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 Rose yellow vein virus in Rosa sp. in New Zealand.

Rose is the top selling cut flower in New Zealand and is the most popular garden plant in the world. Several virus-like diseases have been described in roses, but the causal agents for many remain unknown. Most of the described viruses infecting rose belong to the genera Ilarvirus and Nepovirus. Only recently, a number of new viruses have been or are in the process of being characterized (1,2,3,4). In January 2011, 10 rose samples showing virus-like symptoms were collected from the Wanganui region on the North Island of New Zealand. Total nucleic acid was extracted from these samples using an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). PCR and reverse transcription (RT)-PCR was conducted using specific primers for Arabis mosaic virus (ArMV), Cherry leaf roll virus, Prunus necrotic ringspot virus (PNRSV), Rosa rugosa leaf distortion virus, Rose spring dwarf associated virus, Rose yellow leaf virus, Rose yellow mosaic virus, Rose yellow vein virus (RYVV), and Strawberry latent ringspot virus. Samples were also tested using generic primers for carlavirus, potexvirus, potyvirus, tombusvirus, and phytoplasmas. Two samples (cvs. Pauls Himalayan Musk and Bloomfield) were positive for ArMV, four samples (cvs. Leda, Rosa Mundi, Charles de Mills, and Indica Major) were positive for PNRSV, and two samples (cvs. Leda and Zephirine Drouhin) were positive for RYVV. Samples were negative for all other tested viruses and phytoplasmas. RYVV was detected using two sets of primers (D. Mollov, personal communication) designed to amplify fragments of estimated sizes of 797 bp and 684 bp of the movement protein (MP) and coat protein (CP) genes of RYVV, respectively. RYVV amplicons were sequenced directly (GenBank Accession Nos. JX887423 to JX887426). A BLASTn search of the MP and CP fragments showed the highest nucleotide identity of 98% and 96 to 97%, respectively, with the type isolate of RYVV (JX028536). RYVV has been reported as the causal agent of a vein yellowing disease in rose (2). Symptoms observed in the 'Leda' sample infected with PNRSV and RYVV (vein yellowing and chlorotic mottle in the apex of leaves) were not typical of PNRSV, so they may be caused by RYVV. Symptoms in samples of cv. Zephirine Drouhin (curling of leaves and mottle), observed in both RYVV-positive and -negative samples, may not be associated with RYVV infection. This suggests that vein yellowing may be influenced by cultivar. RYVV has been reported in several rose cultivars, but only in the United States (2). To the best of our knowledge, this is the first report of RYVV infecting rose in New Zealand, where it is likely that the virus has been present for some time. The virus may have a much wider geographical distribution than that reported as the virus was only recently characterized (3). References: (1) B. Lockhart et al. Page 31 in: Program and Abstracts of The 12th International Symposium on Virus Diseases of Ornamental Plants, 2008. (2) D. Mollov et al. Phytopathology 99:S87, 2009. (3) D. Mollov et al. Arch Virol. 158:877, 2012. (4) N. Salem et al. Plant Dis. 92:508, 2008.

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 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.

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.

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.