Occurrence of Hibiscus chlorotic ringspot virus in Hibiscus spp. in New Zealand.

Hibiscus spp. are popular ornamental plants in New Zealand. The genus is susceptible to Hibiscus chlorotic ringspot virus (HCRSV), a member of the genus Carmovirus, which has been reported in Australia, El Salvador, Singapore, the South Pacific Islands, Taiwan, Thailand, and the United States (1-4). In May of 2004, chlorotic spotting and ringspots were observed on the leaves of two H. rosa-sinensis plants in a home garden in Auckland, New Zealand. When inoculated with sap from symptomatic leaves, Chenopodium quinoa and C. amaranticolor developed faint chlorotic local lesions 12 to 15 days later. Phaseolus vulgaris exhibited small necrotic local spots 10 days postinoculation. No symptoms were observed on inoculated plants of Cucumis sativus, Gomphrena globosa, Nicotiana Clevelandii, N. tabacum, or N. sylvestris. Plants of H. rosa-sinensis and the three symptomatic indicator species tested positive for HCRSV using polyclonal antiserum (Agdia Inc., Elkhart, IN) in a double antibody sandwich (DAS)-ELISA. Forward (5'-GGAACCCGTCCTGTTACTTC-3') and reverse (5'-ATCACATCCACATCCCCTTC-3') primers were designed on the basis of a conserved region in the coat protein gene (nt 2722-3278) of HCRSV isolates in GenBank (Accession Nos. X86448 and DQ392986). A product of the expected size (557 bp) was amplified by reverse transcription (RT)-PCR with total RNA extracted from the four infected species. Comparison of the sequence of the amplicon from H. rosa-sinensis (GenBank Accession No. EU554660) with HCRSV isolates from Singapore and Taiwan (GenBank Accession Nos. X86448 and DQ392986) showed 99 and 94% nucleotide identity, respectively. From 2006 to 2008, samples from a further 25 symptomatic hibiscus plants were collected from different locations in the Auckland region. Nineteen, including plants of H. diversifolius, H. rosa-sinensis, and H. syriacus, tested positive for HCRSV by RT-PCR. To our knowledge, this is the first report of HCRSV in New Zealand and of the virus in H. diversifolius and H. syriacus. HCRSV is considered to be widespread in New Zealand. References: (1) A. A. Brunt et al. Plant Pathol. 49:798, 2000. (2) S. C. Li et al. Plant Pathol. 51:803, 2002. (3) H. Waterworth. No.227 in: Descriptions of Plant Viruses. CMI/AAB, Surrey, UK, 1980. (4) S. M. Wong et al. Acta Hortic. 432:76, 1996.

Detection of Poinsettia mosaic virus by RT-PCR in Euphorbia spp. in New Zealand.

Euphorbia pulcherrima (poinsettias) are commonly infected with Poinsettia mosaic virus (PnMV), which resembles the Tymovirus genus in its morphology and viral properties (2) but is closer to the Marafivirus genus at the sequence level (1). Symptoms induced by PnMV range from leaf mottling and bract distortion to symptomless (2). The presence of PnMV in plants imported into New Zealand had never been proven. Leaves of 10 E. pulcherrima samples and six samples from other Euphorbia spp. (E. atropurpurea, E. lambii, E. leuconeura, E. mellifera, E. milii, and E. piscatorial) were collected in the Auckland area, North Island in 2002. Isometric particles of 26 to 30 nm in diameter were observed with electron microscopy in 3 of 10 E. pulcherrima samples. These three samples produced systemic chlorosis and crinkling symptoms on mechanically inoculated Nicotiana benthamiana, which tested PnMV positive by double-antibody sandwich (DAS)-ELISA (Agdia, Elkart, IN). No particles or symptoms on N. benthamiana were observed with the other Euphorbia spp., which were also PnMV-negative by DAS-ELISA. A reverse transcription-polymerase chain reaction (RT-PCR) was developed to further characterize PnMV. Specific primers were designed from the PnMV complete genome sequence (Genbank Accession No. AJ271595) using the Primer3 web-based software (4). Primer PnMV-F1 (5'-CCTGTATTGTCTCTTGCCGTCC-3') and primer PnMV-R1 (5'-AGAGGAAAGGAAAAGGTGGAGG-3') amplified a 764-bp product from nt 5291 of the 5'-end RNA polymerase gene to nt 6082 of the 3'-untranslated region (UTR). Total RNA was extracted from leaf samples using the Qiagen Plant RNeasy Kit (Qiagen Inc., Chastworth, CA). RT was carried out by using PnMV-R1 primer and MMLV reverse transcriptase (Promega, Madison, WI). The PCR was performed in a 20-µl volume reaction containing 2 µl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM PnMV-F1 primer, and 1 U of Taq polymerase (Promega) with a denaturation step (94°C for 5 min), 30 amplification cycles (94°C for 30 s; 55°C for 30 s; 72°C for 1 min), and a final elongation (72°C for 5 min). The sequence of the RT-PCR product (Genbank Accession No. DQ462438) had 98.7% amino acid identity to PnMV. PCR products were obtained from two of three PnMV ELISA-positive E. pulcherrima and three of three PnMV ELISA-positive symptomatic N. benthamiana. The failure to amplify the fragment from all ELISA-positive PnMV is likely because of the presence of inhibitors and latex in E. pulcherrima (3) that make the RNA extraction difficult. Thus, while RT-PCR may be useful for further characterizing PnMV isolate sequences, ELISA may be more reliable for virus detection. In conclusion, to our knowledge, this is the first report of PnMV in E. pulcherrima but not in other Euphorbia spp. in New Zealand. E. pulcherrima plants have been imported into New Zealand for nearly 40 years, and the virus is probably widespread throughout the country via retail nursery trading. References: (1) B. G. Bradel et al. Virology 271:289, 2000. (2) R. W. Fulton and J. L. Fulton. Phytopathology 70:321, 1980. (3) D.-E. Lesemann et al. Phytopathol. Z. 107:250, 1983. (4) S. Rozen and S. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols: Methods in Molecular Biology. S. Krawetz and S. Misener, eds. Humana Press, Totowa, NJ, 2000.

First Report of Spinach latent virus in Tomato in New Zealand.

A Lycopersicon esculentum (tomato) plant from a commercial property in New Zealand was submitted to the Investigation and Diagnostic Centre for diagnosis in 2003. Fruits had faint yellow ringspots but no obvious symptoms were observed on leaves. No virus particles were observed from tomato and symptomatic herbaceous plants crude sap preparations. Mechanically inoculated Nicotiana clevelandii and N glutinosa developed systemic chlorosis, whereas pinpoint necrotic local lesions were observed on Chenopodium amaranticolor. Chlorotic local lesions were also observed on C. quinoa followed by systemic necrosis. No symptoms were observed on Cucumis sativus, Gomphrena globosa, N. benthamiana, N. sylvestris, or N. tabacum cv. White Burley. Total RNA was extracted from N. glutinosa and C. quinoa leaf samples using the Qiagen (Qiagen Inc., Valencia, CA) Plant RNeasy Kit. Reverse transcription (RT) was carried out by using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen, Frederick, MD) followed with PCR using broad-detection primers targeting the genera Carmovirus, Dianthovirus, Ilarvirus, Tospovirus, (Agdia Inc., Elkhart, IN) and Tombusvirus (2). A positive RT-PCR amplification was obtained only with Ilarvirus primers. The 450-bp product (GenBank Accession No. DQ457000) from the replicase gene had a 97.4% nt and 98.6% aa identity with Spinach latent virus (SpLV; Accession No. NC_003808). An RT-PCR protocol was developed for the specific detection of SpLV. Primers were designed from three SpLV RNA sequences (RNA1: NC_003808; RNA2: NC_003809; RNA3: NC_003810) using the Primer3 software (3). Primers SpLV-RNA1-F (5'-TGTGGATTGGTGGTTGGA-3') and SpLV-RNA1-R (5'-CTTGCTTGAGGAGAGATGTTG-3') anneal to the replicase gene from nt 1720 to 2441. Primers SpLV-RNA2-F (5'-GAACCACCGAAACCGAAA-3') and SpLV-RNA2-R (5'-CCACCTCAACACCAGTCATAG-3') bind to the polymerase gene from nt 603 to 1038. Primers SpLV-RNA3-F (5'-GCCTTCATCTTTGCCTTTG-3') and SpLV-RNA3-R (5'-CATTTCATCTGCGGTGGT-3') amplify the movement protein gene from nt 724 to 936. The predicted amplified product sizes were 722, 436, and 213 bp from RNA1, RNA2, and RNA3, respectively. RT was carried out as described above. PCR was performed in a 20-µl reaction containing 2 µl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of forward and reverse primers, and 1 U Taq polymerase (Promega, Madison, WI). The PCR amplification cycle was identical for the three primer pairs: denaturation (95°C for 3 min) followed by 37 cycles of 95°C (20 s), 60°C (30 s), and 72°C (30 s) with a final elongation step (72°C for 3 min). The amplified products were analyzed by gel electrophoresis, stained with SYBR Green, and their identities confirmed by sequencing. The tomato sample was grown from seed imported from the Netherlands where SpLV occurs (4). The virus is of potential importance for the tomato industry because of its symptomless infection and high frequency of seed transmission in many plant species (1,4). SpLV has never been detected in other submitted tomato samples. Consequently, SpLV is not considered to be established in New Zealand. To our knowledge, this is the first report of SpLV in tomato. References: (1) L. Bos et al. Neth. J. Plant Pathol. 86:79, 1980. (2) R. Koeing et al. Arch. Virol. 149:1733, 2004. (3) S. Rozen and H. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols. Humana Press, Totowa, NJ, 2000. (4) Z. Stefenac and M. Wrischer. Acta Bot. Croat. 42:1, 1983.

Partial Characterization of a Carla-Like Virus Infecting Yam (Dioscorea spp.) from China.

Dioscorea opposita (yam) from China was tested for viruses during post-entry quarantine in New Zealand during 2004. No obvious symptoms or virus particles were observed from yam. Mechanically inoculated Nicotiana occidentalis cvs. 37B and P1 produced systemic chlorosis, leaf reduction, and stunting, whereas no symptoms were observed on other tested herbaceous plants (Chenopodium amaranticolor, C. quinoa, Cucumis sativum, Gomphrena globosa, N. benthamiana, N. clevelandii, N. glutinosa, N. sylvestris, and N. tabacum cv. White Burley). Numerous filamentous particles (approximately 600 nm long) were observed by using electron microscopy from symptomatic N. occidentalis. Total RNA was extracted from yam and symptomatic N. occidentalis leaf samples using the Qiagen Plant RNeasy kit (Qiagen, Valencia, CA). Reverse transcription (RT) was carried out using random hexamer primers and SuperScript II RNase H¯ reverse transcriptase (Invitrogen, Carlsbad, CA) followed by polymerase chain reaction (PCR) with different primer pairs. Samples tested negative for Chinese yam necrotic mosaic virus (ChYNMV; genus Macluravirus) with specific primers (supplied by T. Kondo, Aomori Green BioCenter, Aomori, Japan). Negative results were also obtained for the genera Potyvirus, Potexvirus, Capillovirus, Trichovirus, and Foveavirus using RT-PCR with broad detection primers (1,2,4). A positive RT-PCR amplification was obtained from the yam and N. occidentalis samples with universal primers for the genus Carlavirus (Agdia Inc., Elkhart, IN). The 275-bp amplified products from the viral replicase were cloned and sequenced. The yam virus shows a high amino acid similarity with Hop latent virus (87.9%), Aconitum latent virus (86.8%) and Potato virus M (86.8%). Filamentous virus particles belonging to the genera Macluravirus, Potyvirus, and Potexvirus have been reported in yam (3). These virus species are not associated with the carlavirus infection since the virus found in D. opposita tested negative using RT-PCR with primers for these genera. There are no carlaviruses reported to be infecting yams, therefore, it may be considered as a new host-virus association. References: (1) X. Foissac et al. Acta Hortic. 550:37, 2001. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) B. S. M. Lebas. Ph.D. thesis. Greenwich University, Chatham Maritime, UK, 2002. (4) R. A. A. Van der vlugt and M. Berendsen. Eur. J. Plant Pathol. 108:367, 2002.

Development of an RT-PCR for High Plains virus Indexing Scheme in New Zealand Post-Entry Quarantine.

High Plains virus (HPV) causes a potentially serious economic disease of cereals and is of quarantine importance for New Zealand. HPV is transmitted by the wheat curl mite Aceria tosichella, and neither the virus nor its vector is present in New Zealand. Cereal seeds imported to New Zealand are required to be certified HPV-free, as the virus is a regulated pest. A procedure was developed for inspecting plants and testing cereal seedlings in quarantine using reverse transcriptase polymerase chain reaction (RT-PCR) as a detection method. A sample of 50,655 sweet corn seeds was taken from an imported commercial line and germinated in containment. Symptomatic seedlings were collected at 3 and 4 ½ weeks after sowing. Eight out of 27 symptomatic samples tested HPV positive by RT-PCR and were confirmed by enzyme-linked immunosorbent assay (ELISA). Sequence analysis revealed that the HPV isolates had a 99.3 to 100% nucleotide identity and 99.0 to 100% amino acid similarity with the HPV USA isolate (GenBank accession no. U60141). HPV variants were detected by single stranded conformational polymorphism (SSCP) analysis but not by restriction fragment length polymorphism (RFLP).

The effects of cardiopulmonary bypass and cold cardioplegia on coronary flow velocity and the reactive hyperemic response in patients and dogs.

In normal coronary arteries, reactive hyperemic responses to a 20-second occlusion, an index of coronary reserve, usually demonstrate a peak-to-resting flow velocity ratio of 4:1 or more. Most intraoperative studies that have assessed reactive hyperemic responses in bypassed vessels have reported peak-to-resting flow velocity ratios of 2:1 or less following a 20-second occlusion. These decreased reactive hyperemic responses could be due to coronary vasodilatation after cardiopulmonary bypass or to an inadequate physiological result of the surgical procedure. In 14 patients with angiographically normal coronary arteries, the peak-to-resting flow velocity ratio following a 20-second coronary occlusion decreased significantly (p less than 0.05) from 4.4 +/- 0.2 (mean +/- standard error) before bypass to 3.0 +/- 0.3 after bypass. In a similar dog model, the peak-to-resting flow velocity ratio decreased by 36 to 52% during the first hour following one hour of cardiopulmonary bypass and cardioplegia. During the same period, left ventricular perfusion increased 21 to 30%, mean arterial pressure and coronary vascular resistance decreased, and myocardial oxygen consumption was unchanged. In a second group of dogs studied for the effects of duration (200 to 240 minutes) of anesthesia and thoracotomy alone, peak-to-resting flow velocity ratio was significantly lower. These clinical and experimental studies suggest that major coronary vasodilatation occurs early following cardiopulmonary bypass and cold cardioplegia, and may contribute to the blunted coronary reactive hyperemic responses reported during this time. Consequently, an intraoperative peak-to-resting flow velocity ratio of 3:1 for bypassed coronary arteries may represent an excellent physiological result.

First Report of Potato spindle tuber viroid in Tomato in New Zealand.

During May 2000, symptoms resembling those of Potato spindle tuber viroid (PSTVd) infection were observed in glasshouse tomatoes (cv. Daniella) growing on one site in Tuakau, South Auckland, New Zealand. Symptoms appeared 2 to 3 months after planting, were confined to plant tops, and included leaf interveinal chlorosis, epinasty, and brittleness. Affected plants comprised ≍10% of the crop and were located near access points. PSTVd was identified in symptomatic plants by the Dutch Plant Protection Service and confirmed by mechanical transmission and grafting to tomato cv. Rutgers and reverse transcription polymerase chain reaction (2). The sequenced genome of this isolate (Accession AF369530) was 358 nt in length and had the closest homology to a Dutch isolate (Accession X17268). Electron microscopy did not reveal the presence of any viruses in affected plants and specific tests for other tomato pathogens were negative. A survey of 50 tomato glasshouse facilities throughout New Zealand revealed three further infected sites, two located close to the original site and one in Nelson, some 480 km distant. However, a survey of field-grown potato crops within 1.5 km of the original outbreak site did not reveal the presence of the viroid. PSTVd is seed transmitted and was probably introduced in glasshouses by use of infected seed. Glasshouse tomatoes are an important crop in New Zealand and annual production is currently 40,000 tonnes. The yield of affected plants may be decreased by up to 80% if suitable controls are not implemented (1). References: (1) S. Kryczynski et al. Phytopath. Polonica 22:85, 1995. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997.