Occurrence of Four Citrus Viroids in Chongqing, China

Abstract

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.