First Report of Citrus bark cracking viroid and Citrus viroid V Infecting Citrus in China.

Citrus is the most cultivated and highest value crop in the 15 southern provinces and municipalities in South China. Trifoliate orange (Poncirus trifoliata) is the main rootstock for citrus cultivars and is known to be susceptible to citrus viroids. Surveys conducted from 1995 to 2007 revealed 42 symptomatic samples from 33 cultivars (21 from sweet oranges (Citrus sinensis), 6 from mandarins (C. reticulata), 2 from satauma mandarins (C. unshiu), 6 from lemons (C. jambhiri), and 7 from mandarin hybrids). Symptoms included stunting, bark scaling, and cracking on the Trifoliate orange rootstock collected from citrus orchards in the Chongqing municipality, Sichuan, Zhejiang, Jiangxi, Hunan, and Yunnan provinces. Of the 42 samples, 27 were cultivars imported from abroad and 15 were local cultivars. Budwood from infected trees were grafted onto Arizona 861-S1 'Etrog citron' (C. medica) on rough lemon (C. jambhiri) rootstock. After more than 12 months, 39 of 42 samples revealed typical viroid symptoms of stunting, epinasty. and leaf rolling on the Etrog indicator plants. In September 2009, total RNA was extracted with TRIZOL Reagent and a one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), and Citrus dwarfing viroid (CDVd). Also, a one-step RT-PCR protocol using two primer pairs targeting the complete genome sequences was used to detect Citrus bark cracking viroid (CBCVd) (1) and Citrus viroid V (CVd-V) (2). Of the 42 samples, 37 and 35 were positive for HSVd and CDVd, respectively. CEVd and CBLVd were found, respectively, in 14 and 13 of 42 samples. CBCVd was detected in cv. Meishan No. 9 (C. sinensis) from Sichuan Province and cvs. Akemi (C. reticulata) and Nishirokaori (C. reticulata) from Zhejiang Province. CVd-V was detected in cvs. Nishirokaori, Haruka (C. tamuranua), and Kiyomi (C. unshiu × C. sinensis) from Zhejiang, Hunan, and Chongqing Province, respectively. Only Meishan No. 9 is a local cultivar, whereas Akemi, Nishirokaori, Haruka, and Kiyomi are cultivars imported from Japan. Of 42 samples, 3 without typical symptoms on Etrog citrons were infected with HSVd only. Of 42 infected citrus plants, 36 harbored more than one viroid species. RT-PCR products of CBCVd and CVd-V were cloned by standard methods. Eight clones for CBCVd (one from Meishan No. 9 [Accession No. HM042742, 284 bp], three from Akemi [Accession Nos. HM042743-HM042745, 283 to 284 bp], and four from Nishirokaori [Accession Nos. HM042746-HM042749, 286 bp]) and six clones for CVd-V (four from Nishirokaori [Accession Nos. HM042750-HM042753, 294 bp], one from Kiyomi [Accession No. HM042754, 294 bp], and one from Haruka [Accession No. HM042755, 294 bp]) were sequenced and deposited in GenBank. BLAST analysis of the CBCVd (Accession No. HM042742) and CVd-V (Accession No. HM042751) sequences revealed highest nucleotide sequence identity (100 and 96%) to a CBCVd isolate from Cuba (Accession No. AJ630360) and a CVd-V isolate from Spain (Accession No. EF617306), respectively. To our knowledge, this is the first report of CBCVd and CVd-V in China. Our finding emphasizes the need for CBCVd and CVd-V indexing in production and distribution of pathogen-free citrus plants in China. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) P. Serra et al. Phytopathology 98:1199, 2008. (3) X. F. Wang et al. Eur. J. Plant. Pathol. 124:175, 2009.