scholarly journals Practical Field Detection of Citrus Viroids in Florida by RT-PCR

2002 ◽  
Vol 15 (15) ◽  
Author(s):  
S. M. Garnsey ◽  
D. L. Zies ◽  
M. Irey ◽  
P. J. Sieburth ◽  
JS S. Semancik ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Field Detection ◽  
Citrus Viroids ◽  
Practical Field

scholarly journals Detection of Multiple Infections of Citrus exocortis viroid, Citrus viroid III, and Hop stunt viroid Variants in Hunan Province, China

Plant Disease ◽  
2007 ◽  
Vol 91 (9) ◽  
pp. 1205-1205 ◽  
Author(s):  
S. Rizza ◽  
A. Catara ◽  
X. F. Ma ◽  
Z. Deng
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Roll ◽  
Poncirus Trifoliata ◽  
Hunan Province ◽  
Variable Degree ◽  
Rt Pcr ◽  
Stunt Viroid ◽  
Citrus Exocortis Viroid ◽  
Hop Stunt Viroid ◽  
Citrus Viroids

Citrus cultivation in China has increased since the late 1970s, with China now having the largest area of citrus in culture in the world that is spread in 22 provinces and municipalities. Hunan Province has undergone a program to become one of the major citrus producers in China. Poncirus trifoliata is the main rootstock, so citrus viroids are a limiting factor for further citriculture development. In mainland China, only the presence of Citrus exocortis viroid (CEVd) has been reported from Etrog citron indexing, sPAGE (sequential polyacrylamide gel electrophoresis) analysis (2), and reverse transcription (RT)-PCR (3). Three viroid-like RNAs, a1, b1, and d, based on sPAGE patterns were detected years ago in our laboratory in budsticks received from Sichuan Province. To identify different viroids and determine their distribution, a survey has been undertaken. Field trees showing stunting, bark scaling and cracking of the rootstock, and poor yield were tested using biological indexing and PCR for the most frequent citrus viroids. Samples from six trees of a local sweet orange variety and three of a Clementine variety introduced from abroad, both grafted on P. trifoliata and showing a variable degree of bark scaling and cracking, were collected near Changsha and in the County of Xin Ning at the end of summer 2006. Small pieces of bark were inserted in stems of young E. citron budwood grafted on rough lemon and maintained in a warm greenhouse (24 to 32°C). Indexing on E. citron showed mild epinasty and leaf roll typical of citrus viroid infections. To identify specific viroids, bark was ground to a fine powder with liquid nitrogen and total RNA was extracted with TRIZOL Reagent (Invitrogen, San Diego, CA) and tested by RT-PCR to detect CEVd, Hop Stunt viroid (HSVd), and Citrus viroid III (CVd-III), as well as to identify the cachexia variants of HSVd. Four primer pairs were used to test the RNA extracts by RT-PCR (1). All samples were infected by HSVd, eight with CVd-III, and six with CEVd. The cachexia variants of HSVd were detected in four of nine samples. Mixed infections were as follows: one sample had CEVd and HSVd, eight had HSVd and CVd-III, and five were infected by the three viroids. A second sampling 3 months after inoculation gave the same amplification patterns. The results show that at least three viroids are present in citrus orchards in Hunan Province. To our knowledge, this is the first report of cachexia variants of HSVd and CVd-III in China. The common occurrence of these viroids supports the need for proper indexing of mother trees and a specific shoot tip grafting program to create healthy budwood sources to provide healthy plants. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes, 20:105, 2006. (2) L. Han et al. Viroids. CSIRO Publishing, Melbourne, 283, 2003. (3). Q. Hu et al. Acta Bot. Sin. 39:613, 1997.

scholarly journals Field Detection of Tembusu Virus in Western Thailand by RT-PCR and Vector Competence Determination of Select Culex Mosquitoes for Transmission of the Virus

2013 ◽  
Vol 89 (5) ◽  
pp. 1023-1028 ◽  
Author(s):  
Monica L. O'Guinn ◽  
Russell E. Coleman ◽  
Timothy P. Endy ◽  
John S. Lee ◽  
Michael J. Turell ◽  
...  
Keyword(s):  
Vector Competence ◽  
Rt Pcr ◽  
Field Detection ◽  
Culex Mosquitoes ◽  
Tembusu Virus

scholarly journals Molecular Detection and Prevalence of Citrus Viroids in Texas

HortScience ◽  
2007 ◽  
Vol 42 (3) ◽  
pp. 600-604 ◽  
Author(s):  
Madhurababu Kunta ◽  
J.V. da Graça ◽  
Mani Skaria
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Rt Pcr ◽  
Lower Rio Grande Valley ◽  
Biological Indexing ◽  
Hop Stunt Viroid ◽  
Viroid Infection ◽  
Citrus Viroids ◽  
Commercially Important ◽  
Polymerase Chain ◽  
Alternative Detection

Viroids are graft- or mechanically transmissible agents, disseminated through budding. Biological indexing of commercially important citrus cultivars grown in the Lower Rio Grande Valley of Texas showed that many are infected with citrus viroids. Most of these trees carried more than one viroid. In most cases, the infected trees are asymptomatic carriers because sour orange, the predominant rootstock used in Texas, does not show symptoms of viroid infection. Detection of viroids through biological indexing on sensitive indicator plants followed by sequential polyacrylamide gel electrophoresis (sPAGE) is the gold standard but is time-consuming and requires plants to be kept at optimum conditions. A conditional use of reverse transcriptase–polymerase chain reaction (RT-PCR) provides an efficient and alternative detection of viroids for use in the Texas virus-free citrus budwood certification program. RT-PCR could be useful in Texas to help expedite the evaluation for the presence of viroids before conducting the final biologic indexing. Using RT-PCR, we could detect, clone, and sequence full-length viroids of Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) (both cachexia and noncachexia variants), Citrus viroid-III (Citrus dwarfing viroid), and Citrus viroid-IV (Citrus bark cracking viroid) from a collection of viroid-inoculated grapefruit plants. The source plants were previously shown to be viroid-infected by biological indexing on Etrog citron plants. Based on our results, RT-PCR can be a conditional substitute for biological indexing of mother trees in foundation blocks and shoot tip-grafted trees in the virus-free budwood program. A positive RT-PCR result has a serendipitous value because those trees can be discarded from the pool before expensive biological indexing.

A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China

2008 ◽  
Vol 124 (1) ◽  
pp. 175-180 ◽  
Author(s):  
Xuefeng Wang ◽  
Changyong Zhou ◽  
Kezhi Tang ◽  
Yan Zhou ◽  
Zhongan Li
Keyword(s):  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Citrus Viroids ◽  
One Step

A novel RT-PCR approach for detection and characterization of citrus viroids

2006 ◽  
Vol 20 (2) ◽  
pp. 105-113 ◽  
Author(s):  
L. Bernad ◽  
N. Duran-Vila
Keyword(s):  
Rt Pcr ◽  
Citrus Viroids

scholarly journals Occurrence of Four Citrus Viroids in Chongqing, China

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 978-978 ◽  
Author(s):  
X. F. Wang ◽  
C. Y. Zhou ◽  
K. Z. Tang ◽  
Z. A. Li
Keyword(s):  
Rapid Development ◽  
Poncirus Trifoliata ◽  
Rt Pcr ◽  
Genome Sequences ◽  
First Report ◽  
Chongqing Municipality ◽  
Rough Lemon ◽  
Hop Stunt Viroid ◽  
Two Samples ◽  
Citrus Viroids

Chongqing Municipality, located in the superior citrus belt of the upper-middle Yangtze River, is one of the most important citrus-producing areas in China. A survey was performed to evaluate the occurrence and distribution of citrus viroids in this area, where Poncirus trifoliata is the main rootstock. From 2002 to 2006, 72 samples of sweet oranges (Citrus sinensis), lemons (C. jambhiri), mandarins (C. reticulata), and mandarin hybrids, which showed stunting, bark scaling, and cracking symptoms on the rootstock, were collected and graft inoculated into Arizona 861-S1 Etrog citron (C. medica) on rough lemon rootstock and maintained in a greenhouse at 28 to 32°C. Fifty-one of the seventy-two samples were cultivars imported from abroad, and the remaining samples were all local cultivars. Sixty samples induced symptoms typical of citrus viroids on the Etrog indicator plants. To identify the causal agent(s), a one-step reverse transcription (RT)-PCR protocol, using five primer pairs (1) targeting the complete genome sequences, was used to detect Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) (2). All 72 samples were infected by citrus viroids. Sixty-five and thirty-five of the seventy-two samples were positive for HSVd and CVd-III, respectively. CEVd and CBLVd were found, respectively, in 20 and 11 of 72 samples, whereas CVd-IV was not detected. Of 72 samples, 12 without typical symptoms on Etrog citrons were infected by HSVd and CBLVd. Nearly all (70 of 72) infected citrus plants harbored more than one viroid species, and two plants were both infected by CEVd, CBLVd, HSVd, and CVd-III. RT-PCR products were purified and ligated into pGEM T-Easy Vector (Promega, Madison, WI) and three clones for each of the four viroid species were sequenced and deposited in GenBank: CEVd (Accession Nos. EU382202, U382203, and EU382204); CBLVd (Accession Nos. EU382211, EU382212, and EU382213); HSVd (Accession Nos. EU382208, EU382209, and EU382210); and CVd-III (Accession Nos. EU382205, EU382206, and EU382207). BLAST analysis showed that these nucleotide sequences had greater than 94% nucleotide identity to the most similar genome sequences in GenBank. Sweet orange was more frequently infected by viroids than the other citrus cultivars. To our knowledge, this is the first report of CBLVd, HSVd, and CVd-III in Chongqing and the first report of CBLVd in China. The high incidence of citrus viroids in Chongqing necessitates rapid development of a system of propagation and testing to reduce the incidence of viroids and the associated loss of citrus production. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) K. Z. Tang et al. Acta Hortic. Sin. 32:408, 2005.

scholarly journals Characterization and Sap Transmission of Citrus Bent Leaf Viroid in Malaysia

2019 ◽  
Author(s):  
Yasir Iftikhar ◽  
Ying Wei Khoo ◽  
Taneswari Murugan ◽  
Nur Athirah Roslin ◽  
Rabiatul Adawiyah ◽  
...  
Keyword(s):  
Leaf Size ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Specific Primers ◽  
Sequence Identity ◽  
Citrus Viroids ◽  
Citrus Production ◽  
Citrus Orchards ◽  
Citrus Varieties

AbstractA 328 nucleotide (nt) variant of Citrus bent leaf viroid (CBLVd) was characterized from citrus varieties in Malaysia showing leaf bending, stunting and midvein necrosis. CBLVd was detected by RT-PCR assay using CBLVd specific primers in 12 out of 90 samples, collected from six different areas in Malaysia. The viroid was present in five species of citrus namely Citrofortunella microcarpa, Citrus aurantifolia, C. hystrix, C. maxima and C. sinensis. Sequence analysis of the isolates obtained from this study showed 99-100% sequence identity to CBLVd Jp isolate (AB006734). Inoculation of sap obtained from a CBLVd positive C. aurantifolia, inoculated into six months old C. microcarpa seedlings showed the symptoms leaf bending, reduced leaf size of matured leaves and mild mosaic between 4 to 6 months after inoculation. The presence of CBLVd in the inoculated seedlings were confirmed by RT-PCR assay and sequencing.Author SummaryThe authors during a limited survey collected the citrus samples from citrus growing areas of Malaysia to detect the citrus viroids. Citrus viroids are associated with decline in citrus production. Thus, the presence of the citrus viroids and their spread needs to be investigated to facilitate the management of this pathogen in citrus orchards. The authors detected and characterized Citrus bent leaf viroid in Malaysian citrus.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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