Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

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Detection and genome characterization of Potato virus Y isolates infecting potato (Solanum tuberosum L.) in La Union (Antioquia, Colombia)

Agronomía Colombiana ◽

10.15446/agron.colomb.v34n3.59014 ◽

2016 ◽

Vol 34 (3) ◽

pp. 317-328 ◽

Cited By ~ 4

Author(s):

Pablo Gutiérrez S. ◽

Mauricio Marín M. ◽

Daniel Muñoz E.

Keyword(s):

Solanum Tuberosum ◽

Potato Virus ◽

Potato Virus Y ◽

Seed Tuber ◽

Rt Pcr ◽

Potato Plants ◽

Nextgeneration Sequencing ◽

Prevalent Strain ◽

Detailed Molecular Analysis

Potato virus Y (PVY) is one of the most severe viruses affecting the production of potato (Solanum tuberosum) in the world. This study presents a detailed molecular analysis using nextgeneration sequencing (NGS), IC-RT-qPCR and RT-PCR on the PVY isolates infecting seed-tubers and foliage of potato plants cv. Diacol-Capiro in La Union (Antioquia, Colombia). Analysis of incidence by IC-RT-qPCR in 15 random leaf samples of three cultivation plots and fifteen sprouting tuber eye-buds reveal infection levels between 13.4 and 80%; a higher incidence of 86.7% was observed in seed-tuber samples with threshold cycle (Ct) values as low as 24.3. Genome assembly from a bulk of foliage samples resulted in a consensus PVY genome (PVY_LaUnionF) of 9,702 nt and 399 polymorphic sites within the polyprotein ORF; while the assembled genome from sprouts of tubers has 9,704 nt (PVY_LaUnionT) and contained only six polymorphic nucleotide sites. Phylogenetic analysis demonstrates that the PVY isolates from leaf samples are in the recombinant PVYNTN group (sequence identity >99%); while those from tuber sprouts are in the PVYN/NTN group with identities above 95%. Sanger sequencing of viral capsid suggests the presence of a third variant related to PVYO, a prevalent strain reported in potato fields worldwide.

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Prevalence and strain composition of potato virus Y circulating in potato fields in China’s north-central province Shanxi

Plant Disease ◽

10.1094/pdis-09-21-1950-re ◽

2021 ◽

Author(s):

Pengcheng Ding ◽

Dexin Chen ◽

Haixu Feng ◽

Jiao Li ◽

Hui Cao ◽

...

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Potato Production ◽

Central China ◽

Shanxi Province ◽

Rt Pcr ◽

Potato Leaf ◽

Single Strain ◽

North Central ◽

Production Areas

Potato is an important crop in Shanxi province located in north-central China. During 2019-2020, 319 potato leaf samples were collected from eight locations distributed in three major potato production areas in Shanxi. Bio-chip detection kit revealed the presence of several potato viruses, and among them potato virus Y (PVY) was the most common one, reaching the incidence of 87.8% of all symptomatic samples. The immuno-captured multiplex reverse transcription (RT)-PCR was used to identify strains for all 280 PVY-positive samples, unveiling 242 samples infected with a single strain of PVY (86.4%) and 38 (13.6%) with a mixed infection. Of samples with a single-strain infection, PVY -SYR-II accounted for 102 (42.1%), followed by PVYN-Wi (33, 13.6%) , PVY -SYR-I (28, 11.6%), 261-4 (22, 9.1%), PVYNTNa (20, 8.3%), PVYNTNb (19, 7.9%), and PVY -SYR-III (18, 7.4%). Seven isolates representing different recombinants were selected for whole genome sequencing. Phylogenetic and recombination analyses confirmed the RT-PCR based strain typing for all seven strains of PVY found in Shanxi. SXKL-12 is the first SYR-III strain from potato reported from China. However, unlike that in other known SYR-III isolates, the region positioned from 1,764 to1,902 nt in SXKL-12 shared the highest sequence identity of 82.2% with an uncharacterized PVY isolate, JL-23, from China. Interestingly, the PVYN-Wi isolate SXZY-40 also possessed a more divergent sequence for the region positioned from 6,156 to 6,276 nt than other N-Wi isolates known to date, sharing the highest identity of 86.6% with an uncharacterized Chinese PVY isolate, JL-11. Pathogenicity analysis of dominant strains PVY -SYR-II and PVYN-Wi in six local popular potato cultivars revealed that Kexin 13, Helan 15 and Jizhangshu 12 were susceptible to these two strains with mild mottling or mosaic symptoms expression, while three cultivars, Jinshu 16, Qingshu 9, Xisen 6 were found fully resistant.

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The Recombinant Potato virus Y (PVY) Strain, PVYNTN, Identified in Potato Fields in Victoria, Southeastern Australia

Plant Disease ◽

10.1094/pdis-05-20-0961-sc ◽

2020 ◽

Vol 104 (12) ◽

pp. 3110-3114

Author(s):

Mariana Rodriguez-Rodriguez ◽

Mohamad Chikh-Ali ◽

Steven B. Johnson ◽

Stewart M. Gray ◽

Nellie Malseed ◽

...

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Strain Typing ◽

Rt Pcr ◽

Growing Seasons ◽

Fta Cards ◽

Southeastern Australia ◽

Whole Genomes ◽

Pcr Assays

Potato virus Y (PVY) is one of the main viruses affecting potato in Australia. However, molecular characterization of PVY isolates circulating in potato in different states of Australia has not yet been thoroughly conducted. Only nonrecombinant isolates of three biological PVY strains collected from potato were reported previously from Western Australia and one from Queensland. Here, PVY isolates collected from seed potato originating in Victoria, Australia, and printed on FTA cards, were subjected to strain typing by RT-PCR, with three isolates subjected to whole genome sequencing. All the 59 PVY isolates detected during two growing seasons were identified to be recombinants based on two RT-PCR assays. No nonrecombinant PVY isolates were identified. All the RT-PCR typed isolates belonged to the PVYNTN strain. Sequence analysis of the whole genomes of three isolates suggested a single introduction of the PVYNTN strain to Australia but provided no clues as to where this introduction originated. Given the association of the PVYNTN strain with potato tuber damage, growers in Australia should implement appropriate strategies to manage PVYNTN in potato.

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Potato virus Y (PVY) detection in a single aphid by one-step RT-PCR with boiling technique

Entomological Research ◽

10.1111/1748-5967.12170 ◽

2016 ◽

Vol 46 (4) ◽

pp. 278-285 ◽

Cited By ~ 4

Author(s):

Juil Kim ◽

Deok Jea Cha ◽

Min Kwon ◽

Rameswor Maharjan

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Rt Pcr ◽

One Step

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An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽

10.1094/pdis-02-13-0161-sr ◽

2013 ◽

Vol 97 (10) ◽

pp. 1370-1374 ◽

Cited By ~ 31

Author(s):

Mohamad Chikh-Ali ◽

Stewart M. Gray ◽

Alexander V. Karasev

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Rna Extraction ◽

Leaf Tissue ◽

Rt Pcr ◽

Pcr Assay ◽

Potato Leaf ◽

Extraction Step ◽

First Time ◽

Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

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Molecular Characterization of Recombinant Strains of Potato virus Y From Saudi Arabia

Plant Disease ◽

10.1094/pdis-05-15-0562-re ◽

2016 ◽

Vol 100 (2) ◽

pp. 292-297 ◽

Cited By ~ 14

Author(s):

Mohamad Chikh-Ali ◽

Hayam Alruwaili ◽

Dalton Vander Pol ◽

Alexander V. Karasev

Keyword(s):

United States ◽

Saudi Arabia ◽

Seed Potato ◽

Potato Virus ◽

Potato Virus Y ◽

The United States ◽

Tuber Quality ◽

Rt Pcr ◽

First Report ◽

Recombinant Strains

Potato virus Y (PVY) exists as a complex of strains, many of which are recombinants. The practical importance of PVY recombinant strains has increased due to their ability to induce potato tuber necrotic ring spot disease (PTNRD) that seriously affects tuber quality. In Saudi Arabia, potato production has increased fivefold during the last three decades, reaching 460,000 tons per year. Although PVY has been reported as one of the main viruses affecting potatoes, no information is available on PVY strains circulating in the country. In August 2014, a survey was conducted in a seed potato field at Al-Jouf, Saudi Arabia. PVY-positive samples selected based on visual symptoms and serological reactivity were subjected to strain typing using multiplex RT-PCR assays and were determined to represent recombinant PVY strains. Whole genome sequences were determined for two representative isolates, S2 and S9, through direct sequencing of a series of overlapping RT-PCR fragments for each isolate, and found to represent strains PVY-NE11 and PVYZ (SYR-III), respectively. One of the recombinant types, SYR-III, was previously found in nearby Syria and Jordan, but the second recombinant, PVY-NE11, was found before only in the United States. Both recombinants, PVY-NE11 and SYR-III, were previously found associated with PTNRD and thought to be rare. The current identification of PVY-NE11 and SYR-III in seed potato in a new geographic region suggests that these recombinants may not be as rare as previously believed. This is the first report on the occurrence of recombinant strains of PVY in potato in Saudi Arabia, and the first report on the PVY-NE11 strain of PVY found in potato outside of the United States.

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The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVYO, PVYN and PVYC strains using RT-PCR

Journal of Virological Methods ◽

10.1016/s0166-0934(02)00008-3 ◽

2002 ◽

Vol 102 (1-2) ◽

pp. 103-112 ◽

Cited By ~ 58

Author(s):

Neil Boonham ◽

Kathy Walsh ◽

Sarah Preston ◽

Julie North ◽

Penny Smith ◽

...

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Rt Pcr

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First Report of Potato virus Y in Potato in Tajikistan

Plant Disease ◽

10.1094/pdis-03-12-0249-pdn ◽

2012 ◽

Vol 96 (7) ◽

pp. 1074-1074 ◽

Cited By ~ 2

Author(s):

O. J. Alabi ◽

J. M. Crosslin ◽

N. Saidov ◽

R. A. Naidu

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Solanum Tuberosum L ◽

Disease Incidence ◽

Pairwise Comparison ◽

Mixed Infections ◽

Rt Pcr ◽

Fta Cards ◽

Tuber Necrosis ◽

Two Samples

Potato (Solanum tuberosum L.) is widely grown as a staple food and cash crop in Tajikistan and is an important food security crop in the country. In June 2011, we conducted a survey of potatoes in farmers' fields in the Buston and Dushanbe regions (about 200 miles apart) of Tajikistan. Potato plants with stunted growth and leaves showing chlorotic spots, curling, and necrotic spots and rings were observed with the disease incidence monitored in 10 fields each in Buston and Dushanbe areas varying between 10 and 60%. Representative samples from symptomatic plants tested positive for Potato virus Y (PVY) using virus-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from symptomatic plants were collected from Buston and Dushanbe areas, imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to the lab at Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR with primers (PVY/Y4A and PVY/Y3S) specific to the coat protein of PVY (3). Samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as references to validate RT-PCR results. A single DNA product of approximately 480 bp was amplified from potato samples that tested positive with PVY-specific immunostrips. The amplified fragments from two samples from Dushanbe and six from Buston areas were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA) and two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 90 to 100% identity among the cloned amplicons (GenBank Accession Nos. JQ743609 to JQ743616) and 90 to 100% with corresponding nucleotide sequence of reference PVY strains (GenBank Accession Nos. JQ743617 to JQ743621). A global phylogenetic analysis of sequences revealed the presence of PVYO in both samples from Dushanbe and one sample from Buston regions and presence of PVYNTN in the remaining five samples from the Buston region. Because of the possible occurrence of mixed infections of PVY strains (2), further studies are needed to determine the presence of mixed infections of two or more strains of PVY and their specificity to potato cultivars. To our knowledge, this study represents the first confirmed report of two distinct strains of PVY in potato in Tajikistan. The occurrence of PVYNTN, a quarantine pathogen in many countries (2), warrants additional investigations to improve sanitary status of potato fields and to facilitate the availability of virus-free seed in clean plant programs for significant yield increases in Tajikistan. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) S. Gray et al. Plant Dis. 94:1384, 2010. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

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Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽

10.1094/pd-89-0605 ◽

2005 ◽

Vol 89 (6) ◽

pp. 605-610 ◽

Cited By ~ 92

Author(s):

Xianzhou Nie

Keyword(s):

Potato Virus ◽

Reverse Transcription ◽

Potato Virus Y ◽

Dna Amplification ◽

Isothermal Amplification ◽

Rt Pcr ◽

Potato Leaf ◽

Time Saving ◽

Loop Mediated Isothermal Amplification ◽

One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

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