First Report of Bean yellow mosaic virus in Alaska from Clover (Trifolium spp.)

Abstract

In mid-June 2008, distinct mosaic leaves were observed on a cluster of clover (Trifolium spp.) with light pink and white flowers growing at the edge of a lawn in Palmer, AK. Virus minipurification from leaves of affected clover and protein extractions on a polyacrylamide electrophoresis implicated a ~35-kDa putative coat protein (CP). Subsequent western blots and ELISA with a universal potyvirus antiserum (Agdia Inc., Elkhart, IN) confirmed potyvirus identity. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from the same plant were used for reverse transcription (RT)-PCR. Three sets of degenerate primers that targeted potyvirus-specific genes, HC-Pro (helper component protease) and CI (cylindrical inclusion protein) and the genomic 3'-terminus that included a partial NIb (nuclear inclusion), CP (coat protein), and UTR (untranslated region), produced the expected PCR segments (~0.7, ~0.7, and ~1.6 kbp, respectively) on 1% agarose gels (1). Direct sequencing of the HC-Pro (GenBank No. GQ181115), CI (GQ181116), and CP (GU126690) segments revealed 98, 97, and 99% nucleotide identities (no gaps), respectively, to Bean yellow mosaic virus (BYMV)-chlorotic spot (CS) strain, GenBank No. AB373203. The next closest BYMV percent identity comparisons decreased to 79% for HC-Pro (GenBank No. DQ641248; BYMV-W), 79% for CI (U47033; BYMV-S) partial genes, and 96% for CP (AB041971; BYMV-P242). Mechanical inoculations of purified virus preparations produced local lesions on Chenopodium amaranticolor Coste & A. Reyn. (2 of 5) and C. quinoa Willd. (6 of 7), and mosaic on Nicotiana benthamiana Domin (5 of 5). BYMV was specifically confirmed on tester plants using a double-antibody sandwich (DAS)-ELISA BYMV (strain 204 and B25) kit (AC Diagnostics, Inc., Fayetteville, AR) as directed. The absence of another potyvirus commonly found in clover, Clover yellow vein virus (ClYVV), was verified in parallel DAS-ELISA ClYVV assays (AC Diagnostics, Inc). The BYMV isolate was maintained in N. benthamiana, and virion or sap extracts inoculated to the following host range (number of infected/total inoculated plants [verified by BYMV ELISA]): Cucumis sativus L. 'Straight Eight' (0/5), Gomphrena globosa L. (1/4), Nicotiana clevelandii A. Gray (4/7), Phaseolus vulgaris L. 'Bountiful' (1/3), Pisum sativum L. (Germplasm Resources Information Network Accession Nos. -PI 508092 (8/12), -W6 17525 (13/13), -W6 17529 (0/13), -W6 17530 (13/14), -W6 17537 (0/12), -W6 17538 (0/12), and -W6 17539 (0/21), Tetragonia tetragoniodes (2/2), Trifolium pretense L. 'Altaswede' (6/10), T. repens L. 'Pilgrim' (0/8), and Vicia faba L. (1/3). All infected plants had symptoms ranging from systemic mosaic (T. pretense, P. sativum) to leaf distortions (N. clevelandii, Tetragonia tetragoniodes). Interestingly, the host range and genomic sequences of the BYMV Alaskan strain resemble the BYMV-CS (chlorotic spot) strain that was originally isolated from a diseased red clover (T. pretense) plant in Japan more than 40 years ago (2). Although BYMV occurs worldwide and has a wide host range in dictoyledonous and monocotyledonous plants (3), to our knowledge, this is the first report of a natural occurrence of BYMV in Alaska. The incidence and distribution of BYMV in clover and other plant species are not known in Alaska.