First report of Tobacco ringspot virus on Sophora microphylla, a native tree of New Zealand

Sophora microphylla (Kowhai, Fabaceae) is an endemic New Zealand species of considerable cultural, environmental and economic significance. The tree is grown widely as an ornamental in New Zealand and overseas. In September 2008, leaves with mosaic symptoms were collected from a Kowhai tree in central Auckland (Fig. 1). Using a modified protocol of Valverde et al. (1990), double stranded (ds) RNA was isolated from 5 g of leaves from the tree with symptoms and from 5 g of symptomless leaves obtained from a healthy tree. Following electrophoresis on a 5% polyacrylamide gel, two bands (~ 6 kb and 4 kb) were observed for dsRNA isolated from only the leaves with symptoms. DsRNA isolated from leaves with and without symptoms were used in a degenerate oligo-primed PCR (DOP-PCR) (Rott & Jelkmann, 2001). The banding patterns produced from each dsRNA were compared on a 1.5% agarose gel for each DOP primer. For DOP primer 5, three extra bands were observed for the dsRNA isolated from the leaves with symptoms. The three bands were cloned and sequenced. The sequence from one band showed 99% nt identity to Tobacco ringspot virus (TRSV) (GenBank Accession No. AY363727). DsRNA from the tree with symptoms was tested by one-step RT-PCR using primers for the coat protein of TRSV (Jossey & Babadoost, 2006). A 348 bp band was cloned and sequenced. The sequence from the DOP-PCR product and the coat protein PCR product overlapped. The two sequences were assembled (FJ546723) and showed a 93% nt identity to TRSV coat protein (AY363727). Using RT-PCR, TRSV was amplified from a second Kowhai plant with symptoms obtained from a home garden in west Auckland (Fig.2). The sequence from the PCR product (FJ546722) showed 93% nucleotide identity with TRSV (AY363727).The presence of TRSV in both trees showing symptoms was confirmed by DAS ELISA using TRSV-specific antisera (Agdia, Elkhart, USA).

TRSV has a restricted host range in New Zealand, previously reported only in Daphne sp, horseradish (Armoracia rusticana) and grapevines (Vitis vinifera). This is the first report of TRSV in an indigenous plant species in New Zealand, and the first confirmed report of a virus infection in Sophora microphylla. TRSV is a regulated pest in many countries. The susceptibility of Kowhai to infection by TRSV has phytosanitary implications for international movement of plant material.