Comparison of diagnostic techniques for the detection and differentiation of Cherry leaf roll virus strains for quarantine purposes.

Some strains of Cherry leaf roll virus (CLRV) are considered as quarantine pests in New Zealand. CLRV was detected in seven plant host species: Actinidia chinensis, Hydrangea macrophylla, Malus domestica, Plantago major, Ribes rubrum, Rubus idaeus and Rumex sp. collected from New Zealand between 2005 and 2012. Biological, serological and molecular techniques were compared for the detection and differentiation of CLRV isolates. The biological analysis revealed differences in symptomatology and disease severity among the isolates. The five isolates tested by ELISA were serologically related to each other using polyclonal antisera with only one out of four commercially-available antisera successfully detecting all of them. The phylogenetic analysis of sequences obtained from parts of the coat protein, polymerase and 3'-untranslated regions revealed that the New Zealand CLRV isolates clustered into two closely related but distinct phylogenetic groups with some isolates grouping differently depending on the gene studied. The New Zealand CLRV isolates were clearly distinct to overseas isolates found in phylogenetic groups A, D and E. The conventional RT-PCR using primers targeting the CLRV coat protein coding region is recommended for determining sequence differences between strains. These findings will be useful in making regulatory decisions with regard to the testing requirements and the CLRV strains to be regulated in New Zealand.

First Report of Cherry leaf roll virus in Hydrangea macrophylla.

Hydrangea is a popular, summer flowering, ornamental shrub that is native to south and east Asia and North and South America, which is now cultivated throughout the world. Currently, 13 viruses belonging to eight genera have been reported in Hydrangea spp. (1). In April 2011, virus-like disease symptoms, including severe leaf deformation and chlorosis, were observed on two Hydrangea macrophylla 'Sumiko' plants from Australia being held in quarantine in New Zealand. Systemic symptoms of veinal necrosis, necrotic halo spots, and severe leaf deformation were observed on Nicotiana occidentalis '37B' 7 days after inoculation with sap from the symptomatic hydrangea plants. Upon reinoculation with sap of symptomatic leaves from N. occidentalis, necrotic ringspots and tip necrosis, typical of nepovirus infection, were observed on leaves of N. tobacum and Chenopodium quinoa, respectively. Transmission electron microscopy of negatively stained sap from symptomatic leaves of N. occidentalis revealed the presence of isometric particles ~28 nm in diameter. Total nucleic acid was extracted from the symptomatic leaves of N. occidentalis with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). Reverse transcription (RT)-PCR using the reverse primer of Werner et al. (2) and a forward primer, 5'-CGGTGGAGATGCCGGTCCTA-3' (this study), specific to the 3'-untranslated region (3'-UTR) of Cherry leaf roll virus(CLRV) produced an amplicon of ~1,150 bp from N. occidentalis. A consensus sequence of 1,140 bp generated from four clones of the PCR product (GenBank Accession No. JN418885) was 99 and 98% identical at the nucleotide level to a CLRV isolate from Rumex AGBC (GenBank No. AB168099) and Chinese chives (GenBank No. AB168098), respectively. N. occidentalis also tested positive for CLRV using polyclonal antiserum in a double antibody sandwich-ELISA (BIOREBA, Reinach, Switzerland). The presence of CLRV in the original samples and N. occidentalis was confirmed by direct sequencing of the 380-bp amplicons obtained by immunocapture RT-PCR using CLRV-specific primers (2) and the same antiserum. BLASTn analysis of these amplicons (data not submitted to GenBank) also showed 99% nucleotide identity to a New Zealand isolate from a Rubus sp. (GenBank No. AJ877162). The hydrangea plants were released from quarantine because the same strain of CLRV had previously been reported in New Zealand. To our knowledge, this is the first report of CLRV in hydrangea. CLRV is a seed and pollenborne nepovirus and can be transmitted mechanically and by grafting. Since hydrangeas are mainly vegetatively propagated and are less commonly grown from seeds, the natural spread of CLRV will depend on the movement of infected propagation material. It is unknown whether this virus causes reduction in flower quality in hydrangea as reported in other hosts but any impact on flower quality may be of economic significance in commercial nurseries. References: (1) M. Caballero et al. Plant Dis. 93:891, 2009. (2) R. Werner et al. Eur. J. For. Pathol. 27:309, 1997.

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

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.

Dilemmas caused by endogenous pararetroviruses regarding the taxonomy and diagnosis of yam (Dioscorea spp.) badnaviruses: analyses to support safe germplasm movement.

The discovery of endogenous pararetroviral sequences (EPRVs) has had a deep impact on the approaches needed for diagnosis, taxonomy, safe movement of germplasm and management of diseases caused by pararetroviruses. In this article, we illustrate this through the example of yam (Dioscorea spp.) badnaviruses. To enable progress, it is first necessary to clarify the taxonomical status of yam badnavirus sequences. Phylogeny and pairwise sequence comparison of 121 yam partial reverse transcriptase sequences provided strong support for the identification of 12 yam badnavirus species, of which ten have not been previously named. Virus prevalence data were obtained, and they support the presence of EPRVs in D. rotundata, but not in D. praehensilis, D. abyssinica, D. alata or D. trifida. Five yam badnavirus species characterised by a wide host range seem to be of African origin. Seven other yam badnavirus species with a limited host range are probably of Asian-Pacific origin. Recombination under natural circumstances appears to be rare. Average values of nucleotide intra-species genetic distances are comparable to data obtained for other RNA and DNA virus families. The dispersion scenarios proposed here, combined with the fact that host-switching events appear common for some yam badnaviruses, suggest that the risks linked to introduction via international plant material exchanges are high.

First Report of Wheat streak mosaic virus on Wheat in New Zealand.

In August of 2005, seeds of wheat (Triticum aestivum) breeding line 6065.3 tested positive for Wheat streak mosaic virus (WSMV; genus Tritimovirus) by a WSMV-specific reverse transcription (RT)-PCR assay (2). The sequence of the 200-bp amplicon (GenBank Accession No. FJ434246) was 99% identical with WSMV isolates from Turkey and the United States (GenBank Accession Nos. AF454455 and AF057533) and 96 to 97% identical to isolates from Australia (GenBank Accession Nos. DQ888801 to DQ888805 and DQ462279), which belong to the subclade D (1). As a result, an extensive survey of three cereal experimental trials and 105 commercial wheat crops grown on the South Island of New Zealand was conducted during the 2005-2006 summer to determine the distribution of WSMV. Wherever possible, only symptomatic plants were collected. Symptoms on wheat leaf samples ranged from very mild mosaic to symptomless. In total, 591 leaf samples suspected to be symptomatic were tested for WSMV by a double-antibody sandwich (DAS)-ELISA (DSMZ, Braunschweig, Germany). Of the 591 symptomatic samples, 81 tested positive. ELISA results were confirmed by RT-PCR with novel forward (WSMV-F1; 5'-TTGAGGATTTGGAGGAAGGT-3') and reverse (WSMV-R1; 5'-GGATGTTGCCGAGTTGATTT-3') primers designed to amplify a 391-nt fragment encoding a region of the P3 and CI proteins. Total RNA was extracted from the 81 ELISA-positive leaf samples using the Plant RNeasy Kit (Qiagen Inc., Chatsworth, CA). The expected size fragment was amplified from each of the 81 ELISA-positive samples. The positive samples represent 30 of 56 wheat cultivars (54%) collected from 28 of 108 sites (26%) sampled in the growing regions from mid-Canterbury to North Otago. These results suggest that WSMV is widespread in New Zealand both geographically and within cultivars. WSMV is transmitted by the wheat curl mite (Aceria tosichella) (3), which had not been detected in New Zealand despite repeated and targeted surveys. WSMV is of great economic importance in some countries, where the disease has been reported to cause total yield loss (3). Although WSMV is transmitted by seeds at low rates (0.1 to 0.2%) (4), it is the most likely explanation of the spread of the disease in New Zealand. References: (1) G. I. Dwyer et al. Plant Dis. 91:164, 2007. (2) R. French and N. L. Robertson. J. Virol. Methods 49:93, 1994. (3) R. French and D. C. Stenger. Descriptions of Plant Viruses. Online publication. No. 393, 2002. (4) R. A. C. Jones et al. Plant Dis. 89:1048, 2005.

Yams (Dioscorea spp.) from the South Pacific Islands contain many novel badnaviruses: implications for international movement of yam germplasm.

Yam (Dioscorea spp.) samples (n = 690) from seven South Pacific Islands were screened for badnavirus infection by ELISA using two antisera to African badnaviruses. Positive readings were obtained for 26.4-34.6% of samples representing both known (D. bulbifera, D. nummularia and D. pentaphylla) and unreported host species (D. alata, D. esculenta, D. rotundata and D. trifida) in this region. Total DNAs were extracted from 25 ELISA-positive plants and 4 ELISA-negative controls and subjected to PCR amplification with badnavirus-specific primers targeting the reverse transcriptase (RT)-RNaseH genes. All 29 samples yielded the expected size PCR-product for badnaviruses, which were cloned and sequenced. Phylogenetic analyses of the resulting 45 partial (500-527 bp) RT-RNaseH sequences revealed 11 new sequence groups with <79% nucleotide identity to each other or any EMBL sequence. Three sequences (two groups) were highly divergent to the other nine new South Pacific yam badnavirus groups (47.9-57.2% identity) and probably represent either new Caulimoviridae genera or endogenous pararetrovirus sequences. Some sequence groups appeared specific to particular Dioscorea host species. Four 99.9% identical RT-RNaseH sequences possessing nine amino acid deletions from D. esculenta from three islands represent a putative integrated sequence group. The distribution of sequence groups across the islands indicates that badnaviruses have spread extensively between islands and continents through infected germplasm.

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.

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.

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).