WO2020047949A1 - Method for predicting symptom appearance rate and severity of sweet potato viral disease in nursery stage - Google Patents

Abstract

Provided is a method for predicting the symptom appearance rate and severity of a sweet potato viral disease in the nursery stage. The method comprises the following steps: randomly selecting sweet potato seed samples before breeding sweet potatoes; extracting the total DNA and total RNA of the sweet potato seed samples, and detecting the situation of viruses carried by the sweet potato seeds by respectively using PCR and RT-PCR methods; and predicting the symptom appearance rate and severity of a sweet potato viral disease in the nursery stage according to the type of viruses carried by the sweet potato seeds and the virus carrying rate of the sweet potato seeds.

Description

Method for predicting the prevalence and severity of virus disease in sweet potato seedling stage Technical field

The invention relates to a method for predicting the prevalence and severity of virus diseases in sweet potato seedling stage, and belongs to the field of bioengineering technology.

Background technique

Sweet potato is an important food crop and health food for improving the dietary structure of urban and rural residents. At present, sweet potato is also a dominant crop and an important economic cash crop for increasing farmers' income in the adjustment of the agricultural industrial structure in China. China is the world's largest sweet potato producer, with an annual planting area of about 70 million mu, accounting for about 45% of the world's planting area. Virus disease is a kind of important disease on sweet potato, which can cause the decrease of sweet potato yield and degeneration of seeds, which is very harmful to sweet potato production. At present, there are more than 30 types of viruses that infect sweet potatoes in the world. The viruses on sweet potatoes in China are mainly of the following types: (1) Potyvirus, which mainly includes sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC) , Sweet potato virus G (SPVG), sweet potato latent virus (SPLV), sweet potato virus 2 (SPV2), etc. Depending on the species, the Potyvirus virus can cause a yield loss of 5-94%; (2) Sweet potato chlorotic dwarf virus (SPCSV), which can generally cause a yield loss of 15-88% after infecting sweet potatoes, and SPCSV can be used with a variety of The virus forms a symbiotic disease, which causes more severe yield loss, and even crop failure; (3) Sweet potato virus (Sweepoviruses). The Begomovirus virus that infects sweet potatoes is significantly different from the Begomovirus that infects other plants, and this type of virus is called "Sweepoviruses". Sweepoviruses is an important virus on sweet potatoes. According to the 10th report of the International Virus Classification Commission (ICTV), Sweepoviruses contains 13 species, including sweet potato Korean golden vein virus (SPGVKRV), sweet potato leaf curl virus (SPLCV), and sweet potato Ghana. Sharp leaf virus (SPLCCV), Sweet potato Chinese leaf curl virus (SPLCCNV), Sweet potato Georgia leaf curl virus (SPLCGV), Sweet potato Guangxi leaf curl virus (SPLCGV), Sweet potato Henan leaf curl virus (SPLCHnV), Sweet potato Sichuan leaf curl virus 1 (SPLCSiV-1), sweet potato Sichuan leaf curl virus 2 (SPLCSiV-2), sweet potato S. Paulo leaf curl virus (SPLCSPV), sweet potato South Carolina leaf curl virus (SPLCSCV), sweet potato Uganda leaf curl virus (SPLCUV), and sweet potato mottle Virus (SPMV). There are at least 8 species in China. Infection of sweet potatoes by Sweetoviruses can generally cause a yield loss of 11-86%; (4) other viruses, such as cucumber mosaic virus (CMV).

At present, the prevention and control strategy of sweet potato virus disease mainly adopts "planting healthy seedlings" as the core, and the main measures include "early warning of seed potatoes, early elimination of diseased seedlings at the seedling stage" and so on. The main purpose of these measures is to reduce the prevalence and severity of viral disease at the seedling stage, prevent the planting of dominant seedlings into the field, and to reduce the prevalence and yield loss of viral disease at the field stage. In order to achieve the above purpose, it is necessary to establish a method that can predict the risk of virus disease in the seedling stage of sweet potato, which is to predict the possibility and severity of the virus disease in the seedling stage before seedling breeding, evaluate the risk of virus disease, and Seed potatoes with greater risks are treated in time to achieve the purpose of preventing and controlling sweet potato virus disease. It can also effectively reduce the operating risk of seed potato seedling enterprises and the economic losses of potato farmers, and improve the early warning and prevention of sweet potato virus disease in China. The level of control is of great significance.

Summary of the Invention

Aiming at the shortcomings of the prior art, the object of the present invention is to provide a method for predicting the prevalence and severity of virus disease in the sweet potato seedling raising stage. The method detects the virus carried by seed potatoes before seedling raising of sweet potatoes, Species and seed potato virus carry rate, predicting the prevalence and severity of virus disease in sweet potato seedling stage, early warning of the risk of virus disease in sweet potato seedling stage, and providing an effective means for the prevention and control of sweet potato virus disease.

In order to achieve the above objective, the technical solution adopted by the present invention is:

A method for predicting the prevalence and severity of virus disease in the sweet potato seedling raising period. Before sweet potato seedling raising, a random sample of sweet potato seed potatoes is extracted, total DNA and total RNA of the seed potato samples are extracted, and detected by PCR and RT-PCR methods, respectively. According to the virus carried by seed potatoes, according to the types of virus carried by seed potatoes and the virus-carrying rate of seed potatoes, the prevalence and severity of viral diseases at the seedling stage of sweet potatoes are predicted.

Viruses include SPFMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses.

Before the sweet potato breeding, the weight ratio of the sweet potato seed samples was randomly selected to be 8-15%.

The specific primers used in the PCR and RT-PCR methods are as follows:

The PCR reaction system was: 2 × Premix Ex Taq 10.0 μL, 5 pmol / L forward primer and 2.0 μL each, 100-400 ng / μL DNA template 1.5 μL, RNase-Free water to make up to 20.0 μL.

The reaction procedure of PCR is: pre-denaturation at 95 ° C for 5min; denaturation at 95 ° C for 30s, annealing at 53-57 ° C for 30s, extension at 72 ° C for 50s, 35 cycles; extension at 72 ° C for 7min.

The formula for predicting the prevalence of virus disease in the sweet potato seedling stage is:

Predicted virus disease symptom rate (%) = Tested seed potato sample poisoning rate × Poisoned seed potato virus disease onset probability.

The onset probability of virus disease of seed potato virus is 0.797.

The severity of virus disease in sweet potato seedling stage is the moderate and severe symptoms of sweet potato seedling stage. The predicted rate of medium and severe virus disease (%) = the rate of virus infection of SPCSV and Potyvirus in the tested potato samples.

The beneficial effects of the present invention:

The present invention is aimed at the main viruses infecting sweet potatoes in China, such as SPFMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses. By designing specific primers for the above-mentioned viruses, the virus carried by seed potatoes can be detected before breeding of sweet potatoes. The types of virus carried by potato and the virus-carrying rate of seed potato can predict the prevalence and severity of virus disease in sweet potato seedling stage, and provide early warning for the risk of sweet potato virus disease. The prediction method of the present invention is obtained through a large number of field tests and verified by sweet potato seedlings. The actual results are close to the predicted values. The method is reliable and can be applied to the existing sweet potato varieties on the market, especially to commercial potato 19, which has achieved a sweet potato variety. Early Accurate Prediction of the Risk of Diseases in Seedling Stage During Potato Storage. The invention can effectively reduce the operating risk of potato seed and seedling enterprises and the economic loss of potato farmers, and is of great significance for improving the level of early warning and prevention of sweet potato virus disease in China.

detailed description

The specific implementation of the present invention will be further described in detail below with reference to the examples. In the text, Sweepo is the abbreviation of sweetoviruses.

Example 1. Relation between virus types carried by seed potatoes and disease severity at seedling stage

1. Material:

1.1 Test sweet potato seed potatoes: The seed potatoes used in the present invention are seed potatoes harvested in different planting sites in the previous year and stored in a potato cellar. The variety was commercial potato 19 (S19). Randomly selected seed potatoes harvested from 175 different plots were numbered, and then a scalpel was used to dig out the potato skin in the middle of the potato block and its connected potato meat. The liquid potato was ground into powder. , Stored in -70 ℃ ultra-low temperature refrigerator for nucleic acid (DNA and RNA) extraction.

1.2 Seedling cultivation: The potato blocks after sampling are seeded in a nutrient bowl. One potato per bowl is placed in a greenhouse. The time of potato seedling emergence is recorded, and the disease incidence and severity of seedlings are investigated.

2. Primer design and synthesis:

Based on the genomic sequences of SPFMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses in the NCBI GenBank, DNAMAN software was used to perform sequence comparison to design the specific detection primers for the above viruses (Table 1). The primers were prepared by Biotech Bioengineering (Shanghai ) Co., Ltd. Synthesis.

Table 1. Primer sequences and amplified fragment sizes

3. Nucleic acid extraction and PCR / RT-PCR detection:

Total RNA was extracted using Plant Total RNA Purification Kit (Genemark, Taiwan), and total DNA was extracted by Ezup column-type plant genomic DNA extraction kit (Shenggong Biotechnology Co., Ltd., Shanghai, China). 800 ng of RNA sample was taken as a template, and the first strand of cDNA was synthesized by reverse transcription using the RevertAid Reverse Transcriptase Kit (Thermo Scientific, USA). The above specific steps are performed with reference to the corresponding kit instructions. Using the synthesized cDNA and the extracted total DNA as templates, PCR amplification was performed using Ex Taq DNA polymerase (Bao Biological Engineering Co., Ltd., Dalian, China). The reaction system was: 2 × Premix Taq, 10.0 μL, 5 pmol / L forward primer and 2.0 μL each, 100-400 ng / μL total DNA (or cDNA) template 1.5 μL, and RNase-Free water to make up to 20.0 μL. Amplification procedure: pre-denaturation at 95 ° C for 5min; denaturation at 95 ° C for 30s, annealing at 53-57 ° C for 30s, extension at 72 ° C for 50s, 35 cycles; extension at 72 ° C for 7min. The amplified products were detected by 1% agarose gel electrophoresis, and observed with an AlphaImager Mini (Protein Simple, USA) gel imager, and the detection results were recorded.

4. Sequence analysis of amplified fragments:

The PCR amplified product was purified and recovered by Cycle-Pure Kit (Omega Bio-tek, USA), and then entrusted to Bio-Engineering (Shanghai) Co., Ltd. for direct sequencing. The DNAMAN and BLAST online software were used to sort and analyze the sequences obtained after sequencing. The results show that the sequence of each viral nucleic acid fragment obtained by the present invention is more than 98% identical with the corresponding virus sequence, indicating that the designed detection primers are specific primers.

5. Investigation of virus disease in seedling stage: regular surveys are conducted after emergence of sweet potatoes, and the severity of the disease is investigated with potato pieces as a unit. Document the type of symptoms and level of disease. Viral disease classification criteria are as follows:

Grade 0: The plant is normal, and the leaves have no symptoms;

Grade 1: Some leaves of the plant are slightly shrunk or lightly flowered;

Grade 3: All or most of the leaves of the plant are shrunk, yellowed or flowered, the symptoms of new leaves are obvious, and the plants are slightly dwarfed;

Level 5: The whole plant has smaller leaves, slightly deformed, leaf shrinkage, bright veins, or accompanied by yellowing symptoms, and the plant is significantly dwarfed;

Level 7: The whole plant leaves become smaller, the leaves are obviously deformed, slightly leathery, shrunk, bright veins, or with yellowing symptoms, the plant is severely dwarfed;

Grade 9: The whole plant has smaller leaves, severely deformed leaves, leathery texture, severe shrinkage and open veins, curled leaves, and severely stunted plants.

Calculate the prevalence of diseased virus with the following formula:

Symptoms of virus seed potato virus disease (%) = number of virus disease symptoms / total number of virus potato seeds × 100

6. Results analysis

The relationship between the virus types carried by the seed potatoes of the present invention and the severity of viral diseases at the seedling stage of sweet potatoes is shown in Table 2.

Table 2. Relationship between virus types carried in seed potatoes and the severity of seedling disease

It can be seen from Table 2:

(1) The virus carried by seed potatoes is likely to cause virus disease symptoms in the seedling raising period of sweet potatoes: The present invention tested the virus for 175 seed potatoes, of which 153 seeds were tested for virus and 22 viruses were not detected. The seed potato virus carrying rate was 87.4% (153/175). After 153 seed potato seedlings were raised, 122 seed potato seedlings became symptomatic, and 31 seed potato seedlings were not symptomatic. The onset probability of potato virus disease was 79.7% (122/153).

(2) The severity of seedling symptoms is relatively low when seed potatoes carry a single virus: a total of 73 potato pieces containing one virus (including SPCSV, SPFMV, or Sweepoviruses) were detected in the present invention, of which 28 potato pieces (38.4%) Of potato shoots showed no viral symptoms, 39 potato pieces (53.4%) showed mild (grade 1) to moderate (grade 3-5) virus symptoms, and 6 potato pieces (8.2%) Shows severe (grades 7-9) symptoms.

(3) The virus combination carried by seed potatoes affects the severity of viral disease symptoms at seedling stage: The more types of virus carried by seed potatoes, the more severe the virus disease symptoms at seedling stage, especially SPCSV and SPFMV, SPVG, SPVC, and SPLV and other Potyvirus viruses. Compound infection will significantly increase the severity of viral disease. For example, in the 61 SPCSV and Potyvirus infection combinations detected by the present invention, the prevalence rate is 100%, and the severity of symptoms is all moderate (grade 3-5) or severe (grade 7-9).

To sum up: ① the virus carrying seed potatoes is likely to cause symptoms of sweet potato seedlings at seedling stage; ② when seed potatoes carry a virus such as SPCSV, SPFMV or Sweepoviruses, the severity of seedling symptoms is mainly mild (0-1 Grade) or moderate (Grade 3-5); ③ The seed potato carries SPCSV and Potyvirus complex virus, with a 100% onset rate at seedling stage. The severity of symptoms is moderate (Grade 3-5) or severe (Grade 7-9). ).

Based on the above test results, the predicted disease severity and severity (moderate to severe severity) of sweet potato virus disease at the seedling stage are:

Predicted disease rate of virus disease (%) = Toxicity rate of tested seed potato samples × Probability of disease of seed potato virus disease

Predicted Severe Symptom Rate of Viral Disease (%) = Tested SPCSV and Potyvirus Complex Virus Carrying Rate of Seed Potato Samples

Example 2 Application of a Method for Predicting the Symptom Rate and Severity of Virus Diseases in Sweet Potato Seedlings

1. Materials: Randomly select some potato blocks from two batches of seed potatoes from different plots (Luoyang, Xuchang) stored in the potato cellar for virus detection. The variety is Shangshu 19 (S19). The first batch of seed potatoes totaled about 400 kg, and a total of 15 potato pieces (about 7.5 kg) were drawn, and the sampling amount accounted for about 1.9% of the total. The second batch of seed potatoes totaled about 400 kg, and a total of 92 potato pieces were sampled. (Approximately 46kg), and the sampling volume accounts for about 11.5% of the total. The detection method of the virus type is the same as in Example 1.

2. Seedling raising: The conventional two methods are used to raise seedlings in the greenhouse respectively.

3. Disease survey: About 30 days after seed potato seedlings are raised, a survey of disease incidence at the seedling stage is conducted. Five-point sampling was used for plot investigation, and plant disease severity was investigated on a plant basis. The grading standard is the same as in Example 1.

Calculate the predicted rate and severity of viral disease and the actual rate and severity of viral disease according to the following formula:

Predicted virus disease symptom rate (%) = Tested potato sample poisoning rate × diseased potato virus diseased disease probability (same as in Example 1)

Actual virus disease symptom rate (%) = number of virus disease symptomatic strains / total number of surveyed plants × 100

Predicted Severe Symptom Rate of Viral Disease (%) = Tested SPCSV and Potyvirus Complex Virus Carrying Rate of Seed Potato Samples

Actual severe disease rate of severe virus disease (%) = number of severe strains of severe virus disease / total number of surveyed plants × 100

4. Results analysis

The results of the investigation of the severity of the symptoms of the seed potatoes at the seedling stage of the present invention are shown in Table 3.

Table 3. Symptom Severity Survey in Seedling Stage

It can be seen from Table 3:

(1) A total of 15 potato pieces were randomly selected from the first batch of seed potatoes. After virus detection, 7 potato pieces carried the virus with a virus infection rate of 46.7% (7/15); the SPCSV and Potyvirus combined virus infection rate was 6.7 % (1/15); According to the experimental results in Example 1, the virus disease prevalence rate of seed potatoes in this batch is predicted to be 37.2% (46.7% × 0.797); moderate (grade 3-5) or severe (7 Grade -9) was 6.7%.

Seedlings of this batch were seeded. The results of the actual seedling disease investigation showed that the total number of plants investigated was 984, of which 742 were 0, 212 were 1, 30 were 3-5, and 0 were 7-9. The actual viral disease prevalence rate was 24.6%, and the moderate (grade 3-5) or severe (grade 7-9) symptom rate was 3.0% (30/984), which was close to the predicted value.

(2) A total of 92 potato pieces were randomly selected from the second batch of seed potatoes. After virus detection, 85 potato pieces carried the virus, and the infection rate was 92.4% (85/92). The SPCSV and Potyvirus combined virus infection rate was 30.4. % (28/92); According to the experimental results in Example 1, the virus disease prevalence rate of seed potatoes in this batch is predicted to be 73.6% (92.4% × 0.797); moderate (grade 3-5) or severe (7- Grade 9) The onset rate was 30.4%.

Seedlings of this batch were seeded. The actual survey results at the seedling stage showed that the total number of investigated plants was 2745, of which 857 were 0, 323 were 1st, 1062 were 3-5, and 503 were 7-9. The prevalence of viral disease is 68.8%, and the rate of moderate (grade 3-5) or severe (grade 7-9) is 57.0% (1565/2745), which is close to the predicted value, indicating that the prediction method of the present invention is more reliable The prediction result has a high reference value. It can be seen from the test results of the two batches that the larger the sample size, the closer the predicted value to the actual value. Considering the testing cost, workload, and requirements for prediction accuracy, it is recommended that a sampling amount of 8-15% is more appropriate.

The above description is only the best embodiment of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. A method for predicting the prevalence and severity of virus disease in sweet potato seedling stage, characterized in that, before sweet potato seedling breeding, a sample of sweet potato seed potato is randomly selected, and total DNA and total RNA of the seed potato sample are extracted, and PCR and RT are used respectively. -PCR method was used to detect the virus carried by seed potatoes. According to the types of virus carried by seed potatoes and the virus-carrying rate of seed potatoes, the prevalence and severity of viral diseases at the seedling stage of sweet potatoes were predicted.
2. The method according to claim 1, wherein the viruses include SPFMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses.
3. The method for predicting the prevalence and severity of virus disease in sweet potato seedling stage according to claim 1, characterized in that before the sweet potato seedling nursery, the weight ratio of the sweet potato seed potato test sample is randomly selected to be 8-15%.
4. The method for predicting the prevalence and severity of virus disease in sweet potato seedling stage according to claim 1, characterized in that the specific primers used for detection by PCR and RT-PCR methods are as follows:

   
5. The method for predicting the prevalence and severity of virus disease in sweet potato seedling stage according to claim 1, characterized in that the reaction system of PCR is: 2 × Premix Ex Taq 10.0 μL, 5 pmol / L forward primer and reverse primer 2.0 μL each, 100-400 ng / μL DNA template 1.5 μL, RNase-Free water was made up to 20.0 μL.
6. The method for predicting the prevalence and severity of virus disease in sweet potato seedling stage according to claim 1, characterized in that the PCR reaction program is: pre-denaturation at 95 ° C for 5 min; denaturation at 95 ° C for 30s, and annealing at 53-57 ° C for 30s, Extend at 72 ° C for 50s, 35 cycles; extend at 72 ° C for 7 minutes.
7. The method for predicting the prevalence and severity of virus disease at the seedling stage of sweet potato according to claim 1, wherein the calculation formula for predicting the prevalence of virus disease at the seedling stage of sweet potato is:

   Predicted virus disease symptom rate (%) = Tested seed potato sample poisoning rate × Poisoned seed potato virus disease onset probability.
8. The method for predicting the prevalence and severity of virus disease in the seedling raising period of sweet potato according to claim 7, wherein the probability of the disease prevalence of virus seed potato virus disease is 0.797.
9. The method for predicting the prevalence and severity of viral diseases in the seedling stage of sweet potato according to claim 1, characterized in that the severity of the viral disease in the seedling stage of the sweet potato is a moderate to severe symptom in the seedling stage of the sweet potato, and the predicted severe and severe viral disease is Symptom rate (%) = the virus infection rate of SPCSV and Potyvirus of the tested potato samples.