achieve differentiated regulation of the viral inoculation ratio across generations. This design can compensate for the loss of viral resistance during reproductive inheritance and further awaken resistance genes. Because this formula retains viral components across all generations, it also effectively maintains the diversity of the viral population.

[0075] Preferably, the initial inoculum concentration is 10 4 CFU / mL±10%; inoculation volume is 2 μL / strain.

[0076] Based on the first-generation virus inoculation volume of 2μL / strain, the second-generation inoculation volume is 0.15μL / strain, the third-generation inoculation volume is 0.1μL / strain, and the fourth-generation inoculation volume is 0.5μL / strain.

[0077] Preferably, in step S7, after the last group of watermelons bears fruit, watermelon seedlings are grown from watermelon seeds. Growing watermelon seedlings from watermelon seeds is beneficial to the inheritance of virus resistance.

[0078] Preferably, S8, select watermelon seeds for seed preservation whose single watermelon weight is not less than the average weight of the watermelon variety and whose center sugar content is not less than the average center sweetness of the watermelon variety.

[0079] By selecting high-quality seeds of fruits and vegetables for seedling cultivation, we can not only improve the plant's resistance to pesticides, but also help ensure the quality of the fruit.

[0080] Comparative Example:

[0081] The difference from the embodiment is that:

[0082] In S7, the four evolutionary systems are hybridized in pairs to form two evolutionary systems, and the two evolutionary systems are hybridized in pairs to produce offspring as watermelon seedlings for planting;

[0083] That is, remove the step of "inoculating the watermelon plants with the watermelon virus that has been inoculated by their previous donors, and jump to executing S3 until the last group remains" for each evolutionary system generated by hybridization.

[0084] That is, to remove the viral resistance that has been activated through the following generations:

[0085] Preferably, the volume ratio of the viruses applied in the previous generations in step S7 is calculated according to the following formula:

[0086]

[0087] P k : Volume ratio coefficient of the kth generation virus;

[0088] m: number of viruses;

[0089] k: Current virus generation number, which can also be expressed as the serial number of the current virus inoculation (k=1, 2, ..., m).

[0090] Test Example 1:

[0091] Control group 1: 2 mu of "Black Beauty" watermelon seedlings that were not cultivated and treated by the present invention were planted, and the 2 mu were defined as control group 1 and control group 2 respectively.

[0092] Control group 2: 2 mu of “Black Beauty” watermelon seedlings that were cultivated and treated in the control ratio were planted, and the 2 mu were defined as control group 3 and control group 4 respectively.

[0093] Experimental group: 2 mu of first-generation "Black Beauty" watermelon seedlings cultivated according to the embodiment of the present invention were planted, and the 2 mu were defined as experimental group 1 and experimental group 2 respectively.

[0094] There are 6 mu of watermelon fields, and watermelons are planted in the traditional way on each mu with the same number of seedlings.

[0095] Thirty days after planting the seedlings, each virus was tested at a concentration of 10 4 CFU / mL±10%, 2μL / plant, after mixing the four viruses, spray the mixed virus on the watermelon plants between 19:00 and 20:00 in the afternoon.

[0096] The following experimental results were obtained by counting the watermelon yield per mu (single watermelon more than 2kg) and the mortality rate of watermelon plants after virus inoculation:

[0097] Group Mortality rate Watermelon production (single watermelon more than 2kg) Control group 1 71.2％ 351kg Control group 2 69.5％ 241kg Control group 3 35.5％ 1599kg Control group 4 33.6％ 1580kg Experimental group 1 0.5％ 2575kg Experimental Group 2 0.6％ 2555kg

[0098] From the above trial planting results, it can be concluded that when four viruses are used to attack at the same time, the embodiment of applying mixed viruses after optimizing the ratio of successive viruses through cultivation in the embodiment can effectively activate and improve the resistance to viruses without damaging the watermelon plants, and can also maintain a large yield.

[0099] When watermelon seedlings that had been optimized through comparative cultivation were resistant to four viruses at the same time, the disease mortality rate increased significantly after planting and the yield of large fruits decreased significantly because the cultivation process did not involve differentiated regulation of the virus inoculation ratios of different generations to activate antibodies.

[0100] The control groups 1 and 2 had high mortality rates because the seedlings were not treated for toxins, and the remaining watermelon plants were excessively damaged, which affected the quality of the large fruits and led to a strict reduction in the production of large fruits.

[0101] Test Example 2:

[0102] While carrying out Test Example 1, Test Example 2 was carried out simultaneously.

[0103] After self-pollination of the watermelon seedlings cultivated in the embodiment of the present invention, five generations of "Black Beauty" seedlings were cultivated, and the same was trial-planted in 5 seedlings.

[0104] Thirty days after planting the seedlings, each virus was tested at a concentration of 10 4 CFU / mL±10%, 2μL / plant, after mixing the four viruses, spray the mixed virus on the watermelon plants between 19:00 and 20:00 in the afternoon.

[0105] The watermelon yield per mu (single watermelon more than 2 kg) and the mortality rate of watermelon plants after virus inoculation were counted.

[0106] Group Mortality rate Watermelon production (single watermelon more than 2kg) generation 0.5％ 2575kg Second generation 0.6％ 2555kg Three generations 0.6％ 2550kg Four generations 0.5％ 2496kg Five Dynasties 0.9％ 2467kg

[0107] The test results show that the mortality rate of watermelon seedlings cultivated by the present invention increased slightly in the fifth generation, but had no significant effect on yield. Therefore, the watermelon seedlings cultivated by the present invention can be guaranteed to have good drug resistance within five generations.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A watermelon breeding method for improving disease resistance, characterized in that: The following steps are involved: S1. Obtain N types of watermelon viruses and establish multiple parallel evolution systems, each containing a donor region and a validation region; S2, inoculating the i-th virus into the i-th group of donor area plants (i=1, 2...N); S3, after inoculation, the plants were eliminated using the detached leaf identification method; S4. Applying immune inducers to the plants that have not been eliminated and extracting viruses from the dead plants; S5. Inoculate the extracted virus strain and the original virus strain into the watermelon plants in the verification area respectively. If the symptoms of the evolved virus are more severe, define the extracted virus as the evolved virus, replace the original virus with the evolved virus and inoculate it into the donor area, and jump to S3. S6. Self-pollination of surviving plants in the donor area to cultivate watermelon seedlings; S7. Hybridize watermelon plants of different groups in pairs, with one group hybridized only once. Plant the watermelon seedlings cultivated from the two hybridization groups in the new donor area and verification area of ​​the same group. Inoculate the watermelon plants with the watermelon virus that has been inoculated by their previous donors. Jump to S3 and execute until the last group remains.

2. The watermelon breeding method for improving disease resistance according to claim 1, wherein: The detached leaf identification method is performed as follows: (a) Take the top young leaves of the donor area, disinfect their surface, and place them on MS medium containing 2% sucrose; (b) The back of the leaf was inoculated with the same virus as the donor most recently inoculated; (c) culturing at 25°C for 48 h under light; (d) Number of lesions ≥ 4 / cm 2 Or if the lesion area is ≥25%, it will be eliminated.

3. The watermelon breeding method for improving disease resistance according to claim 1, wherein: The immune inducer is a 0.1 mM salicylic acid + 50 μg / mL chitosan oligosaccharide compound solution, and the spraying amount is 5 mL / plant.

4. The watermelon breeding method for improving disease resistance according to claim 1, wherein: In step S5, when the number of lesions on a single leaf caused by the evolved virus increases by more than 15%, the extracted virus is defined as the evolved virus.

5. The watermelon breeding method for improving disease resistance according to claim 4, characterized in that: Watermelon viruses include CMV, WMV, PRSV, and SqMV; After step S7, step S8 is further performed to detect the resistance gene sites SNP_CMV-R, SNP_WMV-R, SNP_PRSV-R, and SNP_SqMV-R, and retain the individual plants containing all four sites.

6. The watermelon breeding method for improving disease resistance according to claim 1, characterized in that: In step S7, before hybridizing the two donor areas, the male flowers of the watermelon plants in one donor area are removed, and the female flowers of the watermelon plants in the other donor area are removed, and artificial directional pollination is performed and the plants are isolated by bagging.

7. The watermelon breeding method for improving disease resistance according to claim 1, characterized in that: The volume ratio of the viruses applied in the previous generations in step S7 was calculated according to the following formula: P k : Volume ratio coefficient of the kth generation virus; m: number of viruses; k: current virus generation number (k=1, 2, ..., m).

8. The watermelon breeding method for improving disease resistance according to claim 7, characterized in that: The inoculum concentration of the latest generation is 10 4 CFU / mL±10%; inoculation volume is 2 μL / strain.

9. The watermelon breeding method for improving disease resistance according to claim 1, characterized in that: In step S7, after the last group of watermelons bears fruit, watermelon seedlings are cultivated using watermelon seeds.

10. The watermelon breeding method for improving disease resistance according to claim 9, characterized in that: S8. Select watermelon seeds for seed preservation whose single watermelon weight is not less than the average weight of the watermelon variety and whose center sugar content is not less than the average center sweetness of the watermelon variety.

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