Citrus Viroid IV Detected in Citrus sinensis and C. reticulata in South Africa

Abstract

Citrus Viroid IV or Bark cracking viroid (CVd-IV) has been reported from various countries, mostly in the Near East, but was unknown in southern Africa. It can cause severe bark cracking of trifoliate orange (Poncirus trifoliata), but symptoms on the indicator, Arizona 861-S-1 Etrog (Citrus medica), are mild and transient, usually leaf epinasty (2–4). CVd-IV was detected for the first time in South Africa during the 2009 biennial indexing of mother trees maintained for the South African Citrus Improvement Scheme (CIS). Symptoms were observed on Etrog indicators budded with material from mother trees of DuRoi Valencia sweet orange (C. sinensis). Initially, leaf bend of a single leaf was noted on one plant, and later, petiole browning developed on all test plants. Reverse transcription (RT)-PCR testing confirmed CVd-IV infection by amplification of a 286-bp PCR product representing the full-length CVd genome using the primers CVd-IV-F3 5′-GGTGGATACAACTCTTGGG-3′ (1) and CVd-IV-SL3 5′-GGGTAGTTTCTATCTCAG-3′ (N. Duran-Vila personal communication). Samples of 78 field and 30 nursery trees of DuRoi Valencia supplied over a 7-year period from the CIS were budded on Etrog and tested 3 months after inoculation. Ninety positive samples were obtained from trees up to 7 years old. These trees were on either Swingle citrumelo (P. trifoliata × C. paradisi) or rough lemon (C. jambhiri) rootstocks and no symptoms of bark-cracking or other abnormalities were noted on these trees. Following the initial interception, a comprehensive screening of the CIS gene source, consisting of 450 cultivars, was initiated and CVd-IV was detected in five other accessions: SB navel (C. sinensis) collected in 1984 in Kwa-Zulu Natal Province of South Africa, Gillemberg navel (C. sinensis) collected in 1986 in Limpopo Province, Tarocco midseason (C. sinensis) imported from Sicily in 1987, Fortuna mandarin (C. reticulata) imported from Spain in 1987, and Westin midseason (C. sinensis) imported from Brazil in 1994. None of these accessions are planted on a commercial scale and field observations are therefore not possible. PCR amplicons of all positive samples were bidirectionally sequenced and sequences submitted to GenBank. Sequence comparisons showed those obtained from DuRoi Valencia (JN903763), Tarocco (JN903764), and SB Navel (JN903765), and GenBank reference sequences AB054633 (Japan) and HM042747 (China) to be identical. Likewise, the South African sequences from Westin (JN903766) and Gillemberg (JN903767) were identical to reference sequences X14638 and NC_003539 (Israel) and GQ260216 (Iran). The Fortuna sequence (JN903762) differed by two base pairs from the latter group. On the basis of the entry dates of the accessions, it is probable that CVd-IV has been in South Africa since at least 1984, but was not detected during indexing because of the erratic symptom expression of CVd-IV on Etrog. Existing CVd-IV-contaminated field material will be monitored for possible disease expression, but thus far the trifoliate hybrid and rough lemon rootstocks do not show the same susceptibility as reported for P. trifoliata (4). The detection of CVd-IV in field and gene-source material emphasizes the importance of intensive and specific indexing to ensure distribution of pathogen-free citrus in South Africa. References: (1) L. Bernad and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) N. Duran Vila et al. J. Gen. Virol. 69:3069, 1988. (3) H. Putcha et al. Nucleic Acids Res. 19:6640, 1991. (4) C. Vernière et al. Plant. Dis. 88:1189, 2004.