Ultrastructural Localization of Bean Common Mosaic Virus in Dormant and Germinating Seeds of Phaseolus vulgaris

HOCH, H. C., and R. PROVVIDENTI. 1978. Ultrastructural localization of bean common mosaic virus in dormant and germinating seeds of Phaseolus vulgaris. Phytopathology 68: 327-330. The ultrastructural localization of bean common mosaic of undulated filaments. Following 48 hr of water imbibition virus (BCMV) was determined for mature dehydrated and and growth, meristem cells of the root apex were free of virus germinated Phaseolus vulgaris seeds. The BCMV particles particles and virus-related inclusions. Pinwheel inclusions appeared only as linear arrays in most cells of mature with plates were abundant beyond the 10th cell back from the dehydrated seeds. Inclusions were present only as "scrolls". root meristem initials. Individually dispersed BCMV Other virus-related inclusion material appeared as particles became apparent in older cells of germinated seeds. amorphous or fibrillar areas as well as paracrystalline arrays In nature, bean common mosaic virus (BCMV) has a electron microscopy of nongerminated seeds, the limited host range and depends upon aphid and seed embryonic axes were excised from the cotyledons, cut intransmission for its survival. The virus is known to be to 1-mm lengths, then split longitudinally. Several 1-mm seed-borne in common bean (Phaseolus vulgaris L.), cross sections from the cotyledon midregion also were phasemy bean [Macroptilium lathyroides (L.) Urb.], taken. These specimens were fixed in 4.0% tepary bean (P. acutifolius Gray, var. latifolius Freeman), glutaraldehyde, buffered with either 0.1 M P0 4 (K+), pH mung bean [Vigna radiata (L.) Wilczek], and scarlet 6.8, or 0.1 M cacodylate (Na+), pH 7.0, for 6 hr. runner bean (P. coccineus L.) (1, 4, 11, 12, 13). For the Consecutive 1-mm segments from the first 5 mm of the first three species, there is evidence that BCMV is carried root tip and 1-mm segments from the midroot, hypocotyl, in the embryo (3, 11, 12, 14). The purpose of this cotyledon-embryonic axis attachment, primary leaves, investigation was to localize BCMV at the cellular and and apical bud of germinated seeds were similarly fixed. tissue level in cotyledons and embryonic axes of dormant Next, the tissues were rinsed for 2 hr in corresponding and germinating seeds of P. vulgaris, using electron buffer solutions and postfixed with buffered 2.0% OS04 microscopy. for 4 hr. The material was dehydrated via an acetone series and embedded in Spurr's medium (16). Serial thin MATERIALS AND METHODS sections, stained with aqueous uranyl acetate and lead citrate, were examined with a JEOL 100B electron Bean seeds were derived from plants of cultivar microscope operating at either 60 or 80 KV. Near-median Michelite 62 that either had been inoculated at the longitudinal sections were made whenever possible. At primary leaf stage with the NY 68-95 strain of BCMV (12) least six individual dormant seeds or germinated seeds or had remained noninoculated. Prior to their use, viruswere examined for each stage of development. infected and virus-free seeds were stored at 8 C and 45% Indexing bean seed prior to germination and relative humidity for about 3 mo. preparation for electron microscopy provided a means of From each seed, approximately one-fifth of the assuring that the material was either infected with BCMV cotyledons furthest from the embryonic axis (epicotyl, or free of BCMV. Thus, the cytological aspects of this hypocotyl, and radicle) was excised with a sterilized study dealt only with seeds of known virus infectivity. jeweler's saw and indexed for the presence of BCMV. The Concurrently, some of the adjacent longitudinally-split tissue was triturated in 0.05 M PG4 (K+), pH 7.0, and segments were assayed for BCMV using Black Turtle 2 rubbed onto primary leaves of Black Turtle 2 and VCand VC-1822 beans. Several samples, from 1822 beans, systemic and local-lesion hosts of BCMV, corresponding positions on different plants, were pooled respectively (12). to make enough tissue to grind and to inoculate indicator Seeds found to be infected with BCMV and those from plants. This was necessary, particularly when working healthy controls were divided into two groups. The first with the split terminal 1 mm of root apices. group was left dehydrated, whereas those of the second group were allowed to imbibe distilled water and RESULTS germinate for periods of 3 hr to 7 days at 22 C. For Several distinct configurations of virus particle 00032-949X/78/000 054$03.00/0 Copyright © 1978 The American Phytopathological Society, 3340 arrangement and virus-associated materials were Pilot Knob Road, St. Paul, MN 55121. All rights reserved, observed in BCMV-infected tissues: (i) typical flexuous