First Molecular Evidence of Citrus psorosis virus and Citrus viroid III from Citrus spp. in New Zealand

Abstract

In December 2008, a collection of Citrus spp. in Kerikeri, New Zealand was surveyed for virus and viroid diseases. Symptoms characteristic of virus or viroid infection were not observed other than Citrus tristeza virus (CTV)-associated stem pitting when examined with the bark removed. Total RNA was extracted from bark samples of 273 trees using RLT buffer (Qiagen Inc., Chatsworth, CA) on a KingFisher mL workstation (Thermo Scientific, Waltham, MA) and tested by reverse transcription (RT)-PCR). Samples from three trees, two from sweet orange, Citrus × sinensis (L.) Osbeck (pro sp.) (maxima × reticulate) and one from tangerine, Citrus reticulata Blanco, tested positive for Citrus psorosis virus (CPsV), and two samples, one each from lemon, Citrus × limon (L.) Burm. F. (pro sp.) (medica × aurantifolia) and sweet orange, tested positive for Citrus viroid III (CVd-III) using previously published primers and PCR cycling conditions (2,4) in a one-step RT-PCR system. The 20-μl RT-PCR reaction was done with Verso Reddymix reagents (Thermo Scientific) containing 250 nM of specific primers and 300 μg/μl of bovine serum albumin (Sigma-Aldrich, St. Louis, MO). The CVd-III genome was completed using specific internal primers (forward: 5′-AACGCAGAGAGGGAAAGGGAA-3′, reverse: 5′-TAGGGCTACTTCCCGTGGTC-3′) with the following cycling conditions: 50°C for 15 min, 94°C for 2 min, then 40 cycles of 94°C for 10 s, 57°C for 30 s, and 68°C for 30 s. The three CPsV amplicons of 419 bp from the RNA-dependent RNA polymerase gene (GenBank Accession Nos. GQ388241 to GQ388243) had 96 to 100% nucleotide identity to each other. A 276-bp (nt position 48 to 323) fragment of the 419-bp sequence was used for comparison with sequences available on GenBank. The three 276-bp CPsV sequences had 89 to 97% nucleotide identity to other CPsV available in GenBank at the time of the analysis. The CVd-III genomes of 291 bp (GenBank Accession Nos. HQ219183 and JF521494) are identical and showed 94 to 99% nucleotide identity to other CVd-III available in GenBank. The presence of CPsV was confirmed in the three samples by a CPsV-specific double-antibody sandwich-ELISA kit (Agritest S.r.l., Valenzano, Italy), while the presence of CVd-III was confirmed only in the lemon sample by r-PAGE (3). The concentration of the viroid in the sweet orange sample may have been below the detection limit of the test. The incidence of the diseases is probably low since CPsV and CVd-III were detected in only a few trees which were planted between 1998 and 2002 at Kerikeri from budwoods of unknown sources imported between the 1970s and 1990s. New Zealand's growing conditions generally do not favor viroid replication in plants, whereas the temperatures may be suitable for CPsV disease. However, symptom characteristics to CPsV and CVd-III have never been observed on the infected trees. This is most likely because of the presence of CTV in the trees (data not shown). CPsV symptoms were thought to have been observed in the 1950s in New Zealand (1) but the causal agent had not been identified. To our knowledge, this is the first molecular and serological evidence of CPsV and the first report of the presence of CVd-III in New Zealand. References: (1) W. A. Fletcher. Orchard. N. Z. 30:33, 1957. (2) T. Ito et al. J. Virol. Methods 106:235, 2002. (3) C. Jeffries and C. James. OEPP/EPPO Bull. 35:125, 2005. (4) S. Martin et al. J. Gen. Virol. 87:3097, 2006.