Real-Time PCR Assays for the Detection of Puccinia psidii.

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and ß-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae.

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.

First Report of Leaf Spot of Dracaena reflexa Caused by Burkholderia gladioli Worldwide.

In December 2008 (austral summer), a new disease of Dracaena reflexa Lam. cv. Anita was observed in a postentry quarantine greenhouse near Auckland, New Zealand on plants imported from Costa Rica. Symptoms included rust-colored, water-soaked lesions with chlorotic margins approximately 5 by 10 mm. When the disease was first noticed, incidence approached 80%, but subsequent reduction in greenhouse temperature dramatically reduced symptom expression and lesions were only visible on some leaf tips. Bacteria consistently isolated from the lesions on King's medium B (KB) were cream-colored, shiny, and produced a yellow, diffusible, nonfluorescent pigment. All isolates were able to rot onion slices. On the basis of BIOLOG (Hayward, CA) carbon utilization profiles, isolates were initially identified as Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 with a probability index of 100% and a similarity index of 0.85. For molecular identification, a near full-length sequence of the 16S rDNA gene was amplified from all isolates using primers fD2 and rP1 (1), obtaining a PCR product of approximately 1,500 bp. The nucleotide sequences were 100% identical to a number of B. gladioli GenBank entries, including Accession Nos. EF193645 and EF088209. To confirm pathogenicity, three isolates (two isolated prior to greenhouse temperature reduction and one after) were used. Three D. reflexa plants were inoculated per bacterial isolate by wounding three young fully expanded leaves on each plant (four wounds per leaf) and spraying the leaves with a bacterial suspension in sterile distilled water at 108 CFU/ml. At the same time, Gladiolus nanus plants were inoculated in a similar manner. Control plants (D. reflexa and G. nanus) were wounded and sprayed with sterile distilled water. All inoculated plants were covered with plastic bags to maintain humidity and placed in a growth chamber at 25°C. At 3 days, all inoculated plants began to show water soaking and reddish coloration around the inoculation sites, and by 7 days, the lesions had expanded to resemble natural infection. Bacteria isolated on KB from the leading edge of each lesion were morphologically identical to the initial isolates. No bacteria were recovered from the wound sites on the control plants. The 16S rDNA sequences of selected isolates from inoculated plants showed 100% identity to the sequences of the initial isolates, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of B. gladioli causing leaf spot of D. reflexa in the world. Reference: (1) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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.

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.

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 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.

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).

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.