Abstract. Seed is the main source of infection of narrow-leafed lupin (Lupinus angustifolius) crops by cucumber mosaic virus (CMV). The ELISA procedure is ...
Aust. J. Agric. Res., 1993, 44, 41-51
A Polymerase Chain Reaction Assay for Cucumber Mosaic Virus in Lupin Seeds
S. lie,^ C. R. W i l ~ o n , R. ~ >A~. C. ones^*^ and M. G. K. ~ o n e s ~ 9 ~ A Centre for Agricultural Biotechnology, School of Biological and Environmental Sciences, Murdoch University, Perth, W.A. 6150. Western Australian Department of Agriculture, Baron Hay Court, South Perth, W.A. 6151. Cooperative Research Centre for Legumes in Mediterranean Agriculture, University of Western Australia, Nedlands, W.A. 6009. Current address: Dept of Primary Industry, Fisheries & Energy, Newtown Research Laboratories, Hobart, Tas. 7001.
Abstract Seed is the main source of infection of narrow-leafed lupin (Lupinus angustifolius) crops by cucumber mosaic virus (CMV). The ELISA procedure is currently used for large-scale, routine testing of lupin seed samples, but a more sensitive, reliable and labour-saving assay is needed which detects levels of seed infection as low as 0.1%. A Polymerase Chain Reaction (PCR) using ground dry seed samples was developed for this purpose. Primers based on concensus sequences of eight published CMV coat protein cDNAs (RNA3) of CMV subgroups 1 and 2 were used. The assay involved (1) a reverse transcription step for cDNA synthesis and (2) amplification of a specific fragment (482-501 bp depending on the strain) by PCR. Two methods of extracting virus from infected lupin material were used: (i) a rapid procedure which was effective for samples with higher levels of infection, e.g. infected leaves and 20.5% infected seed; (ii) a phenol-chloroform procedure, which led to greater sensitivity, enabling reliable detection of 0.1% seed infection. It detected CMV in 16 commercial seed samples (0.1-8% seed infection) belonging to seven cultivars from 12 different localities. Both methods were suitable for routine testing of the flour derived from grinding dry seed. On dissection of infected seeds, CMV was detected in the cotyledons and embryo and usually in or on the testa. The PCR assay detected virus from both CMV subgroups, but only subgroup 2 was found in lupin seed samples. The two CMV subgroups can be distinguished by digestion of amplified DNA with the restriction enzyme EcoRI; only CMV strains of subgroup 2 are digested to yield two fragments of size 330 and 170 bp. Keywords: Cucumber Mosaic Virus (CMV), Polymerase Chain Reaction (PCR), Lupinus angustifolius, lupin seed, routine testing.
Introduction Narrow-leafed lupin (Lupinus angustifolius) is the most widely grown grain legume in Australia. About 80% of the national crop is produced in Western Australia, where it is grown in rotation with cereals or ley pastures. Cucumber mosaic cucumovirus (CMV) is a threat to the lupin industries in both Western and south-eastern Australia (Bowyer and Keirnan 1981; Alberts et al. 1985; Jones 1987, 1988). Infection with CMV causes individual crop failures and widespread losses, and disrupts both breeding and trialing programs, and certified seed production (Jones 1988, 1991; Jones and McLean 1989). CMV epidemics depend on a source of the virus and presence of aphid vectors. CMV is seed-borne and
S. Wylie et al.
seed-infected plants constitute the primary infection source from which spread develops. Planting of seed stocks with little or no infection is the most important control measure. Sowing seed with
