CN108950089B - Method for predicting virus disease manifestation rate and severity of sweet potato in seedling raising period - Google Patents
Method for predicting virus disease manifestation rate and severity of sweet potato in seedling raising period Download PDFInfo
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Abstract
The invention discloses a method for predicting the disease rate and severity of virus diseases in seedling stage of sweet potatoes, which comprises the steps of randomly extracting sweet potato seed samples before sweet potato seedling, extracting total DNA and total RNA of the sweet potato seed samples, detecting the virus conditions carried by the sweet potatoes by respectively adopting PCR (polymerase chain reaction) and RT-PCR (reverse transcription-polymerase chain reaction) methods, and predicting the disease rate and severity of the virus diseases in seedling stage of the sweet potatoes according to the types of the virus carried by the sweet potatoes and the virus carrying rate of the sweet potatoes. The prediction method is obtained through a large number of field tests, the actual result is close to the predicted value through sweet potato seedling culture verification, the method is reliable, the method can be suitable for the existing sweet potato varieties in the market, and is particularly suitable for commercial sweet potatoes 19, the early accurate prediction of the disease occurrence risk in the seedling culture period in the storage period of the sweet potato seeds is realized, and early warning is provided for the occurrence risk of sweet potato virus diseases. The method can effectively reduce the operation risk of potato seedling enterprises and the economic loss of potato farmers, and has important significance for improving the early warning, prevention and control level of the sweet potato virus diseases in China.
Description
Technical Field
The invention relates to a method for predicting virus disease manifestation rate and severity of sweet potato in seedling raising period, belonging to the technical field of bioengineering.
Background
The sweet potato is an important food crop in China and a health care food for improving the dietary structure of urban and rural residents. At present, sweet potatoes are also superior crops in the structural adjustment of agricultural industry in China and important benefit type economic crops for increasing the income of farmers. China is the biggest sweet potato producing country in the world, and the planting area of the country is about 7000 ten thousand mu throughout the year, which accounts for about 45% of the planting area of the world. The virus disease is an important disease on the sweet potatoes, can cause the yield reduction and the seed nature degradation of the sweet potatoes, and has great harm to the production of the sweet potatoes. At present, more than 30 kinds of viruses infecting sweet potatoes are reported all over the world, and the viruses on the sweet potatoes in China mainly comprise the following types: (1) potyvirus viruses mainly comprise sweet potato feathery mottle virus (SPCMV), Sweet Potato Virus C (SPVC), Sweet Potato Virus G (SPVG), Sweet Potato Latent Virus (SPLV), sweet potato virus 2(SPV2) and the like. Potyvirus viruses can generally cause 5-94% yield loss depending on the species; (2) sweet Potato Chlorotic Stunt Virus (SPCSV) generally causes 15-88% yield loss after infecting sweet potatoes, and the SPCSV can form synergistic diseases with various viruses to cause more serious yield loss and even no harvest; (3) sweet potato geminivirus (sweet potato geminiviruses). The Phaseolus viridis (Begomovirus) infecting sweet potatoes is significantly different from Begomovirus infecting other plants, and is called as "Sweepoviruses". Sweepoviruses are a class of important viruses on sweet potatoes, and according to the tenth report of the International Committee for Virus Classification (ICTV), Sweepoviruses contain 13 species, which are sweet potato Korean vein Virus (SPGVHRV), Sweet Potato Leaf Curl Virus (SPLCV), sweet potato Canali leaf curl 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 (SPLCiV-1), sweet potato Sichuan leaf curl Virus 2 (SPLCiV-2), sweet potato Bauloro leaf curl Virus (SPLCPV), sweet potato south Carolina leaf curl Virus (SPLCCV), sweet potato Wuta leaf curl Virus (SPLCUV) and Sweet Potato Mottle Virus (SPMV), respectively. At least 8 species exist in China, and sweet potato infection by sweet potatoes generally can cause 11-86% yield loss; (4) other viruses such as Cucumber Mosaic Virus (CMV) and the like.
At present, the prevention and control strategy of 'planting healthy seedlings' as the core is mainly adopted for preventing and controlling the sweet potato virus diseases, and the main measures comprise 'early warning of potato planting, early elimination of diseased seedlings in the seedling raising period' and the like. The main purposes of the measures are to reduce the disease rate and severity of the virus diseases in the seedling stage and prevent the disease-developing seedlings from being planted in the field so as to reduce the disease rate and yield loss of the virus diseases in the field. In order to achieve the purpose, a method capable of predicting the occurrence risk of the virus diseases of the sweet potatoes in the seedling raising period is needed to be established, namely the possibility and the severity of the occurrence of the virus diseases of the seedling raising period are predicted before the sweet potatoes are raised, the occurrence risk of the virus diseases is evaluated, the seed potatoes with higher risk are timely treated, the purpose of preventing and controlling the occurrence of the virus diseases of the sweet potatoes is achieved, the operational risk of potato seed seedling enterprises and the economic loss of potato farmers can be effectively reduced, and the method has important significance for improving the early warning and preventing and controlling level of the virus diseases of the sweet potatoes in China.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for predicting the disease manifestation rate and the severity of the virus diseases in the seedling stage of sweet potatoes.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for predicting the disease rate and severity of virus diseases in the seedling raising stage of sweet potatoes comprises the steps of randomly extracting sweet potato seed samples before sweet potato seedling raising, extracting total DNA and total RNA of the sweet potato seed samples, detecting the virus conditions carried by the sweet potatoes by adopting PCR (polymerase chain reaction) and RT-PCR (reverse transcription-polymerase chain reaction) methods respectively, and predicting the disease rate and severity of the virus diseases in the seedling raising stage of the sweet potatoes according to the types of the virus carried by the sweet potatoes and the virus carrying rate of the sweet potatoes.
Viruses include SPFMMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses.
Before sweet potato seedling, randomly extracting sweet potato seed samples with the weight ratio of 8-15%.
The specific primers used for the detection by PCR and RT-PCR methods are as follows:
the reaction system of PCR is: 2.0. mu.L of 2 XPimmix Ex Taq, 5pmol/L of each of the forward and reverse primers, 2.0. mu.L of each of the forward and reverse primers, 1.5. mu.L of 100 and 400 ng/. mu.L of the DNA template, and RNase-Free water to 20.0. mu.L.
The reaction procedure for PCR was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 53-57 ℃ for 30s, extension at 72 ℃ for 50s, and 35 cycles; extension at 72 ℃ for 7 min.
The calculation formula for predicting the virus disease manifestation rate of the sweet potato in the seedling raising period is as follows:
the predicted virus disease manifestation rate (%). the virus carrying rate of the detected seed potato sample is multiplied by the manifestation probability of the virus disease of the seed potato.
The apparent probability of the virus-carrying potato virus disease is 0.797.
The severity of the virus disease in the sweet potato seedling raising period is the severe symptom showing condition in the sweet potato seedling raising period, and the predicted moderate severe symptom showing rate (%) of the virus disease is the virus carrying rate of the detected compound virus SPCSV and Potyvirus of the potato seed sample.
The invention has the beneficial effects that:
aiming at main viruses SPFMMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV and sweet potato viruses infecting sweet potatoes in China, the invention designs specific primers aiming at the viruses, detects the virus condition carried by seed potatoes before sweet potato seedling raising, predicts the disease manifestation rate and severity of the virus diseases during sweet potato seedling raising according to the types of the viruses carried by the seed potatoes and the virus carrying rate of the seed potatoes, and provides early warning for the occurrence risk of the sweet potato virus diseases. The prediction method is obtained through a large number of field tests, the actual result is close to the predicted value through the sweet potato seedling raising verification, the method is reliable, the method can be suitable for the existing sweet potato varieties in the market, and is particularly suitable for the commercial sweet potato 19, and the early accurate prediction of the disease occurrence risk in the seedling raising period in the storage period of the sweet potato seeds is realized. The method can effectively reduce the operation risk of potato seedling enterprises and the economic loss of potato farmers, and has important significance for improving the early warning, prevention and control level of the sweet potato virus diseases in China.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail. Herein, sweet is an abbreviation of sweet viruses.
Example 1 relationship between types of viruses carried by seed potatoes and severity of diseases at seedling stage
1. Materials:
1.1 testing of seed sweet potato: the seed potatoes used in the invention are harvested in different planting places in the last year and stored in the potato cellar. The variety is commercial potato 19(S19), the seed potatoes harvested in 175 different plots are randomly selected and numbered, then part of the potato skins in the middle of the potato plots and the potato flesh connected with the potato skins are dug by a scalpel, ground into powder by liquid nitrogen and stored in an ultralow temperature refrigerator at-70 ℃ for nucleic acid (DNA and RNA) extraction.
1.2 seedling culture: and (3) seedling the sampled potato blocks in nutrition pots, placing one potato in each pot in a greenhouse, recording the seedling emergence time of the potato blocks, and investigating the disease manifestation rate and severity in the seedling stage.
2. Designing and synthesizing a primer:
based on the genome sequences of SPFMMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV and Sweepoviruses in NCBI GenBank, DNAMAN software was used to perform sequence comparison, and specific detection primers (Table 1) for the above viruses were designed, which were synthesized by Biotechnology engineering (Shanghai) GmbH.
TABLE 1 primer sequences and amplified fragment sizes
3. Nucleic acid extraction and PCR/RT-PCR detection:
total RNA was extracted using a Plant Total RNA Purification Kit (Genemark, Taiwan), and Total DNA was extracted using an Ezup column type Plant genomic DNA extraction Kit (Biotechnology engineering Co., Ltd., Shanghai, China). Taking 800ng of an RNA sample as a template, carrying out Reverse transcription by using a RevertAID Reverse Transcriptase kit (Thermo Scientific, USA) to synthesize a first cDNA chain, and referring to the corresponding kit instruction for the specific operation steps. The synthesized cDNA and the extracted total DNA were used as templates, respectively, and Ex Taq DNA polymerase (Takara Bio Inc., China, Dalian, China) was used for PCR amplification. The reaction system is as follows: 2.0. mu.L of 2 XPimmix Ex Taq, 5pmol/L of each of the forward and reverse primers, 1.5. mu.L of 100 and 400 ng/. mu.L of the total DNA (or cDNA) template, and 20.0. mu.L of RNase-Free water. And (3) amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 53-57 ℃ for 30s, extension at 72 ℃ for 50s, and 35 cycles; extension at 72 ℃ for 7 min. The amplification products were detected by electrophoresis on a 1% agarose gel, visualized on an AlphaImager Mini (protein simple, USA) gel imager and the results recorded.
4. Sequence analysis of the amplified fragments:
the PCR amplification product was purified and recovered by Cycle-Pure Kit (Omega Bio-tek, USA), and then subjected to direct sequencing by Competition Bioengineering (Shanghai) Co., Ltd. The sequences obtained after sequencing were collated and analyzed using DNAMAN and BLAST online software. The result shows that the consistency of each virus nucleic acid fragment sequence obtained by the invention and the corresponding virus sequence is more than 98 percent, which indicates that the designed detection primers are all specific primers.
5. Investigation of virus diseases in seedling stage: the investigation is carried out regularly after the seedlings of the sweet potatoes emerge, and the disease severity is investigated by taking the sweet potato blocks as units. The symptom type and disease grade are recorded. The viral disease grading criteria are as follows:
level 0: the plant is normal, and the leaves have no symptoms;
level 1: the leaves of the plant parts are slightly shriveled or slightly mosaic;
and 3, level: all or most leaves of the plants are shriveled, yellowed or mosaic, the symptoms of bright veins of new leaves are obvious, and the plants are slightly dwarfed;
and 5, stage: the whole plant leaves are small and slightly deformed, the leaves are shriveled and have bright veins or are accompanied with yellowing symptoms, and the plants are obviously dwarfed;
and 7, stage: the leaves of the whole plant are reduced, the leaves are obviously deformed, slightly tanned, shrunken and bright, or are accompanied with yellowing symptoms, and the plant is seriously dwarfed;
and 9, stage: the whole plant leaves become small, the leaves are seriously deformed, the plant is tanned, the shrinkage and the bright pulse are serious, the leaves are curled, and the plant is seriously dwarfed.
Calculating the virus disease manifestation rate of the potato with the virus seeds according to the following formula:
the apparent disease probability (%) of potato virus with virus seeds is ═ the number of virus disease apparent disease blocks/total number of potato blocks with virus seeds is multiplied by 100
6. Analysis of results
The relationship between the virus types carried by the sweet potatoes and the severity of the sweet potato seedling virus diseases is shown in Table 2.
TABLE 2 relationship between the types of viruses carried by the seed potatoes and the severity of the disease during the seedling stage
As can be seen from table 2:
(1) the virus carried by the seed potato is likely to cause virus symptoms at the seedling raising stage of the sweet potato: the virus detection is carried out on 175 potato seeds, wherein 153 potato seeds carry virus, 22 potato seeds do not detect virus, the virus carrying rate of the potato seeds is 87.4 percent (153/175), 122 potato seedlings of the potato seeds are obvious diseases after 153 potato seeds with virus are cultivated, and 31 potato seedlings of the potato seeds are not obvious diseases, so the obvious disease probability of the virus carrying potato seedlings is 79.7 percent (122/153).
(2) The severity of seedling stage symptoms is relatively light when the seed potatoes carry a single virus: the present invention co-detected 73 potato pieces containing one virus (containing SPCSV, SPFMV or sweet potatoes), of which 28 potato pieces (38.4%) showed no viral symptoms, 39 potato pieces (53.4%) showed mild (grade 1) to moderate (grade 3-5) viral symptoms, and 6 potato pieces (8.2%) showed severe (grade 7-9) symptoms.
(3) The virus combination carried by the seed potatoes affects the severity of the symptoms of the virus disease at the seedling stage: the more virus types carried by the potato seeds, the more serious the symptoms of the virus diseases in the seedling stage, and particularly, the complex infection of the SPCSV and Potyvirus viruses such as SPCMV, SPVG, SPVC, SPLV and the like can obviously increase the severity of the virus diseases. For example, the incidence of infection of 61 SPCSVs with Potyvirus virus detected in the present invention was 100%, and the severity of symptoms was moderate (grade 3-5) or severe (grade 7-9).
In conclusion, ① potatoes carrying viruses are likely to cause potato seedling emergence at the seedling stage of the sweet potatoes, ② potatoes carrying SPCSV, SPFMMV or Sweepoviruses and other viruses have the symptoms of mild (grade 0-1) or moderate (grade 3-5) severity at the seedling stage, ③ potatoes carrying SPCSV and Potyvirus compound viruses have the symptoms of 100% emergence rate at the seedling stage and the symptoms of moderate (grade 3-5) or severe (grade 7-9).
According to the detection result, the predicted virus disease manifestation rate and severity (medium-severity manifestation rate) of the sweet potato seedling raising stage are as follows:
predicted virus disease manifestation rate (%). the virus-carrying rate of the seed potato sample and the manifestation probability of the virus-carrying seed potato virus disease
Predicted severe manifestation rate (%) of virus disease detected seed potato sample SPCSV and Potyvirus compound virus carrying rate
Embodiment 2 application of method for predicting virus disease manifestation rate and severity of sweet potato in seedling raising period
1. Materials: randomly extracting partial potato blocks from two batches of seed potatoes stored in the potato cellar and derived from different plots (Luoyang, Schang) respectively as samples for virus detection, wherein the variety is commercial potato 19 (S19). The total amount of the first seed potatoes is about 400kg, 15 potato blocks (about 7.5kg) are extracted, and the sampling amount accounts for about 1.9 percent of the total amount; the total amount of the second batch of potatoes is about 400kg, 92 potatoes (about 46kg) are extracted totally, and the sampling amount accounts for about 11.5 percent of the total amount. The virus species were detected in the same manner as in example 1.
2. Seedling culture: the two batches of potato seeds are respectively cultivated in a greenhouse by a conventional method.
3. And (4) disease investigation: and (5) about 30 days after the seed potatoes are cultivated, and investigation on the disease condition in the seedling stage is carried out. The plot survey adopts 5-point sampling, and the disease severity of plants is surveyed in units of plants. The grading criteria were the same as in example 1.
And calculating the predicted virus disease manifestation rate and severity and the actual virus disease manifestation rate and severity according to the following formulas:
predicted virus disease manifestation rate (%) of seed potato sample with virus band x manifestation probability of seed potato virus disease with virus (same as example 1)
Actual rate of virus manifestation (%) is the number of virus manifestation strains/total number of investigated strains × 100
Predicted severe manifestation rate (%) of virus disease detected seed potato sample SPCSV and Potyvirus compound virus carrying rate
Actual rate of moderate-to-severe virus disease development (%) - (number of moderate-to-severe virus disease development strains/number of total investigated strains × 100
4. Analysis of results
The severity survey results of the seedling stage symptoms of the potato seeds are shown in Table 3.
TABLE 3 severity survey of seedling stage symptoms of seed potatoes
As can be seen from table 3:
(1) 15 potato blocks are randomly extracted from the first batch of seed potatoes, and virus detection shows that 7 potato blocks carry viruses with the virus carrying rate of 46.7 percent (7/15); the virus carrying rate of the SPCSV and Potyvirus compound virus is 6.7% (1/15); according to the experimental result of example 1, the virus morbidity of the seed potatoes of the batch is predicted to be 37.2% (46.7% × 0.797); the mean (grade 3-5) or severe (grade 7-9) incidence of the disease is predicted to be 6.7%.
The seed potatoes of the batch are cultivated, and the actual seedling disease investigation result shows that the total number of the investigated plants is 984 plants, wherein 0 grade 742 plants, 1 grade 212 plants, 3-5 grade 30 plants and 7-9 grade 0 plants are investigated. The actual virus disease manifestation rate is 24.6%, and the moderate (grade 3-5) or severe (grade 7-9) manifestation rate is 3.0% (30/984), which is close to the predicted value.
(2) Randomly extracting 92 potato blocks from the second batch of potato seeds, detecting viruses to ensure that 85 potato blocks carry viruses and the virus carrying rate is 92.4% (85/92); the virus carrying rate of the SPCSV and Potyvirus compound virus is 30.4% (28/92); according to the experimental result of example 1, the virus disease incidence rate of the potato seeds of the batch is predicted to be 73.6% (92.4% × 0.797); the rate of moderate (grade 3-5) or severe (grade 7-9) symptoms is 30.4%.
The seed potatoes of the batch are cultivated, and the actual disease investigation result in the seedling stage shows that the total number of investigated plants is 2745, wherein 857 plants at the 0 level, 323 plants at the 1 level, 1062 plants at the 3-5 level and 503 plants at the 7-9 level. The virus disease manifestation rate is 68.8 percent, the moderate (grade 3-5) or severe (grade 7-9) manifestation rate is 57.0 percent (1565/2745), and the prediction value is close to the predicted value, which shows that the prediction method of the invention is more reliable, and the prediction result has high reference value. From the two-batch sampling detection results, the larger the sampling amount is, the closer the predicted value is to the actual value, the detection cost, the workload and the requirement on the prediction precision are comprehensively considered, and 8-15% of the sampling amount is recommended to be more appropriate.
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (4)
1. A method for predicting the disease rate and severity of virus diseases in seedling raising of sweet potatoes is characterized in that sweet potato seed samples are randomly extracted before sweet potato seedling raising, total DNA and total RNA of the sweet potato seed samples are extracted, the conditions of the virus carried by the sweet potatoes are detected by adopting PCR (polymerase chain reaction) and RT-PCR (reverse transcription-polymerase chain reaction) methods respectively, and the disease rate and severity of the virus diseases in seedling raising of the sweet potatoes are predicted according to the types of the virus carried by the sweet potatoes and the virus carrying rate of the sweet potatoes;
the viruses include SPCMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV and Sweepoviruses;
the calculation formula for predicting the virus disease manifestation rate of the sweet potato in the seedling raising period is as follows:
the predicted virus disease manifestation rate is equal to the virus carrying rate of the detected seed potato sample multiplied by the manifestation probability of the virus disease of the seed potato;
wherein the manifestation probability of the virus-bearing potato virus disease is 0.797;
the severity of the virus disease in the sweet potato seedling raising period is the severe symptom showing condition in the sweet potato seedling raising period, and the predicted severe symptom showing rate of the virus disease is the virus carrying rate of the detected compound virus of the seed potato sample SPCSV and Potyvirus;
before sweet potato seedling, the weight ratio of the detection sample of the sweet potato seed is randomly extracted to be 8-15%.
2. The method for predicting the virus disease manifestation rate and severity of sweet potato seedling raising period according to claim 1, wherein specific primers used for detection by PCR and RT-PCR methods are as follows:
3. the method for predicting the virus disease manifestation rate and severity of sweet potato seedling raising period according to claim 1, wherein the reaction system of PCR is as follows: 2.0. mu.L of 2 XPimmix Ex Taq, 5pmol/L of each of the forward and reverse primers, 2.0. mu.L of each of the forward and reverse primers, 1.5. mu.L of 100 and 400 ng/. mu.L of the DNA template, and RNase-Free water to 20.0. mu.L.
4. The method for predicting the virus disease manifestation rate and severity of sweet potato seedling raising period according to claim 1, wherein the reaction program of PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 53-57 ℃ for 30s, extension at 72 ℃ for 50s, and 35 cycles; extension at 72 ℃ for 7 min.
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| PCT/CN2018/110578 WO2020047949A1 (en) | 2018-09-03 | 2018-10-17 | Method for predicting symptom appearance rate and severity of sweet potato viral disease in nursery stage |
| JP2020539058A JP6889337B2 (en) | 2018-09-03 | 2018-10-17 | How to predict the incidence and severity of viral diseases during the sweet potato seedling raising period |
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| CN110367123B (en) * | 2019-08-21 | 2022-08-02 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Resistance identification method for sweet potato leaf curl virus disease |
| CN111334584B (en) * | 2020-03-04 | 2022-08-16 | 河南省农业科学院植物保护研究所 | Method for predicting SPCSV (Spot Summit syndrome Virus) toxicity rate of sweet potato seeds |
| CN113462814A (en) * | 2021-05-21 | 2021-10-01 | 中国热带农业科学院热带生物技术研究所 | RPA primer, probe, kit and detection method for detecting sweet potato golden vein virus |
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| WO2018107103A1 (en) * | 2016-12-09 | 2018-06-14 | The Broad Institute, Inc. | Crispr-systems for modifying a trait of interest in a plant |
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| CN102108419A (en) * | 2010-11-30 | 2011-06-29 | 河南省农业科学院植物保护研究所 | Multiple RT-PCR (reverse transcription-polymerase chain reaction) detection method for SPVD (sweet potato virus disease) |
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| JP2021506332A (en) | 2021-02-22 |
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