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

ABSTRACT

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. 93332, 2009.