Lupine accessions from the Cool Season Food Legume Seed Collection are grown for seed regenerations in Pullman, WA by the Agricultural Research Service, Western Regional Plant Introduction Station. Selected seed was germinated in the greenhouse and assayed by indirect ELISA using antiserum for potyvirus group detection (Agdia, Inc., Elkhart, IN). Healthy transplants were grown for seed collection on outside plots. In July of 2005, more than 90% of 307 Lupinus luteus L. transplants developed severe yellowing, necrosis, and stunting with an estimated 5% plant death. Plants were heavily infested with aphids and leaf sap was serologically positive for potyvirus. Partially purified virus preparations from infected plants contained filamentous particles and a 35-kDa protein that reacted with universal potyvirus antiserum on western blots. Reverse transcription (RT)-PCR using potyvirus universal primers (2) and cDNA derived from virion RNA generated a ~1.7-kbp product that was cloned and sequenced. The sequenced portion of the genomic RNA contained 1,610 nucleotides (nt) on its 3′-terminus (GenBank Accession No. EU144223) that included a partial nuclear inclusion protein, NIb, (1 to 637 nt) with the conserved amino acid (aa) replicase motif GDD (131 to 139 nt), the coat protein (CP) gene of 821 nt (638 to 1,459 nt), and a 171-nt untranslated region (1,460 to 1,630 nt) attached to a poly(A)tail. The CP sequence contained a NAG motif instead of the DAG motif commonly associated with aphid transmission. Searches in the NCBI GenBank database revealed that the CP aa and nt sequences contained conserved domains with isolates of Bean yellow mosaic virus (BYMV). A pairwise alignment (ClustalX) (4) of the CP aa from 20 BYMV isolates with the BYMV-Pullman isolate revealed identities from 96% (BYMV-S, U47033) to 88% (BYMV-MI [X81124)] -MI-NAT [AF434661]). This meets the species demarcation criteria of more than ~80% identity for inclusion with BYMV (1). Virion mechanical inoculations resulted in local lesions on Chenopodium amaranticolor Coste et Reyn and C. quinoa Willd., necrotic blotches on Phaseolus vulgaris L., and yellow spots and systemic movement in L. succulentus Douglas ex. K. Koch, L. texensis ‘Bluebonnet’, and L. texensis ‘Maroon’; BYMV was confirmed by western blots and ELISA. The experimental inoculations represent the first documented report of BYMV in the annual L. succulentus and biennial L. texensis species. Since BYMV is seedborne and transmitted by many aphid species (3), it is possible that several lupine transplants escaped potyvirus detection, and secondary transmission of BYMV to plants occurred by aphids. During the 1950s, BYMV was confirmed in several annual lupines grown as crops in the southeastern United States (3). To our knowledge, this is the first report of BYMV occurring naturally in a lupine species in Washington. BYMV is a destructive virus to lupine species worldwide and has a wide host range in Fabaceae. This research directly contributes toward the maintenance of virus-free lupine seed for distribution to scientists focusing on lupine research.
References: (1) P. H. Berger et al. Family Potyviridae. Page 819 in: Virus Taxonomy: Eighth Report of the ICTV. C. M. Fauquet et al. eds., 2005. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) R. A. C. Jones and G. D. Mclean, Ann. Appl. Biol. 114:609, 1989. (4) J. D. Thompson et al. Nucleic Acids Res. 24:4878, 1997.