Sensitive detection of potato viruses, PVX, PLRV and PVS, by RT-PCR in potato leaf and tuber
M. Peiman A B and C. Xie A CA Key Laboratory of Horticultural Plant Biology (Huazhong Agricultural University), Ministry of Education, National Center for Vegetable Improvement (Central China), Wuhan 430070, China.
B Takestan Islamic Azad University, Shami Shop, Talestan, Iran.
C Corresponding author. Email: xiech@mail.hzau.edu.cn
Australasian Plant Disease Notes 1(1) 41-46 https://doi.org/10.1071/DN06017
Accepted: 27 October 2006 Published: 8 November 2006
Abstract
A multiplex reverse transcription polymerase chain reaction (M-RT-PCR) was developed for the simultaneous detection of three major potato viruses, Potato virus X (PVX), Potato virus S (PVS) and Potato leaf roll virus (PLRV), in potato leaves and tubers (stored and dormant). Primers were designed from the coat protein gene of all three viruses and were able to detect virus in leaf RNA dilutions of 1 : 1024 to 1 : 4096 and from tuber RNA dilutions of 1 : 256 to 1 : 1024. The sensitivity of virus detection in leaves was higher than in tubers. All three viruses were successfully detected from tissues sampled from the tuber cortex, perimedulla and pith, with all PCR bands being strongest from cortex tissues. The reliability of the M-RT-PCR to detect PVX, PVS and PLRV in tubers was determined by testing 95 samples of field, greenhouse and in vitro potato plantlets from Wuhan, China and comparing the results with enzyme-linked immunosorbent assays (ELISA). M-RT-PCR detected all infections found by ELISA in leaf and stored tubers. In freshly harvested tubers, M-RT-PCR proved more sensitive than ELISA, as indicated by an additional 3.2% of infections detected with the M-RT-PCR method.
Additional keywords: cDNA, disease, random primer, survey.
Acknowledgements
We are very grateful to Dr Luis Salazar, International Potato Center (CIP) for his gift of antisera. Also, special thanks to him and Prof. Gad Leobenstein, Agricultural Research Organization (ARO), The Volcani Center for correcting the English and his critical reading of our manuscript. We also thank Prof. Liu Jun for her advice on technical matters. Two anonymous reviewers also made valuable comments. This work is supported by a post-doctoral fellowship from the Chinese government.
Bariana HS,
Shannon AL,
Chu PWG, Waterhouse PM
(1994) Detection of five seedborne legume viruses in one sensitive multiplex polymerase chain reaction test. Phytopathology 84, 1201–1205.
(verified 25 October 2006)
Heldak J
(2001) detection of potato viruses by RT-PCR in potato regenerants derived from in vitro heat-treated shoot tips. Acta Fytotechnica et Zootechnica 4, 275–277.
Higgins D,
Thompson J,
Gibson T,
Thompson JD,
Higgins DG, Gibson TJ
(1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.
| PubMed |
James D,
Varga A,
Pallas V, Candresse T
(2006) Strategies for simultaneous detection of multiple plant viruses. Canadian Journal of Plant Pathology 28, 16–29.
Liu J, Xie C
(2001) Correlation of cell division and cell expansion to potato microtuber growth in vitro. Plant Cell, Tissue and Organ Culture 67, 159–164.
| Crossref | GoogleScholarGoogle Scholar |
Meunier A,
Schmit JF,
Stas A,
Kutluk N, Bragard C
(2003) Multiplex reverse transcription-PCR for simultaneous detection of beet necrotic yellow vein virus, beet soilborne virus, and beet virus Q and their vector Polymyxa betae KESKIN on sugar beet. Applied and Environmental Microbiology 69, 2356–2360.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Mumford RA,
Walsh K,
Barker L, Boonham N
(2000) Detection of potato mop top virus and tabacco rattle virus using a multiplex-real-time fluorescent reverse-transcription polymerase chain reaction assay. Phytopathology 90, 448–453.
Nie X, Singh RP
(2002) A new approach for the simultaneous differentiation of biological and geographical strains of potato virus Y by uniplex and multiplex RT-PCR. Journal of Virological Methods 104, 41–54.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Park KS,
Bae YJ,
Jung EJ, Kang SJ
(2005) RT-PCR-based detection of six garlic viruses and their phylogenetic relationships. Journal of Microbiology and Biotechnology 15, 1110–1114.
Petrunak DM,
Gildow FE, Christ BJ
(1991) Incidence and distribution of six viruses infecting potatoes in Pennsylvania. Plant Disease 75, 644.
Sharman M,
Thomas JE, Dietzgen RG
(2000) Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. Journal of Virological Methods 89, 75–88.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Singh RP
(1998) Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids. Journal of Virological Methods 74, 125–138.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Singh RP, Nie X
(2003) Multiplex virus and viroid detection and strain separation via multiplex reverse transcription polymerase chain reaction. Canadian Journal of Plant Pathology 25, 127–134.
Singh RP, Singh M
(1998) Specific detection of potato virus A in dormant tubers by reverse-transcription polymerase chain reaction. Plant Disease 82, 230–234.
Singh RP,
McLaren DL,
Nie X, Singh M
(2003) Possible escape of a recombinant isolate of Potato virus Y by serological indexing and methods of its detection. Plant Disease 87, 679–685.
Yang JW,
Song BT,
Li YJ, Liu J
(2006) A simple and efficient method for RNA extraction from potato tuber. Journal of Agricultural Biotechnology 14, 297–298.
