Real time RT‐PCR assay for quantitative detection of Citrus viroid III in plant tissues

Abstract

A rapid and sensitive real time reverse transcription‐PCR (RT‐PCR) assay based on SYBR Green I chemistry was developed for the quantitative detection of Citrus viroid III (CVd‐III) in citrus samples. CVd‐III titre was determined at different times in green bark of sour orange, Troyer citrange, trifoliate orange and alemow seedlings inoculated with a CVd‐IIIb source. Ten weeks after inoculation the viroid was detected in the four species, without substantial differences in viroid titre among them. Nine weeks later an overall increase of viroid titre was observed. The copy number of CVd‐III in sour orange and Troyer citrange was monitored up to 52 weeks after inoculation and a further increase of viroid titre was observed at 35 weeks. For validation purposes, field samples were tested from 58 citrus trees with mixed infections of CVd‐III, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), as well as from healthy controls. Based on the sensitivity (100%), specificity (96·7%), accuracy (99·2%) and repeatability (Cohen's kappa index 0·98) of the assay, it is suggested that its employment in breeding programmes would be helpful in the evaluation of host resistance and viroid accumulation in plants.