scholarly journals Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses

Plant Disease ◽  
2010 ◽  
Vol 94 (10) ◽  
pp. 1248-1254 ◽  
Author(s):  
T. Maoka ◽  
S. Sugiyama ◽  
Y. Maruta ◽  
T. Hataya
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Colorimetric Detection ◽  
Simultaneous Detection ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Virus S ◽  
Cdna Macroarray ◽  
Potato Viruses ◽  
Homologous Virus

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

scholarly journals Macroarray Detection of Eleven Potato-Infecting Viruses and Potato spindle tuber viroid

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 730-740 ◽  
Author(s):  
Bright Agindotan ◽  
Keith L. Perry
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Tobacco Rattle Virus ◽  
Alfalfa Mosaic Virus ◽  
Potato Virus X ◽  
Potato Spindle Tuber Viroid ◽  
Apparent Competition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Potato Virus S

A macroarray was developed for the detection of 11 potato viruses and Potato spindle tuber viroid. The 11 viruses detected included those commonly found or tested for in North American potato seed certification programs: Alfalfa mosaic virus, Cucumber mosaic virus, Potato mop top virus, Potato leafroll virus, Potato latent virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, and Tobacco rattle virus. These viruses were detected using oligonucleotide 70-mer probes and labeled targets prepared by a random primed amplification procedure. Potato plants analyzed included those infected with 12 reference virus stocks and 36 field isolates. Results from the macroarray were entirely consistent with those obtained using a standard serological assay (enzyme-linked immunosorbent assay). Four isolates of Potato spindle tuber viroid, in mixed infection with one or more viruses, also were detected in the array, although strong hybridization signals required amplification with viroid-specific primers in combination with anchored-random primers. In individual plants, up to four viruses, or a viroid plus two viruses, were detected, with no apparent competition or inhibition. Macroarrays are a cost-effective approach to the simultaneous diagnostic detection of multiple pathogens from infected plants.

scholarly journals Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control

Plant Disease ◽  
2006 ◽  
Vol 90 (2) ◽  
pp. 185-189 ◽  
Author(s):  
ZhiYou Du ◽  
JiShuang Chen ◽  
Chuji Hiruki
Keyword(s):  
Potato Virus ◽  
Internal Control ◽  
18S Rrna ◽  
Degradation Kinetics ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Potato Viruses ◽  
Das Elisa

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

scholarly journals Presence and distribution of economically important potato viruses in Montenegro

2011 ◽  
Vol 26 (2) ◽  
pp. 117-127
Author(s):  
Jelena Zindovic
Keyword(s):  
Potato Virus ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Leaf ◽  
Potato Virus S ◽  
Potato Varieties ◽  
Potato Viruses ◽  
Das Elisa

The research was carried out, in the period 2002-2004 in order to determine the presence and distribution of potato viruses at 12 different locations and on 9 different potato varieties grown in Montenegro. The research included collecting of samples in seed potato crops and testing of six economically important potato viruses: Potato leaf roll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS), Potato virus A (PVA) i Potato virus M (PVM). Using the direct enzyme-linked immunosorbent assay (DAS-ELISA) and commercial antisera specific for six potato viruses, it was found that PVY was the most frequent virus during the three-year research period. The second frequent virus was PVS, followed by PVA, PLRV, PVM and PVX. Single and mixed infections were detected, and the most prevalent were the single infections of PVY. Also, in the period 2002-2004, PVY had the highest distribution and the number of present viruses was different at different localities and on different potato varieties. Further investigations were related to detailed characterization of the most prevalent virus (PVY), which is at the same time economically the most important one. Serological characterization of PVY was performed utilizing DAS-ELISA kit with commercial monoclonal antibodies specific for detection of the three strain groups of PVY, and the two strain groups - necrotic (PVYN/PVYNTN) and common (PVYO), were identified. Necrotic strains were prevalent in 2002 and 2004, while in 2003 PVYO was the most frequent strain in virus population. The presence of stipple streak strain (PVYC) was not detected in any of the tested samples.

scholarly journals Incidence of Viruses Infecting Tomato and Their Natural Hosts in the Southeast and Central Regions of Iran

Plant Disease ◽  
2009 ◽  
Vol 93 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hossain Massumi ◽  
Mehdi Shaabanian ◽  
Akbar Hosseini Pour ◽  
Jahangir Heydarnejad ◽  
Heshmetollah Rahimian
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Tomato Mosaic Virus ◽  
Tobacco Streak Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Streak Virus ◽  
Arabis Mosaic Virus ◽  
Potato Virus S ◽  
Chlorotic Spot ◽  
Central Regions

A survey was conducted to determine the incidence of Cucumber mosaic virus (CMV), Beet curly top virus (BCTV), Tomato yellow leaf curl virus (TYLCV), Tomato chlorotic spot virus (TcSV), Potato virus Y (PVY), Potato virus S (PVS), Tomato spotted wilt virus (TSWV), Tomato ringspot virus (TRSV), Tomato aspermy virus (TAV), Arabis mosaic virus (ArMV), Tobacco streak virus (TSV), Tomato bushy stunt virus (TBSV), Tobacco mosaic virus (TMV), and Tomato mosaic virus (ToMV) on tomato (Solanum lycopersicum) in the major horticultural crop growing areas in the southeast and central regions of Iran. A total of 1,307 symptomatic leaf samples from fields and 603 samples from greenhouses were collected from January 2003 to July 2005 in five southeastern and central provinces of Iran. Samples of symptomatic plants were analyzed for virus infection by enzyme-linked immunosorbent assay (ELISA) using specific polyclonal antibodies. ArMV and CMV were the most frequently found viruses, accounting for 25.6 and 23.4%, respectively, of the collected samples. BCTV, TSWV, TMV, PVY, ToMV, and TYLCV were detected in 6.1, 5.8, 5.6, 5, 4.8, and 1.6% of the samples, respectively. TBSV, TAV, TSV, PVS, and TRSV were not detected in any of the samples tested. Double and triple infections involving different combination of viruses were found in 13.9 and 1.7% of samples, respectively. This is the first report of PVY and ArMV as viruses naturally infecting tomato in Iran. Infection of tomato plants with PVY and ArMV was confirmed. Six out of 20 plant species belonging to six genera, growing in tomato fields or in the nearby areas, were found infected with TSWV, TMV, PVY, and CMV.

scholarly journals Incidence and Distribution of Important Viral Pathogens in Some Iranian Potato Fields

Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 609-615 ◽  
Author(s):  
R. Pourrahim ◽  
Sh. Farzadfar ◽  
A. R. Golnaraghi ◽  
A. Ahoonmanesh
Keyword(s):  
Potato Virus ◽  
Alfalfa Mosaic Virus ◽  
Potato Virus X ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mixed Infections ◽  
Virus Infections ◽  
Potato Virus S ◽  
Leafroll Virus ◽  
Almost All ◽  
Biological Methods

From a total of 8,135 potato leaves collected from 132 fields in 11 provinces of Iran, the incidence and distribution of Alfalfa mosaic virus (AlMV), Eggplant mottled dwarf virus (EMDV), Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus M (PVM), Potato virus S(PVS), Potato virus X (PVX), Potato virus Y (PVY), and Tomato yellow fruit ring virus (TYFRV) were assessed using serological and biological methods. Based on enzyme-linked immunosorbent assay (ELISA) results, viruses in decreasing order of incidence in potato were PVS (35.9%), PVY (34.4%), PVA (27.0%), PVX (20.8%), PLRV (13.9%), PVM (9.0%), AlMV (7.0%), TYFRV (5.9%), and EMDV (5.1%). All 132 fields surveyed had some degree of virus infection, ranging from 28.8 to 98.6%, with an overall incidence of 75.2%. The highest and lowest incidence of virus infections among the surveyed provinces occurred in Kerman (93.2%) and Ardabil (56.7%), respectively. Overall, 25.0 and 50.2% of the collected potato samples had single or mixed infections, respectively. High levels of mixed infections were found between PVX and PVS (8.6%), and PVX and PVY (7.6%). Moreover, co-infection of samples with PVS and PVY, PVA and PVS, and PVA and PVY, the aphid-vectored virus/virus combinations, occurred at the highest incidence in almost all provinces surveyed, 15.3, 13.8, and 12.8%, respectively. In this study, Beet curly top virus was detected in symptomatic potato samples collected from some fields in the Kermanshah province.

scholarly journals Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

scholarly journals Epidemic of Potato virus Y and Cucumber mosaic virus in Henan Province Tobacco

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
Author(s):  
X. D. Li ◽  
Y. Q. Li ◽  
H. G. Wang
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Early Stage ◽  
Potato Virus X ◽  
Henan Province ◽  
Enzyme Linked Immunosorbent Assay ◽  
Resistant Varieties ◽  
Vein Necrosis

Flue-cured tobacco is an important crop in Henan Province, China. During the 2000 growing season, many tobacco plants showed various degrees of mottling, mosaic, vein clearing, or vein necrosis in most of the counties. Some plants even died at an early stage of growth. A survey was conducted in May-June in several tobacco-growing counties, and the incidence of symptomatic plants in individual fields ranged from 10 to 85%. The most widely planted tobacco varieties, NC89, K326, and K346, were highly susceptible. Symptomatic plants were collected from Jiaxian and Xiangcheng counties and samples were tested by enzyme-linked immunosorbent assay for Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), and Potato virus X (PVX). Of 65 samples tested, 21 were positive for only PVY, 16 positive for only CMV, one each was positive for only TMV or PVX. Nineteen samples were doubly infected with various combinations of these viruses and six were infected with combinations of three viruses. The causal agent(s) in the remaining sample could not be determined. In total, CMV was detected in 40 samples, PVY in 38, PVX in 10, and TMV in 7 samples. TMV and CMV used to be the most important viruses and PVY occurred only rarely. But PVY has become prevalent in Henan and in neighboring Shandong province (2). CMV and TMV were reported to be the most prevalent viruses in Shanxi (1) and Fujian Provinces (3). Because resistant varieties are not available, and mixed infections are more common, the results presented here explain why huge damage is occurring in tobacco crops in recent years. Some varieties are partially resistant to TMV and CMV but the varieties commonly grown are highly susceptible to PVY. Therefore, breeding for resistance to viruses, especially to PVY, is urgent to control the occurrence of tobacco viral diseases. References: (1) J. L. Cheng et al. Acta Tabacaria Sin. 4:43, 1998. (2) J. B. Wang et al. Chinese Tobacco Sci. 1:26, 1998. (3) L. H. Xie et al. Acta Tabacaria Sin. 2:25, 1994.

scholarly journals Multiple DNA markers for evaluation of resistance against Potato virus Y, Potato virus S and Potato leafroll virus

2018 ◽  
Vol 54 (No. 1) ◽  
pp. 30-33 ◽  
Author(s):  
M. Naderpour ◽  
L. Sadeghi
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Simultaneous Detection ◽  
Potato Leafroll Virus ◽  
Potato Seed ◽  
Breeding Programs ◽  
Potato Virus S ◽  
Sequence Characterized Amplified Region ◽  
Leafroll Virus ◽  
Resistance To Viruses

Molecular markers within or close to genes of interest play essential roles in marker-assisted selection. PCR-based markers have been developed for numerous traits in different plant species including several genes conferring resistance to viruses in potato. In the present work, rapid and reliable approaches were developed for the simultaneous detection of Ryadg and Ry-fsto, Ns, and PLRV.1 genes conferring resistance to Potato virus Y, Potato virus S and Potato leafroll virus, respectively, on the basis of previously published and newly modified markers. The sequence characterized amplified region (SCAR) markers for Ryadg, Ns and PLRV1 and the newly modified cleaved amplified polymorphic sequences (CAPS) marker for Ry-fsto were amplified in one PCR reaction which could simply characterize Ryadg and PLRV.1 resistance. Additional digestion of amplicons with EcoRV and MfeI for genotyping the Ry-fsto and Ns resistance genes, respectively, was needed. The effectiveness of genotyping in triplex and tetraplex PCRs was tested on 35 potato varieties used for potato seed production and breeding programs.  

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt

2018 ◽  
Vol 73 (11-12) ◽  
pp. 423-438 ◽  
Author(s):  
Engy E. Abdel Aleem ◽  
Radwa M. Taha ◽  
Faiza A. Fattouh
Keyword(s):  
Phylogenetic Analysis ◽  
Nucleotide Sequence ◽  
Mosaic Virus ◽  
Complete Nucleotide Sequence ◽  
Leaf Roll ◽  
Alfalfa Mosaic Virus ◽  
Potato Leaf Roll Virus ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Potato Viruses

Abstract Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

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