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

Citrus cachexia is an economically important disease of citrus hosts caused by specific variants of Hop stunt viroid (HSVd) that are usually referred to as Citrus cachexia viroid (CCaVd). Eight cachexia-associated HSVd isolates were collected from six citrus growing areas of China, where citrus cachexia had not been reported previously. Forty-seven independent cDNA clones were used for genetic diversity and phylogenetic analysis. There were no sequence variant-cultivar correlation and no distinct regional specificity among or within the cachexia-associated HSVd populations analysed. Three clusters consisting of three major HSVd variants were identified by phylogenetic analysis, suggesting that most Chinese isolates contain a mixture of cachexia and non-cachexia variants. Biological properties of eight CCaVd isolates were determined by inoculating ParsonÕs Special mandarin (Citrus reticulata). Our results suggest that the interactions between CCaVd and non-CCaVd variants might play an important role in suppressing cachexia symptom expression.

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“…In addition to HSVd, most of the 38 isolates contained several other viroid species (Wang et al. 2009b).…”

Section: Resultsmentioning

confidence: 99%

Molecular, Biological and Phylogenetic Analysis of Chinese Isolates ofHop stunt viroidAssociated with Citrus Cachexia Disease

Wang

Zhou

et al. 2010

Journal of Phytopathology

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“…Selection of suitable viroid-free seeds and planting materials seems to be the practicable method of containing the viroid diseases. As these viroids are transmitted through propagation materials strict enforcement of certification regulations would be of great importance for containing the spread of these viroids (Wang et al 2009). …”

Section: Detection Of Viroids In the Environmentmentioning

confidence: 99%

Detection of Viral and Viroid Plant Pathogens in the Environment

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Plant viruses and viroids with obligate parasitism as the mode of existence, have to be in association with living cells of plant hosts always. However, some highly stable viruses have been detected in a free state in soil, water or air. Other viruses depend on the vectors such as insects, mites, nematodes or fungi present in the environment for their spread from infected plants to healthy plants which may be available in the same field or far away. The role of alternative/additional host plant species functioning as sources of virus infection has been demonstrated by applying various diagnostic techniques. The presence of viruses has been detected by employing biological, immunological and nucleic acid-based techniques. The effectiveness and reliability of detection varies significantly. However, molecular techniques have been shown to be more sensitive, specific and fast in providing the results, although they are unable to indicate the level of pathogenic potential of the viruses detected in the environmental samples. Viroids lacking natural vectors and stability outside living cells have been detected in several additional/alternative plant species which may function as reservoir of viroid infection.

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“…Several strategies have been successfully employed for the simultaneous detection and identification of several viruses in a single PCR by using either multiple primer pairs (multiplex RT-PCR) or a polyvalent primer pairs (polyvalent PCR) (James 1999;Bertolini et al 2001;Ito et al 2002;Foissac et al 2001;Roy et al 2005;Sanchez-Navarro et al 2005;Gambino and Gribaudo 2006;Wang et al 2009). Real-time PCR is known to be more sensitive, faster and relatively easier to perform compared with conventional PCR (reduced cycle times and steps, no need for electrophoresis, staining and gel documentation, reduced risk of contamination).…”

Section: Introductionmentioning

confidence: 99%

Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

Loconsole

Saponari²,

Savino

2010

Eur J Plant Pathol

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Citrus, one of the most economically important crops, is susceptible to a number of arthropod-and graft-transmissible pathogens. Rapid and reliable methods for detecting multiple pathogens are important for routine diagnosis by reducing time, labour and costs. To this end, primers and TaqMan probes for Citrus psorosis virus (CPsV) and Citrus variegation virus (CVV) detection by singleplex realtime (q) reverse transcription (RT)-PCR were initially designed. Further optimizations included the development of a multiplex (m) RT-qPCR assay to detect simultaneously CPsV, CVV, and Citrus tristeza virus (CTV) in a single reaction. When 10-fold serial dilutions prepared using total RNAs from CPsV-and CVV-infected plants were tested, RT-qPCR assays proved to be 100 and 1000 times more sensitive than conventional RT-PCR, respectively. The target viruses were effectively identified by mRT-qPCR in fieldinfected clementine and sweet orange trees. The optimized multiplex assay proved to be as sensitive as the singleplex tests, thus providing a valuable alternative tool for detection of these citrus viruses.

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

Abstract: Citrus plants are natural hosts of five viroid species and large numbers of sequence variants.

Cited by 47 publications

References 18 publications

Molecular, Biological and Phylogenetic Analysis of Chinese Isolates ofHop stunt viroidAssociated with Citrus Cachexia Disease

Molecular, Biological and Phylogenetic Analysis of Chinese Isolates ofHop stunt viroidAssociated with Citrus Cachexia Disease

Detection of Viral and Viroid Plant Pathogens in the Environment

Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

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