Zantedeschia mild mosaic virus, a new widespread virus in calla lily, detected by ELISA, dot-blot hybridization and IC-RT-PCR

Yams (Dioscorea spp.) are widely grown in China as vegetables and herbal medicine. However, studies on viral diseases on yams are still limited. As a pilot project of a government initiative for improving yam productivity, a small study was conducted in Guangxi, a southern province of China, on viral disease in yams. Incidence of virus-like disease for the three extensively grown D. alata cultivars, GH2, GH5, and GH6, were 12 to 40%, 12 to 29%, and 11 to 25%, respectively, as found in a field survey with a five-plot sampling method in 2010. A total of 112 leaf samples showing mosaic or mottling or leaves without symptoms were collected from the cvs. GH2, GH5, GH6, and seven additional cultivars (D. alata cvs. GY2, GY23, GY47, GY69, GY62, GY72, and D. batatas cv. Tiegun). To determine if the symptoms were caused by Yam mild mosaic virus (YMMV; genus Potyvirus, family Potyviridae), total RNA was extracted from leaves with a commercial RNA purification kit (TIANGEN, Beijing, China), and reverse-transcription (RT)-PCR was conducted with a YMMV-specific primer pair (4) that amplifies the 3′-terminal portion of the viral genome. A PCR product with the predicted size of 262 bp was obtained from samples of GH5 (number testing positive of total number of leaves = 5 of 12), GH6 (24 of 42), and GY72 (1 of 1), but not from asymptomatic leaves. PCR products from a GH5 sample (YMMV-Nanning) and a GH6 sample (YMMV-Luzhai) were cloned and sequenced using an ABI PRISM 3770 DNA Sequencer. The two PCR products were 97% identical at nucleotide (nt) level and with the highest homology (89% identity) to a YMMV isolate (GenBank Accession No. AJ305466). To further characterize the isolates, degenerate primers (2) were used to amplify viral genome sequence corresponding to the C-terminal region of the nuclear inclusion protein b (NIb) and the N-terminal region of the coat protein (CP). These 781-nt fragments were sequenced and a new primer, YMMV For1 (5′-TTCATGTCGCACAAAGCAGTTAAG-3′) corresponding to the NIb region, was designed and used together with primer YMMV UTR 1R to amplify a fragment that covers the complete CP region of YMMV by RT-PCR. These 1,278-nt fragments were sequenced (GenBank Accession Nos. JF357962 and JF357963). CP nucleotide sequences of the YMMV-Nanning and YMMV-Luzhai isolates were 94% similar, while amino acid sequences were 99% similar. BLAST searches revealed a nucleotide identity of 82 to 89% and a similarity of 88 to 97% for amino acids to sequences of YMMV isolates (AF548499 and AF548519 and AAQ12304 and BAA82070, respectively) in GenBank. YMMV is known to be prevalent on D. alata in Africa and the South Pacific, and has recently been identified in the Caribbean (1) and Colombia (3). To our knowledge, this is the first report of the natural occurrence of YMMV in China and it may have implications for yam production and germplasm exchange within China. References: (1) M. Bousalem and S. Dallot. Plant Dis. 84:200, 2000. (2) D. Colinet et al. Phytopathology 84:65, 1994. (3) S. Dallot et al. Plant Dis. 85:803, 2001. (4) R. A. Mumford and S. E. Seal. J. Virol. Methods 69:73, 1997.

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Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽

10.1094/pdis.1998.82.1.84 ◽

1998 ◽

Vol 82 (1) ◽

pp. 84-88 ◽

Cited By ~ 24

Author(s):

R. H. Li ◽

G. C. Wisler ◽

H.-Y. Liu ◽

J. E. Duffus

Keyword(s):

Western Blot ◽

Blot Hybridization ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Dot Blot ◽

Tomato Plants ◽

Dot Blot Hybridization ◽

Field Samples ◽

Infected Tomato ◽

Tomato Infectious Chlorosis Virus

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

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Comparison of direct-RT-PCR and dot-blot hybridization for the detection of Potato spindle tuber viroid in natural host plant species

European Journal of Plant Pathology ◽

10.1007/s10658-012-0061-y ◽

2012 ◽

Vol 134 (4) ◽

pp. 859-864 ◽

Cited By ~ 2

Author(s):

Nikon Vassilakos ◽

Oxana Kektsidou ◽

Maria Papaeythimiou ◽

Christina Varveri

Keyword(s):

Host Plant ◽

Plant Species ◽

Blot Hybridization ◽

Potato Spindle Tuber Viroid ◽

Natural Host ◽

Rt Pcr ◽

Dot Blot ◽

Host Plant Species ◽

Dot Blot Hybridization

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An Integrated Diagnostic Approach for Citrus Exocortis Viroid

10.21203/rs.3.rs-768766/v1 ◽

2021 ◽

Author(s):

Chih-Hsu Lin ◽

Ting-Hsuan Hung ◽

I Hu ◽

Ta-Hsin Ku ◽

Chun-Yi Lin ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Severe Disease ◽

Blot Hybridization ◽

Rt Pcr ◽

Dot Blot ◽

Tomato Cultivar ◽

Dot Blot Hybridization ◽

Reverse Transcription Pcr ◽

One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

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Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

PLoS ONE ◽

10.1371/journal.pone.0248581 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0248581

Author(s):

Clyde S. Manuel ◽

Cassandra Suther ◽

Matthew D. Moore ◽

Lee-Ann Jaykus

Keyword(s):

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Blot Hybridization ◽

Molecular Techniques ◽

Rt Pcr ◽

Dot Blot ◽

Dot Blot Hybridization ◽

One Step

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.

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Dot-blot hybridization and RT-PCR detection of extra small virus (XSV) associated with white tail disease of prawn Macrobrachium rosenbergii

Diseases of Aquatic Organisms ◽

10.3354/dao058083 ◽

2004 ◽

Vol 58 ◽

pp. 83-87 ◽

Cited By ~ 30

Author(s):

J Sri Widada ◽

V Richard ◽

Z Shi ◽

D Qian ◽

JR Bonami

Keyword(s):

Blot Hybridization ◽

Macrobrachium Rosenbergii ◽

Pcr Detection ◽

Rt Pcr ◽

Dot Blot ◽

Dot Blot Hybridization ◽

Small Virus

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First Report of Ranunculus mild mosaic virus on Ranunculus asiaticus in Yunnan Province, China

Plant Disease ◽

10.1094/pdis-92-11-1585a ◽

2008 ◽

Vol 92 (11) ◽

pp. 1585-1585 ◽

Cited By ~ 1

Author(s):

J. H. Wang ◽

S. Zhao ◽

X. M. Yang

Keyword(s):

Mosaic Virus ◽

Yunnan Province ◽

Disease Incidence ◽

Viral Disease ◽

Mild Mosaic ◽

Rt Pcr ◽

Potential Threat ◽

First Report ◽

Ranunculus Asiaticus ◽

Mild Mosaic Virus

In June 2007, a new viral disease occurred in commercial fields of Ranunculus asiaticus in the Yunnan Province of China. Infected plants exhibited mosaic symptoms and growth abnormalities. Viral disease incidence for this ornamental crop host in the Yunnan Province was estimated to range from 10 to 20%. Electron microscopic examination of negatively stained leaf-dip preparations from symptomatic plants identified long, flexuous linear particles (approximately 800 nm). The samples were tested using indirect antigen-coated plate (ACP)-ELISA. ACP-ELISA results showed that the leaf samples from symptomatic plants reacted positively to the potyvirus group antibody (Agdia Inc., Eklhart, IN). Total nucleic acid extracted from symptomatic plants was tested using reverse transcription (RT)-PCR with primers (S 5′-GGNAAAAYAGYGGNCARCC-3′; M4: 5′-GTTTTCCCAGTCACGAC-3′ [N = A, G, C, or T; Y = C or T; and R = A or G]) designed to amplify the 3′ terminal region of genomic RNA of the genus Potyvirus (1). RT-PCR produced a 1,650-bp amplification product that was cloned and sequenced (GenBank Accession No. EU684747). The sequenced portion showed 90 and 99% identity with the Ranunculus mild mosaic virus (RMMV) isolates (GenBank Accession Nos. DQ152191 and EF445546) from Italy and Israel, respectively (2). To our knowledge, this is the first report of RMMV in China. Infection from this virus may cause losses for cut-flower production of Ranunculus asiaticu and it is also a potential threat for international trade of Ranunculus germplasm. References: (1) J. Chen and J. P. Chen. Chin. J. Virol. 18:371, 2002. (2) M. Turina et al. Phytopathology 96:560, 2006.

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Identification of Resistance to Potato yellow mosaic virus-Trinidad Isolate (PYMV-TT) Among Lycopersicon Species

Plant Disease ◽

10.1094/pdis.2003.87.6.686 ◽

2003 ◽

Vol 87 (6) ◽

pp. 686-691 ◽

Cited By ~ 7

Author(s):

S. N. Rampersad ◽

P. Umaharan

Keyword(s):

Mosaic Virus ◽

Germ Plasm ◽

Blot Hybridization ◽

Yellow Mosaic Virus ◽

Dot Blot ◽

Breeding Lines ◽

Dot Blot Hybridization ◽

Lycopersicon Species ◽

Commercial Cultivars ◽

Leaf Curl Virus

Three studies were carried out with the objective of identifying resistance to Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) among Lycopersicon species through field screening and using field-inoculated infector rows. The presence of PYMV-TT was confirmed using dot blot hybridization assays. In the first study, eight commercial cultivars of tomato were tested for resistance. In a subsequent study, 11 breeding lines showing high levels of resistance to Tomato mottle virus (ToMoV-Florida) and six lines resistant to Tomato yellow leaf curl virus (TYLCV-Old World Begomovirus) were screened for resistance to PYMV-TT. All breeding lines and the commercial cultivars tested were susceptible to PYMV-TT infection. The third study involved screening 92 accessions from a representative core collection of wild Lycopersicon germ plasm. PYMV-TT was not detected in individuals of 22 accessions.

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