First report of Citrus bark cracking viroid and Citrus viroid V infecting citrus in China.

摘要

Citrus viroid surveys conducted from 1995 to 2007 revealed 42 symptomatic samples from 21 sweet orange (Citrus sinensis), 6 mandarin (C. reticulata), 2 from satsuma mandarins (C. unshiu), 6 from lemons (C. jambhiri) and 7 from mandarin hybrids. Symptoms included stunting, bark scaling and cracking on the trifoliate orange (Poncirus trifoliata) rootstock collected from citrus orchards in Chongqing, Sichuan, Zhejiang, Jiangxi, Hunan and Yunnan, China. 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 and a one-step multiplex RT-PCR assay 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 2 primer pairs targeting the complete genome sequences was used to detect Citrus bark cracking viroid (CBCVd) and Citrus viroid V (CVd-V). 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, and the cultivars Akemi and Nishirokaori (C. reticulata), from Zhejiang. CVd-V was detected in the cultivars Nishirokaori, Haruka (C. tamurana) and Kiyomi (C. unshiu x C. sinensis) from Zhejiang, Hunan and Chongqing, respectively. 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 and 6 clones for CVd-V were sequenced and deposited in GenBank. BLAST analysis of the CBCVd and CVd-V sequences revealed highest nucleotide sequence identity (100 and 96%) to a CBCVd isolate from Cuba and a CVd-V isolate from Spain, respectively. This is thought to be the first report of CBCVd and CVd-V in China.