First Report of a Carlavirus in Fuchsia spp. in New Zealand.

The genus Fuchsia has 110 known species and numerous hybrids. These ornamental plants with brightly colored flowers originate from Central and South America, New Zealand, and Tahiti, but a wider variety are now grown all over the world. Few viruses have been reported in Fuchsia spp.: a carlavirus, Fuchsia latent virus (FLV) (1-3), a cucumovirus, Cucumber mosaic virus (CMV) (3), and two tospoviruses, Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (4). In August 2009, five plants, each representing a different cultivar of Fuchsia hybrid, from home gardens in the Auckland and Southland regions of New Zealand, displayed variable symptoms including mild chlorosis, mild mottle, or purple spots on leaves. Plants tested negative for CMV, INSV, and TSWV using commercial ImmunoStrips (Agdia Inc., Elkhart, IN); however, flexuous particles of ~650 to 700 nm were found by electron microscopy in all samples. Local lesions were also observed on Chenopodium quinoa plants 4 weeks after sap inoculation. Total RNA was extracted from all plants with a RNeasy Plant Mini Kit (Qiagen Inc., Doncaster, Australia) and tested by reverse transcription (RT)-PCR using two generic sets of primers (R. van der Vlugt, personal communication) designed to amplify fragments of ~730 and 550 bp of the replicase and coat protein genes of carlaviruses, respectively. Amplicons of the expected size were obtained for all samples, cloned, and at least three clones per sample were sequenced. No differences within clones from the same samples were observed (GenBank Accession Nos. HQ197672 to HQ197681). A BLASTn search of the viral replicase fragment showed the highest nucleotide identity (76%) to Potato rough dwarf virus (PRDV) (EU020009), whereas the coat protein fragment had maximum nucleotide identity (70 to 72%) to PRDV (EU020009 and DQ640311) and Potato virus P (DQ516055). Sequences obtained were also pairwise aligned using the MegAlign program (DNASTAR, Inc., Madison, WI) and results showed that the isolates had 83 to 97% identity to each other within each genome region. Further sequences (HQ197925 and HQ197926) were obtained from a Fuchsia plant originating from Belgium, a BLASTn analysis showed high nucleotide identity (84 to 99%) to the New Zealand isolates. The low genetic identity to other Carlavirus members suggests that these isolates belong to a different species from those previously sequenced. On the basis of electron microscopy and herbaceous indexing, the isolates had similar characteristics to a carlavirus reported from Fuchsia in Italy (1) and FLV reported in Canada (2). The Italian carlavirus isolate was obtained and tested with the same primers by RT-PCR. Pairwise analysis of the Italian sequences (HQ197927 and HQ197928) with the New Zealand and Belgian sequences showed between 84 and 95% similarity within each genome region. These results suggest that the carlavirus infecting these plants is the same virus, possibly FLV. To our knowledge, this is the first report of this carlavirus infecting Fuchsia spp. in New Zealand, but the virus has probably been present for some time in this country and is likely to be distributed worldwide. References: (1) G. Dellavalle et al. Acta Hortic. 432:332, 1996. (2) L. J. John et al. Acta Hortic. 110:195, 1980. (3) P. Roggero et al. Plant Pathol. 49:802, 2000. (4) R. Wick and B. Dicklow. Diseases in Fuchsia. Common Names of Plant Diseases. Online publication. The American Phytopathological Society, St. Paul, MN, 1999.

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.

First Report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in Grapevine (Vitis vinifera) in New Zealand.

In February 2009, grapevines (Vitis vinifera) in a commercial vineyard in Auckland were showing shortened, spindly canes with tiny leaves. Approximately 10% of the vines were affected. An RNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used to isolate total RNA from leaves collected from six symptomatic (cvs. BAC0022A and Syrah) and eight symptomless vines (cvs. BAC0022A, Syrah, and Chardonnay). RNA was tested by reverse transcription-PCR for the presence of Australian grapevine viroid, Citrus exocortis viroid, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2, and Hop stunt viroid (HSVd). Four of the six symptomatic and all the symptomless vines tested positive for GYSVd-1 using primers 5'-TGTGGTTCCTGTGGTTTCAC-3' and 5'-ACCACAAGCAAGAAGATCCG-3', which amplify the complete genome (368 bp), and published primers PBCVd100C/194H (3), which amplify a 220-bp region of the genome. Amplicons from each PCR were transformed into a pCR 4-TOPO vector (Invitrogen, Carlsbad, CA), cloned, and sequenced. Sequence from both PCRs aligned identically to generate a consensus sequence (GenBank Accession No. HQ447056), which showed 99% nt identity to GYSVd-1 (GenBank No. X87906) by BLASTN analysis. All symptomatic and symptomless vines also tested positive for HSVd using primers C/H-HSVd (4) and HSVd-C60/H79 (1), which amplify the complete genome (298 bp). Amplicons from each isolate were cloned and sequenced. Sequence from both PCRs were aligned. Clones from all isolates, with the exception of one, aligned identically to create a consensus sequence (GenBank No. HQ447057) that showed 99% nt identity to Chinese HSVd isolates from grapevine (GenBank Nos. DQ371436-59) by BLASTN analysis. Sequence from the remaining isolate (GenBank No. HQ447056) was identical to a German Riesling grape isolate of HSVd (GenBank No. X06873). The presence of each viroid was further confirmed in PCR-positive plants by dot-blot hybridization with digoxigenin-labeled synthetic ssRNA probes specific to the full-length genomes of GYSVd-1 and HSVd (S. Harper and L. Ward, unpublished data). To our knowledge, this is the first report of GYSVd-1 and HSVd in V. vinifera in New Zealand. Since both viroids were detected in symptomatic and symptomless plants, the symptoms observed in the vineyard cannot be attributed to viroid infection. Symptoms described for GYSVd-1 include leaf spots and flecks, but no disease symptoms have been reported in grapes as a result of HSVd (2). Viruses found in the vines include Grapevine leaf roll virus-3, Grapevine viruses A and B, and Rupestris stem pitting associated virus, but these are not thought to be the cause of the symptoms. Two sequence types of HSVd were found, suggesting at least two separate introductions of HSVd into the vineyard. The vineyard is more than 40 years old so both viroids may have been present for some years. Export of wine from New Zealand was worth 1 billion dollars in 2009, so there is potential for these viroids to have an economic impact if symptoms are expressed. HSVd has been reported from China, Europe, Japan, Middle East, Pakistan, and the United States. GYSVd-1 has been reported from Australia, China, East Mediterranean, Europe, Japan, and the United States. References: (1) A. Hadidi et al. Acta Hortic. 309:339, 1992. (2) A. Hadidi et al., eds. Viroids. CSIRO Publishing, Collingwood, Australia, 2003. (3) R. Nakaune and M. Nakano. J. Virol. Methods 134:244, 2006. (4) A. M. Shamoul et al. J. Virol. Methods 105:115, 2002.

Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications.

Xylella fastidiosa is a regulated plant pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, a loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) assay were developed to the rimM gene of X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than existing published assays for detection of X. fastidiosa when screened against 20 isolates representing the four major subgroups of the bacterium from a range of host species. No cross-reaction was observed with DNA from healthy hosts or other bacterial species. The LAMP and real-time assays could detect 250 and 10 copies of the rimM gene, respectively, and real-time sensitivity was comparable with an existing published real-time PCR assay. Hydroxynapthol blue was evaluated as an endpoint detection method for LAMP. When at least 500 copies of target template were present, there was a noticeable color change indicating the presence of the bacterium. Techniques suitable for DNA extraction from plant tissue in situ were compared with a standard silica-column-based laboratory extraction method. A portable PickPen and magnetic bead system could be used to successfully extract DNA from infected tissue and could be used in conjunction with LAMP in the field.

First Report of Helleborus net necrosis virus in Hellebore in New Zealand.

Hellebores (Helleborus spp.) are widely grown in gardens for their winter and early spring flowers. They are extremely hardy and will grow easily in many different environments. In April 2009, black streaks on the leaves and stems were observed on approximately 3 to 5% of hellebores in a home garden in the Waikato Region, New Zealand. The symptoms appeared similar to those of 'black death', determined to be associated with a newly characterized carlavirus termed Helleborus net necrosis virus (HeNNV) (1). Transmission electron microscopy revealed the presence of typical carlavirus-like particles (slightly flexuous filaments ≈700 nm long) in crude sap extracts. Total nucleic acid was extracted separately from the leaves and stem from one of the symptomatic plants with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). One-step reverse transcription (RT)-PCR using carlavirus group-specific primers (Agdia Inc., Elkhart, IN) produced an amplicon of approximately 300 bp in both the leaf and stem samples. The PCR product was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and three clones were sequenced. BLAST analysis of the consensus sequence (GenBank Accession No. GQ499837) showed the highest nucleotide identity (78%) to the replicase polyprotein genes of HeNNV strains I6 and G5 (GenBank Accession Nos. FJ196837 and FJ196836, respectively). A fragment of 376 bp was also amplified from the symptomatic plant by RT-PCR with primers HCV8484c and HCV8109, designed specifically to amplify the capsid protein genes and putative nucleic acid binding protein genes of HeNNV (1) and sequenced directly (GenBank Accession No. GQ499838). BLAST analysis showed the highest nucleotide identity (85%) with HeNNV strain G5 (GenBank Accession No. FJ196835) followed by 84% nucleotide identity with HeNNV strains H6 and I6 (GenBank Accession Nos. FJ196836 and FJ196837, respectively). Three asymptomatic hellebore plants purchased from a nursery tested negative by RT-PCR using the carlavirus group-specific and HeNNV-specific primers as described above. To our knowledge, this is the first report of HeNNV infecting hellebores in New Zealand. Hellebores are regularly imported into New Zealand as tissue cultures or nursery stock. The import requirement for acceptance of plants in tissue culture is visual inspection at the border. Nursery stock must be grown in a post-entry quarantine facility for 6 months during which time they are inspected for pests and diseases. The long latent period of HeNNV (1) and the recent discovery of the etiology of 'black death' could have resulted in accidental introduction of diseased plants. Knowledge of the causal agent of 'black death' is beneficial to growers who have already implemented cultural control strategies to reduce the spread of the disease. Most carlaviruses are aphid transmitted but no vector of HeNNV has been identified, and therefore, it is unknown if the virus spreads naturally in New Zealand. Reference: (1) K. C. Eastwell et al. Plant Dis. 93:332, 2009.

First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) in New Zealand.

In February 2009, 10 cape gooseberry plants (Physalis peruviana) grown from seed on a domestic property in Christchurch, New Zealand, showed severe leaf distortion, fasciation and etiolation of growing tips, and weak flowering. Symptoms were first observed in the emerging seedlings. No virus particles were observed in sap from infected plants with the electron microscope. Total RNA was isolated from leaves of the 10 plants with a Qiagen RNeasy Plant Mini Kit (Valencia, CA). All 10 plants tested positive for Potato spindle tuber viroid (PSTVd) by real-time reverse transcription (RT)-PCR (1) and by RT-PCR with PSTVd-specific primers (3) and generic pospiviroid primers (4). For both conventional PCRs, the expected 359-bp amplicons were sequenced directly and sequences were aligned together to create a consensus sequence (GenBank Accession No. FJ797614). BLASTn analysis showed 98% nucleotide identity to PSTVd (EU862231, DQ308556, X17268, and AY532801-AY532804). Sap from one of the infected plants was mechanically inoculated onto healthy P. peruviana, Solanum lycopersicum 'Rutgers', Chenopodium amaranticolor, C. quinoa, Cucumis sativum 'Crystal Apple', Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis '37B', N. tabacum 'WB', N. sylvestris, and Phaseolus vulgaris 'Prince'. After 4 weeks, the leaves of the 'Rutgers' tomato plants were showing severe distortion, purpling, and necrosis of mid-veins and P. peruviana plants were showing distortion of newly emerging apical leaves. Healthy control P. peruviana were asymptomatic. Symptoms appeared milder than that observed in the original P. peruviana plants, but this may be related to different environmental conditions or age or growth stage of the plants when inoculated. All other indicator plants were symptomless, but along with P. peruviana, tested positive for PSTVd by real-time RT-PCR (1). The presence of PSTVd was further confirmed in one original symptomatic and the mechanically inoculated P. peruviana plants and in the indicator plants by dot-blot hybridization with a digoxygenin-labeled synthetic ssRNA probe specific to the full-length PSTVd genome. PSTVd has been reported in New Zealand previously in commercial glasshouse crops of tomatoes and peppers (2), but was eradicated and so remains a regulated pest. The plants were grown from seeds imported from Germany and it is possible that the infection was seedborne. PSTVd was reported in young cape gooseberry seedlings in Germany and Turkey but the infection was asymptomatic (5). Symptoms were associated with the PSTVd-infected cape gooseberry in New Zealand. To our knowledge, this is the first report of the viroid in domestically grown plants in New Zealand, and only the second report of PSTVd in cape gooseberry worldwide. Our findings suggest that this species is an emerging host for PSTVd and that dissemination of seed may provide a pathway for international movement of the viroid. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) B. S. M. Lebas et al. Australas. Plant Pathol. 34:129, 2005. (3) A. M. Shamoul et al. Can. J. Plant Pathol. 19:89, 1997. (4) J. T. H. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (5) J. T. H. Verhoeven et al. Plant Dis. 93:316, 2009.

First Report of 'Candidatus Phytoplasma australiense' in Potato.

In January of 2009, potato plants (Solanum tuberosum) from a commercial crop in the Waikato Region, New Zealand were observed to have symptoms of upward rolling and purpling of the leaves. The symptoms appeared similar to those of "zebra chip", a disorder of potato recently found to be associated with 'Candidatus Liberibacter solanacearum' in New Zealand and the United States (4). Total DNA from the leaf midveins and tubers from one of the symptomatic plants was separately extracted with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). DNA extracted from leaf midveins and tubers tested negative for 'Ca. L. solanacearum' by nested-PCR using primer pair OA2/OI2c (4) followed by Lib16S01F/Lib16S01R (5'-TTCTACGGGATAACGCACGG-3' and 5'-CGTCAGTATCAGGCCAGTGAG-3'), which amplifies a 580-bp region of the 16S rRNA gene. However, DNA extracted from the tuber tissue tested positive for phytoplasma by TaqMan real-time PCR (3). No phytoplasma was detected in the DNA extracted from leaf tissue. The 16S rRNA gene, 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene of the phytoplasma were amplified with primers P1/P7 (1). The PCR product was cloned into the pCR 4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ943262). BLAST analysis showed 100% identity to 'Ca. Phytoplasma australiense' (16SrXII, Stolbur group). A fragment of approximately 850-bp of the Tuf gene was also amplified (2) and sequenced directly (GenBank Accession No. FJ943263). BLAST analysis showed 100% identity to Tuf gene variant IX of 'Ca. P. australiense' (2). An additional 14 plants showing similar leaf symptoms and also production of aerial tubers were collected from seven different potato fields from the Auckland and Waikato regions. Total DNA from the leaf midveins, stem, and tubers were separately extracted from each of the plants. The samples were tested for phytoplasma by nested-PCR using primer pair R16F2/R16R2, followed by NGF/NGR (1), and tested for 'Ca. L. solanacearum' by nested-PCR as described above. Seven plants tested positive only for phytoplasma, three tested positive for only 'Ca. L. solanacearum', and four plants tested positive for both pathogens. The pathogens were most commonly detected in samples extracted from the stem with 9 and 5 of the 14 samples testing positive for phytoplasma and liberibacter, respectively. Six of each of the leaf and tuber samples tested positive for phytoplasma. Liberibacter was detected in one of the leaf samples and in four of the tuber samples. 'Ca. P. australiense' has only been reported from New Zealand and Australia. The only other known hosts of 'Ca. P. australiense' in New Zealand are strawberry and native plants belonging to the genera Cordyline, Coprosma, and Phormium (2). In Australia, 'Ca. P. australiense' is associated with Australian grapevine yellows and Papaya dieback (2). To our knowledge, this is the first report of 'Ca. P. australiense' infecting potato as well as the first report of phytoplasma and 'Ca. L. solanacearum' mixed infections in potato. References: (1) M. T. Andersen et al. Plant Pathol. 47:188, 1998. (2) M. T. Andersen et al. Phytopathology 96:838, 2006. (3) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (4) L. W. Liefting et al. Plant Dis. 93:208, 2009.

First Report of Narcissus degeneration virus, Narcissus late season yellows virus, and Narcissus symptomless virus on Narcissus in New Zealand.

In September 2008, Narcissus plants originating from commercial nurseries in Taranaki (TK) in New Zealand's North Island and Canterbury (CB) in the South Island were received showing leaf mottling, flower distortion, and color break. The CB plant also showed stunting. Filamentous virus particles (700 to 900 nm long) were seen in crude sap of both plants with a transmission electron microscope. Total RNA was isolated from the leaves of both plants with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA), and cDNA was synthesized by Superscript III (Invitrogen, Carlsbad, CA). cDNA was used in PCR to test for viruses in the following genera: Allexivirus, Carlavirus, Cucumovirus, Nepovirus A and B, Potyvirus, Potexvirus, Tospovirus, and Tobravirus. Both plants tested positive for potyvirus using generic potyvirus primers (3). Amplicons from both plants were directly sequenced. The forward and reverse sequence from the CB plant matched sequences in the GenBank database for Narcissus late season yellows virus (NLSYV) and Narcissus degeneration virus (NDV), respectively. The potyvirus amplicon from the CB plant was cloned and sequenced. Sequence from independent clones was obtained for NLYSV only (No. FJ546721), and this sequence showed 97% nucleotide identity to NLYSV No. EU887015. The CB plant was tested with a second set of generic potyvirus primers using forward (PV1SP6) (2) and reverse primers (U335) (1). BLASTN analysis of the sequence obtained from independent clones (No. FJ543718) matched sequence for NDV only (97% nucleotide identity to No. AM182028). BLASTN analysis of the potyvirus obtained for the TK plant (No. FJ546720) showed 97% nucleotide identity to NLSYV (No. EU887015). The TK plant also tested positive for a carlavirus using commercial primers (Agdia, Elkhart, IN) and unpublished generic carlavirus primers (A. Blowers, personal communication). Amplicons from both PCRs were cloned and sequenced. BLASTN analysis of both sequences (Nos. FJ546719 and GQ205442) showed 94% nucleotide identity to Narcissus symptomless virus (NSV) No. AM182569. Both plants were also tested for NLSYV, Narcissus virus Q, Narcissus latent virus, and Narcissus yellow stripe virus by indirect ELISA (Neogen, Lansing, MI). Results confirmed the presence of NLSYV in both plants but the plants were negative for the other viruses. NLSYV has been detected previously from Narcissus pseudonarcissus L. (daffodil) (D. Hunter, personal communication); however, to our knowledge, this is the first official report of NDV, NLSYV, and NSV in New Zealand. Since both plants tested negative for several other viruses by PCR and ELISA, this would suggest that the symptoms observed may have been caused by NSV, NLSYV, NDV, or as a result of a mixed infection. However, symptoms were not confirmed using Koch's postulate. NSV has been reported in the literature as symptomless. NLYSV has been reported to be a possible cause of leaf chlorosis and striping and NDV has been associated with chlorotic leaf striping in N. tazetta plants (4). Since Narcissus is an important flower crop for domestic production in New Zealand, the reduction in flower quality observed when these viruses are present may be of economic significance in commercial nurseries. References: (1) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (2) A. M. Mackenzie et al. Arch Virol. 143:903, 1998. (3) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000. (4) W. P. Mowat et al. Ann. Appl. Biol. 113:531, 1988.

Detection of Sweet potato virus 2 in Sweet Potato in New Zealand.

In New Zealand, sweet potato (Ipomoea batatas) is a crop of cultural importance and an important food source; it is grown mainly in the districts of Kaipara, Auckland, and the Bay of Plenty in the North Island. In January of 2008, virus symptoms that included chlorotic spots, ring spots, and mottling were observed on the leaves of commercial sweet potato crops (cvs. Beauregard, Owairaka Red, and Toka Toka Gold) growing in the three main production areas. A survey was done to determine the extent of virus infection in these crops. Fifty to one hundred leaves were collected randomly from each of 26 different fields. Leaves from each field were bulked into groups of 10, giving a total of 173 composite samples. All samples tested negative for Cucumber mosaic virus, C-6 virus, Sweet potato caulimo-like virus, Sweet potato chlorotic fleck virus, Sweet potato chlorotic stunt virus (SPCSV), Sweet potato latent virus, and Sweet potato mild specking virus by nitrocellulose membrane enzyme-linked immunosorbent assays (International Potato Center-CIP, Lima, Peru). Total nucleic acid was extracted from all 173 composite samples and used in real-time PCR assays specific for Sweet potato leaf curl virus (SPLCV) and real-time reverse transcription (RT)-PCR specific for SPCSV, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2; synonym Sweet potato virus Y) (1). No samples were positive for SPLCV or SPCSV, but 107 and 138 samples tested positive for SPFMV and SPVG, respectively. SPFMV and SPVG have been reported previously in New Zealand (2,3). Sixty four samples from 16 different fields tested positive for SPV2. Of the 64 samples, 52 were also infected with SPVG and SPFMV, and 10 were co-infected with SPVG but not SPFMV; no samples were co-infected with SPV2 and SPFMV when SPVG was absent. From a representative SPV2 positive sample, the 70-bp amplicon obtained by the real-time RT-PCR primers was cloned and sequenced A BLAST search showed 100% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050887 and AY178992). Subsequently, primers (V2-F1c: 5'-AGAACAGGACAAACTCAACC-3'; V2-R1: 5'-TAATCACCCTTCACACCTTC-3') were designed to amplify an approximately 434-bp fragment within the SPV2 coat protein gene. One-step RT-PCR was done on four of the SPV2 positive samples and amplicons of the expected size were sequenced directly (GenBank Accession No. FJ461774). Sequence comparison showed 99% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050886, AM050887, AY178992, and EF577437). SPV2 is a member of the genus Potyvirus but the virus has not been fully characterized. It is known that single-potyvirus infections cause mild or no symptoms in sweet potato, and consequently, no significant yield reduction is observed generally. However, co-infection with other viruses such as SPCSV produces a synergistic effect and more severe disease symptoms (4). To our knowledge, this is the first report of SPV2 infecting sweet potato in New Zealand. References: (1) C. D. Kokinos and C. A. Clark. Plant Dis. 90:783, 2006. (2) M. N. Pearson et al. Australas. Plant Pathol. 35:217, 2006. (3) M. Rännäli et al. Plant Dis. 92:1313, 2008. (4) M. Untiveros et al. Plant Dis. 91:669, 2007.

First Report of the Detection of 'Candidatus Liberibacter' Species in Zebra Chip Disease-Infected Potato Plants in the United States.

Zebra chip (ZC), an emerging disease causing economic losses to the potato chip industry, has been reported since the early 1990s in Central America and Mexico and in Texas during 2000 (4). ZC was subsequently found in Nebraska, Colorado, New Mexico, Arizona, Nevada, California, and Kansas (3). Severe losses to potato crops were reported in the last few years in Mexico, Guatemala, and Texas (4). Foliar symptoms include purple top, shortened internodes, small leaves, enlargement of the stems, swollen axillary buds, and aerial tubers. Chips made from infected tubers exhibit dark stripes that become markedly more visible upon frying, and hence, are unacceptable to manufacturers. Infected tubers may or may not produce plants when planted. The causal agent of ZC is not known and has been the subject of increased investigation. The pathogen is believed to be transmitted by the potato psyllid, Bactericera cockerelli, and the association of the vector with the disease is well documented (3). Following the report of a potential new liberibacter species in solanaceous crops in New Zealand, we sought to identify this liberibacter species in plants with symptoms of the ZC disease. Six potato plants (cv. Russet Norkota) exhibiting typical ZC symptoms were collected in Olton, TX in June of 2008. DNA was extracted from roots, stems, midribs, and petioles of the infected plants using a FastDNA Spin Kit and the FastPrep Instrument (Qbiogene, Inc., Carlsbad, CA). Negative controls from known healthy potato plants were included. PCR amplification was carried out with 'Candidatus L. asiaticus' omp primers (1), 16S rDNA primers specific for 'Ca. L. asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' (1), and 16S rDNA primers OA2 (GenBank Accession No. EU834130) and OI2c (2). Amplicons from 12 samples were directly sequenced in both orientations (McLab, San Francisco CA). PCR amplifications using species-specific primers for the citrus huanglongbing liberibacter were negative. However, 1.1- and 1.8-kb amplicons were obtained with the OA2/OI2C and omp primers, respectively. The sequences for the rDNA were submitted to NCBI GenBank (Accession Nos. EU884128 and EU884129). BLASTN alignment of the 16S rDNA sequences obtained with primers OA2 and OI2c revealed 99.7% identity with a new species of 'Ca. Liberibacter' identified in New Zealand affecting potato (GenBank Accession No. EU849020) and tomato (GenBank Accession No. EU834130), 97% identity with 'Ca. L. asiaticus', and 94% with 'Ca. L. africanus' and 'Ca. L. americanus'. The neighbor-joining phylogenetic tree constructed using the 16S rDNA fragments delineated four clusters corresponding to each of the liberibacter species. These results confirm that 'Ca. Liberibacter' spp. DNA sequences were obtained from potatoes showing ZC-like symptoms, suggesting that a new species of this genus may be involved in causing ZC disease. To our knowledge, this is the first report of the detection of 'Ca. Liberibacter' spp. in potatoes showing ZC disease in the United States. References: (1) C. Bastianel et al. Appl. Environ. Microbiol. 71:6473, 2005. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) G. A. Secor and V. V. Rivera-Varas. Rev. Latinoamericana de la Papa (suppl.)1:1, 2004.

Association of 'Candidatus Liberibacter solanacearum' with Zebra Chip Disease of Potato Established by Graft and Psyllid Transmission, Electron Microscopy, and PCR.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with 'Candidatus Liberibacter' sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of 'Ca. L. solanacearum' previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of 'Ca. L. solanacearum' with potatoes affected by ZC in the United States, Mexico, and Guatemala.

A New 'Candidatus Liberibacter' Species in Solanum betaceum (Tamarillo) and Physalis peruviana (Cape Gooseberry) in New Zealand.

A new 'Candidatus Liberibacter' species was recently identified in tomato, capsicum, and potato in New Zealand. The tomato/potato psyllid, Bactericera cockerelli, is thought to be the vector of this species of liberibacter. During studies to determine additional host plants of the pathogen, leaves of Solanum betaceum (tamarillo, also known as tree tomato) and leaves and stems of Physalis peruviana (cape gooseberry) were collected from a home garden in South Auckland, New Zealand in July of 2008. These plants were not showing any obvious disease symptoms. They were located close to a commercial glasshouse site containing known liberibacter-infected tomatoes, and many psyllids were observed on the tamarillo tree over the summer and until late autumn. Total DNA was extracted from four tamarillo and two cape gooseberry samples with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples from tamarillo that were used for the extraction were taken from the midveins of old and young leaves and from young petioles. For cape gooseberry, samples were from the leaf midveins and the stems. The samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (1). These primers amplify a 1,160-bp fragment of the 16S rRNA gene of the new liberibacter species. Amplicons of the expected size were obtained from all four tamarillo samples, with no amplification from negative control tamarillo plants grown from seed in an insect-proof glasshouse. Almost the entire length of the 16S rRNA gene was amplified using primer pairs fD2 (3)/OI2c and OA2/rP1 (3), and the 16S-23S rRNA intergenic spacer was amplified with primer pair OI2/23S1 (2). These amplicons, along with that from the OA2/OI2c primer pair, were directly sequenced, and overlapping fragments were assembled using the SeqMan software of the LaserGene package (DNASTAR, Inc., Madison, WI) (GenBank Accession No. EU935004). A 650-bp fragment of the ß operon was also amplified and sequenced directly (GenBank Accession No. EU935005). BLAST analysis showed 100% nt identity to the liberibacter of tomato (GenBank Accession Nos. EU834130 and EU834131) and potato (GenBank Accession Nos. EU849020 and EU919514). The two cape gooseberry samples produced amplicons of the expected size with the 16S rRNA and ß operon primers and the origin of the fragments were confirmed by direct sequencing with BLAST analysis showing 100% nt identity to isolates from tomato, potato, and tamarillo. To determine the distribution of disease, 53 samples of 10 leaves each (representing two leaves from five plants) were collected randomly from a commercial tamarillo crop in South Auckland. Small sections of the midveins were removed from each of the 10 leaves, bulked, and DNA was extracted as described above. The samples were tested by PCR using primer pair OA2/OI2c. Amplicons of the expected size were obtained from 2 of the 53 samples. To our knowledge, this is the first report of a liberibacter in tamarillo and cape gooseberry. It is unknown if the liberibacter induces symptoms in these species or if they act as symptomless reservoirs of the pathogen. The infected plants will be observed for symptom development over the course of a growing season. References: (1) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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.

First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand.

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5'-GCACGGCAGATAAACACGAC-3') and reverse (5'-GTGGGCAACCTTCAACTGTG-3') primers designed to amplify the 3' untranslated region (3'UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3'UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

First Report of Wisteria vein mosaic virus on Wisteria sinensis in New Zealand.

Wisteria vein mosaic virus (WVMV) is a member of the Potyvirus genus. The virus has been reported in Wisteria spp. in Australia, China, the United States, and a number of European countries (2). In 2006, several W. sinensis plants with mottling and mosaic symptoms were observed in a commercial plant nursery in Whenuapai, north of Auckland, New Zealand. These plants had been propagated from a nursery in the New Plymouth area of New Zealand. Sap from the symptomatic Wisteria plants was examined with an electron microscope and elongated and flexuous potyvirus-like particles approximately 750 nm long were observed. RNA was extracted from the symptomatic plants with a Qiagen RNeasy Plant Mini Kit (Doncaster, Australia). The RNA was initially tested using general potyvirus primers, PV1/SP6 (4) and U335 (3), with the cycling conditions of 94°C for 5 min followed by 40 cycles of 94°C for 45 s, 50°C for 45 s, 72°C for 90 s, and a final extension of 72°C for 7 min. The polymerase chain reaction (PCR) product (695 bp) was directly sequenced (GenBank Accession No. EU580146) and a BLAST search in GenBank showed 98% nucleotide identity with WVMV (GenBank Accession no. AF484549). The RNA was then tested using WVMV-specific primers, WVMVF1 and WVMVR1, and the published cycling conditions (2). PCR amplicons of 701 bp were obtained. PCR products were directly sequenced (GenBank Accession No. EU308592), and a BLAST search in GenBank showed 98% nucleotide identity with published sequences of WVMV (GenBank Accession Nos. AF484549 and AY656816). In addition, RNA was extracted from the original isolate of WVMV that was reported in the Netherlands (1; supplied by R. van der Vlugt, Plant Research International) and the RNA was amplified using the WVMV-specific primer pair. The sequence obtained from PCR amplicons of the type isolate (GenBank Accession No. EU308593) showed a 98% nucleotide identity with the New Zealand WVMV isolate and with published sequences of WVMV (as shown above). From the symptomatology, particle morphology, and nucleotide sequences, it is concluded that WVMV is present in New Zealand. The distribution of the virus in New Zealand is not known, but the affected plants at the New Plymouth nursery may have been imported into New Zealand as many as 30 years ago. Although WVMV infection can reduce the quality of commercial plants, the disease is not economically significant in New Zealand. References: (1) L. Bos. Neth. J. Plant Pathol. 76:8, 1970. (2) G. R. G. Clover et al. Plant Pathol. 52:92, 2003. (3) S. A. Langeveld et al. J. Gen Virol. 72:1531, 1991. (4) A. M. Mackenzie et al. Arch Virol. 143:903, 1998.

A New 'Candidatus Liberibacter' Species in Solanum tuberosum in New Zealand.

Symptoms resembling "zebra chip" disease (3) were observed in potato (Solanum tuberosum) tubers harvested from a breeding trial in South Auckland, New Zealand in May 2008. The tubers had necrotic flecking and streaking that became marked when the potatoes were fried. Affected plants generally senesced early, at the beginning of April. The mean yield was approximately 60% less than expected and harvested tubers had less dry matter (13%) than normal (19%). Large numbers of the psyllid Bactericera cockerelli were observed on the crop during the summer. Total DNA was extracted from the vascular tissue of five symptomatic tubers and seven volunteers collected from the affected field with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (2). These primers amplify a 1,160-bp fragment of the 16S rRNA sequence of a 'Candidatus Liberibacter' species identified in tomato and capsicum in New Zealand. No fragment was amplified from healthy plants, but amplicons of the expected size were obtained from all symptomatic tubers and one plant. A 650-bp fragment of the ß operon was also amplified from symptomatic tubers. The amplicons were directly sequenced (GenBank Accession Nos. EU849020 and EU919514). BLAST analysis showed 100% identity to the tomato/capsicum liberibacter (GenBank Accession Nos. EU834130 and EU834131). From a commercial potato field adjoining the breeding trial, groundkeeper tubers were collected and separated into those that were asymptomatic and those that exhibited a range of symptoms. Total DNA was extracted and tested by PCR using the OA2/OI2c primers. In the first category, 6 of 10 tubers tested positive, whereas the 10 tubers in the second category tested negative. Two phytoplasmas seem to be involved in the "zebra chip" disease complex (4) but were not detected in the samples in this study. To our knowledge, this is the first report of a liberibacter associated with disease in potato. From transmission electron microscope observations, previous researchers have hypothesized that a bacterium-like organism may cause "zebra chip" (1) and B. cockerelli is associated with the disease (3). "Zebra chip" was first reported in Mexico in 1994, since then it has caused significant economic damage in Guatemala, Mexico, and the southwestern United States. The economic impact of the disease in New Zealand is yet to be determined. References: (1) S. H. De Boer et al. Page 30 in: New and Old Pathogens of Potato in Changing Climate. A. Hannukkala and M. Segerstedt, eds. Online publication. Agrifood Research Working Paper 142, 2007. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) G. A. Secor et al. Plant Dis. 90:377, 2006.

First Report of Passiflora latent virus in Banana Passionfruit (Passiflora tarminiana) in New Zealand.

Passiflora latent virus (PLV) naturally infects cultivated and wild Passiflora species in Australia, Germany, Israel and the United States (1-3). In March 2004, chlorotic lesions were observed on leaves of three vines of Passiflora tarminiana on one site in Auckland, New Zealand. Chenopodium amaranticolor and C. quinoa inoculated with sap from symptomatic leaves developed chlorotic local spots, followed by systemic leaf chlorosis and necrosis. Local symptoms appeared more quickly on C. quinoa (12 days) than on C. amaranticolor (20 days). No symptoms were observed on inoculated plants of Nicotiana benthamiana, N. clevelandii, N. occidentalis, N. tabacum, or Phaseolus vulgaris. Electron microscopy of crude sap preparations from infected C. quinoa, C. amaranticolor, N. occidentalis, and P. tarminiana showed flexuous, filamentous virus particles approximately 650 nm long. Plants of P. tarminiana and the three inoculated indicator species containing virus particles tested positive by PLV polyclonal antiserum in double-antibody sandwich (DAS)-ELISA (DSMZ, Braunschweig, Germany) and immunosorbent electron microscopy (Stephan Winter, DSMZ, personal communication). Nucleic acid was extracted from leaves of plants of each of the four viruliferous species with an RNeasy Plant Mini Kit (Qiagen, Doncaster, Australia) and then used in reverse transcription (RT)-PCR tests with novel forward (5'-CGAGACACACGCAAACGAA-3') and reverse (5'-CAGCAAAGCAAAGACACGA-3') primers specific to a 523-bp fragment of the PLV polyprotein. PCR products of the expected size were obtained, and an amplicon from P. tarminiana was directly sequenced (GenBank Accession No. EU257510). A BLAST search in GenBank showed 94% nucleotide sequence identity with a PLV isolate from Israel (GenBank Accession No. DQ455582). To our knowledge, this is the first finding of PLV in P. tarminiana and the first report of the virus in New Zealand. Chenopodium spp. have been reported previously as experimental hosts (2,3), and this study revealed that N. occidentalis also can be infected latently with PLV. P. tarminiana is a weed in New Zealand and subject to active control measures to manage the species. Economically important species such as P. edulis and P. ligularis are potentially susceptible to the virus. These species are not grown commercially in the surrounding area but are common in domestic Auckland gardens. Infected vines were removed from the site and destroyed, and symptomatic vines have not been observed at other sites. References: (1) R. D. Pares et al. Plant Dis. 81:348, 1997. (2) S. Spiegel et al. Arch. Virol. 152:181, 2007. (3) A. A. Stihll et al. Plant Dis. 76:843, 1992.

First Report of Apium virus Y in Celery in New Zealand.

Apium virus Y (ApVY) has been detected for the first time in New Zealand. In January 2006, leaf mosaic and vein-banding symptoms were observed on cultivated celery (Apium graveolens cv. Tongo) in Wanganui, New Zealand. Symptoms were widespread and seen in several paddocks. Similar symptoms were also observed in poison hemlock (Conium maculatum), a weed commonly found growing along the edges of the crop. Chenopodium amaranticolor and C. quinoa plants inoculated with leaf sap from a single, symptomatic celery or hemlock plant developed necrotic local spots after 9 and 12 days, respectively. Six Nicotiana spp. did not develop symptoms and were not tested further. Electron microscopy of sap from the celery, hemlock, and C. quinoa plants revealed the presence of elongated flexuous virus particles, 650 to 850 nm long. Symptomatic plants of these three species were tested for ApVY by reverse transcription (RT)-PCR using novel forward (5'-ATGATGCGTGGTTTGAAGG-3') and reverse (5'-CTTGGTGCGTGAGTTCTTG-3') primers specific to the coat protein gene (GenBank Accession No. AF203529). Amplicons of the expected size (approximately 425 bp) were obtained from all samples, and an amplicon from celery was sequenced (GenBank Accession No. EU127499). Comparison with ApVY sequences in GenBank confirmed the identity of the product, which had 97 to 99% nucleotide identity with GenBank Accession Nos. AF 203529, AF207594, and AY049716. The effect of ApVY on celery is unknown. ApVY has recently been described and infects three species of Apiaceae in Australia (2). In this study, diseased celery, but not the hemlock plants, were found to be coinfected with Celery mosaic virus (CeMV) by enzyme-linked immunsorbent assays with CeMV-specific antibodies (Loewe Biochemica GmbH, Sauerlach, Germany). Therefore, the symptoms observed in celery may be induced by ApVY or CeMV. CeMV is a serious disease of celery in New Zealand (1) and CeMV symptoms may mask the presence of ApVY. References: (1) P. R. Fry and C. H. Procter. N. Z. Commer. Grower 24:23, 1968. (2) J. Moran et al. Arch. Virol. 147:1855, 2002.

The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Beet necrotic yellow vein virus (BNYVV). The two assays designed were a broad-spectrum one that detected RNA 2 from all types and a second designed to detect types containing RNA 5. The assays were validated against a range of different isolates from Europe and the Far East. These real-time assays were compared to a conventional RT-PCR assay for the detection of RNA 5. Sensitivity comparisons showed that for the detection of RNA 5, TaqMan was 10,000 times more sensitive than the conventional RT-PCR assay. Further improvements were made to the test procedure by using post-ELISA virus release (VR), as an alternative to RNA extraction. This significantly increased the speed of processing samples and reduced the staff input required, allowing the TaqMan assay to be used routinely as part of an annual survey of UK field samples.

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.