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WO2020047949A1 - Procédé de prédiction du taux d'apparition de symptômes et de la gravité d'une maladie virale de patate douce en stade de culture en pépinière - Google Patents

Procédé de prédiction du taux d'apparition de symptômes et de la gravité d'une maladie virale de patate douce en stade de culture en pépinière Download PDF

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Publication number
WO2020047949A1
WO2020047949A1 PCT/CN2018/110578 CN2018110578W WO2020047949A1 WO 2020047949 A1 WO2020047949 A1 WO 2020047949A1 CN 2018110578 W CN2018110578 W CN 2018110578W WO 2020047949 A1 WO2020047949 A1 WO 2020047949A1
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Prior art keywords
sweet potato
virus
severity
potato
disease
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Ceased
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PCT/CN2018/110578
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English (en)
Chinese (zh)
Inventor
张振臣
赵付枚
王爽
田雨婷
乔奇
秦艳红
王永江
张德胜
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Institute of Plant Protection of Henan Academy of Agricultural Sciences
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Institute of Plant Protection of Henan Academy of Agricultural Sciences
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Priority to JP2020539058A priority Critical patent/JP6889337B2/ja
Publication of WO2020047949A1 publication Critical patent/WO2020047949A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • 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.
  • 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).
  • SPFMV sweet potato feathery mottle virus
  • SPVC sweet potato virus C
  • SPVG Sweet potato virus G
  • SPLV sweet potato latent virus
  • SPV2 sweet potato virus 2
  • the Potyvirus virus can cause a yield loss of 5-94%
  • SPCSV Sweet potato chlorotic dwarf virus
  • 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.
  • ICTV International Virus Classification Commission
  • SPLCCV Sharp leaf virus
  • SPLCCNV Sweet potato Chinese leaf curl virus
  • SPLCGV 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-2 Sweet potato Sichuan leaf curl virus 2
  • SPLCSPV sweet potato South Carolina leaf curl virus
  • SPLCUV sweet potato Kenya leaf curl virus
  • SPMV sweet potato mottle Virus
  • 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.
  • 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.
  • 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.
  • the weight ratio of the sweet potato seed samples was randomly selected to be 8-15%.
  • the PCR reaction system was: 2 ⁇ Premix Ex Taq 10.0 ⁇ L, 5 pmol / L forward primer and 2.0 ⁇ L each, 100-400 ng / ⁇ L DNA template 1.5 ⁇ L, RNase-Free water to make up to 20.0 ⁇ L.
  • 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:
  • 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 present invention is aimed at the main viruses infecting sweet potatoes in China, such as SPFMV, SPVC, SPVG, SPLV, SPV2, CMV, SPCSV, and Sweepoviruses.
  • 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.
  • 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.
  • Sweepo is the abbreviation of sweetoviruses.
  • Example 1 Relation between virus types carried by seed potatoes and disease severity at seedling stage
  • 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 °C ultra-low temperature refrigerator for nucleic acid (DNA and RNA) extraction.
  • DNA and RNA nucleic acid
  • 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.
  • 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.
  • the reaction system was: 2 ⁇ Premix Taq, 10.0 ⁇ L, 5 pmol / L forward primer and 2.0 ⁇ L each, 100-400 ng / ⁇ L total DNA (or cDNA) template 1.5 ⁇ L, and RNase-Free water to make up to 20.0 ⁇ L.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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). ).
  • Seedling raising The conventional two methods are used to raise seedlings in the greenhouse respectively.
  • Example 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.
  • Example 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.

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Abstract

L'invention concerne un procédé pour prédire le taux d'apparition de symptômes et la gravité d'une maladie virale de patate douce en stade de culture en pépinière. Le procédé comprend les étapes suivantes : sélection aléatoire d'échantillons de semences de patate douce avant amélioration des patates douces; extraction de l'ADN total et de l'ARN total des échantillons de semences de patate douce, et détection de la situation de virus portés par les semences de patate douce en utilisant respectivement des procédés PCR et RT-PCR; et prédiction du taux d'apparition de symptômes et de la gravité d'une maladie virale de patate douce en stade de culture en pépinière selon le type de virus portés par les semences de patate douce et le taux de transport de virus des semences de patate douce.
PCT/CN2018/110578 2018-09-03 2018-10-17 Procédé de prédiction du taux d'apparition de symptômes et de la gravité d'une maladie virale de patate douce en stade de culture en pépinière Ceased WO2020047949A1 (fr)

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JP2020539058A JP6889337B2 (ja) 2018-09-03 2018-10-17 サツマイモの育苗期におけるウイルス性疾患の発症率と重症度の予測方法

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CN201811019008.7A CN108950089B (zh) 2018-09-03 2018-09-03 一种预测甘薯育苗期病毒病显症率和严重度的方法

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CN110367123B (zh) * 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) 一种甘薯卷叶病毒病的抗性鉴定方法
CN111334584B (zh) * 2020-03-04 2022-08-16 河南省农业科学院植物保护研究所 一种预测甘薯种薯spcsv带毒率的方法
JP7698836B2 (ja) * 2021-02-12 2025-06-26 国立大学法人 宮崎大学 Ｒｔ－ｌａｍｐ法によるサツマイモ病原ウイルスの診断方法の開発
CN113462814A (zh) * 2021-05-21 2021-10-01 中国热带农业科学院热带生物技术研究所 一种检测甘薯金脉病毒的rpa引物和探针、试剂盒及检测方法
CN117461522A (zh) * 2023-11-06 2024-01-30 江苏省农业科学院 一种植物病虫害的指示植物的筛选方法和指示方法

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WO2018107103A1 (fr) * 2016-12-09 2018-06-14 The Broad Institute, Inc. Systèmes crispr pour modifier un caractère d'intérêt chez une plante

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