Studies on seed transmission of subterranean clover mottle virus and its detection in clover seed by ELISA and RT-PCR

Abstract

Plants of 5 naturalised annual clover species that occur within subterranean clover (Trifolium subterraneum) pastures were infected with 3 isolates of subterranean clover mottle virus (SCMoV), their seed harvested and sown, and the seedlings tested for SCMoV presence by enzyme-linked immunosorbent assay (ELISA). Seed transmission was detected in 3 species, but always occurred at low levels (0·1–0·5%). With T. cernuum, seed transmission was obtained with 3 isolates, but with T. campestre and T. tomentosum, it was detected only with one. Seed transmission rates in 3 subterranean clover cultivars were similar (0·1–0·4%). Together with subterranean clover, T. campestre, T. cernuum, and T. tomentosum probably play a role in persistence of the virus in annual pastures through the dry summer period via infection of dormant seed.

ELISA was used to test both subterranean clover leaf samples and seed samples from SCMoV-infected swards for the virus. When leaf samples and whole seeds were tested, SCMoV was detected at dilutions up to 1/512 in leaf sap and 1/64 in seed extracts. When seeds were separated into seed coat and cotyledons/embryo components, the virus was detected in both. However, pre-treatment of seeds with trisodium phosphate before separation into the 2 components eliminated assayable SCMoV from the cotyledons/embryos, whilst the virus was then only detectable in extracts of seed coats if left undiluted. This suggests that most of the SCMoV in seeds is associated with the seed coat and is destroyed by pre-treatment with trisodium phosphate.

Part of the genome of SCMoV was cloned and sequenced to develop primers specific for SCMoV for reverse transcriptase polymerase chain reaction assay (RT-PCR). Detection of 2 SCMoV isolates by the RT-PCR assay was confirmed by restriction analysis of the specific 596 base pair RT-PCR product. RT-PCR detected SCMoV at higher dilutions than ELISA in both leaf and whole seed extracts. The RT-PCR assay developed is suitable for sensitive routine testing of bulked seed samples of subterranean clover for presence of seed-borne SCMoV.