A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

Seed germination is the initial step of seedling development in plants. Seed priming with salts has been used to synchronise seed germination. In general, a long-term treatment with a relatively high salt concentration, such as 1 M NaCl, is employed. To improve the e ciency of this treatment, we examined the e ect of seed priming with a lower NaCl concentration than conventional method in tomato (Solanum lycopersicum). Tomato seeds were soaked for 24 h at 25°C in the dark in 100-1000 mM of NaCl solution (NaCl-priming) or distilled water (hydro-priming). To estimate the e ect of NaCl-priming on seed germination and subsequent seedling growth, the germination rate, seedling emergence, plant height, and hypocotyl and root length were investigated under NaCl-, hydro-and non-priming treatments. At 4 d a er sowing, the seedling emergence was markedly promoted by 300 mM of NaCl-priming. e NaCl-priming also signi cantly enhanced the seed germination rate at 48 h a er sowing. Seedling growth, as indicated by plant height, stem diameter and hypocotyl and root length, was promoted by NaCl-priming. ese results suggest that priming with low saline has similar e ects as conventional priming methods. A comprehensive gene expression analysis showed that the genes related to seedling growth and stress responses were up-regulated by NaCl-priming at 144 h a er the start of the treatment, followed by advanced and uniform seed germination. e seedlings exhibited an increased tolerance to Ralstonia solanacearum, the causative agent of bacterial wilt of tomato, compared with the hydro-primed and non-primed seedling.

A viral isolate, designated N-1 and obtained from a gentian (Gentiana scabra) plant that exhibited mosaic symptoms, was transmitted mechanically to nine plant species in six families. These plants are known as hosts of fabaviruses. The N-1 isolate was composed of isometric particles 30 nm in diameter and included two RNA molecules of approximately 6.0 and 3.6 kb in length, as estimated by agarose gel electrophoresis. The RNAs were encapsidated separately in two of the three types of particle. Each particle contained two distinct proteins with Mr values of 39.3 x 10(3) and 26.6 x 10(3), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of complete nucleotide sequences of the RNAs suggested that each encoded a single large polyprotein, in which putative functional proteins were arranged in a manner similar to those in Broad bean wilt virus 1 (BBWV-1) and Broad bean wilt virus 2 (BBWV-2), which are members of the genus Fabavirus (family Comoviridae). Analysis of the deduced amino acid sequences of the proteins indicated that those of isolate N-1 shared 38 to 66% identity with those of BBWV-1 and BBWV-2 but only 16 to 42% identity with those of a comovirus, Cowpea mosaic virus. Phylogenetic analysis, based on the amino acid sequences of RNA polymerase, placed isolate N-1 in a separate lineage from BBWV-1 and BBWV-2. In indirect-enzyme-linked immunosorbent assay, isolate N-1 exhibited distant serological relationship to BBWV-1, BBWV-2, and Lamium mild mosaic virus, another fabavirus. Our results suggest that N-1 represents a new species of Fabavirus. We propose the name Gentian mosaic virus for this new species.

An attenuated isolate of Bean yellow mosaic virus (BYMV), designated B-33, that caused barely any mottling on the leaves of gentian plants (Gentiana scabra) was isolated from dwarf gentian plants. Infection with this isolate did not cause severe symptoms in several leguminous plants or in prairie gentian (Eustoma grandiflorum) plants. Dwarf gentian plants that had been infected with an inoculum of partially purified B-33 were propagated vegetatively from cuttings without difficulty. The B-33 isolate could be distinguished from other, more virulent, isolates by comparing symptoms on prairie gentian plants and by examining restriction fragment length polymorphism after reverse transcription and amplification by the polymerase chain reaction. In cross-protection tests, dwarf gentian plants that had been infected with B-33 were protected from infection with the virulent BYMV isolate 35-1. Potted plants that had been inoculated with B-33 grew larger and produced more flowers than unprotected plants. Thus, B-33 is an effective agent for controling mosaic and atrophic diseases caused by BYMV in clonal dwarf gentian plants.

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One-step reverse transcription-polymerase chain reaction (one-step RT-PCR) was successfully adapted to detect Bean yellow mosaic virus in the crude sap of infected dwarf gentian plants (Gentiana scabra). The virus was detected directly from crude sap at dilutions up to 10 6 -fold (W/ V), which is 100 times more sensitive than indirect enzymelinked immunosorbent assay or bioassay after mechanical inoculation, and equally as sensitive as immunocapture RT-PCR. This one-step RT-PCR method using crude sap was also useful for detecting 16 virus species from ten genera with isometric, filamentous, and rod-shaped particles from 32 plant species in 15 families.

An isometric virus was isolated from a cultivated Adonis plant (A. ramosa). The purified virus particle is 28 nm in diameter and is composed of a single coat protein and a single RNA genome of 3,991 nucleotides. Sequence analysis showed that the virus is closely related to carnation mottle virus. The virus was used to mechanically infect healthy A. ramosa plants, resulting in mosaic and leaf curl symptoms; however, attempts to inoculate carnation plants did not result in infection. We propose the virus as a new carmovirus and have named it adonis mosaic virus (AdMV).