Kaoru Hanada

Three distinct groups of isolates of Tomato yellow leaf curl virus in Japan and construction of an infectious clone

Complete nucleotide sequences of eight Japanese isolates of Tomato yellow leaf curl virus (TYLCV) were determined and compared with four TYLCV isolates already reported. These isolates separated into three groups – Shizuoka (Sz), Aichi (Ai), Nagasaki (Ng) – and had 99% identities within the groups. Full-length molecules of DNA-A of group Sz consist of 2791 nt and those of group Ai contain 2787 nt. Both were closely related to TYLCV-Is.M, although those of group Ng had 2793 nt and were more closely related to TYLCV-Is. Comparison of common sequences of isolates belonging to groups Sz and Ai had substitutions of 4 nt in the intergenic region and nonsynonymous substitutions at open reading frames between the groups. None of the isolates tested had DNAβ molecules. Agroinfection of four plant species with a DNA-A dimeric infectious clone of TYLCV-SzY, a member of group Sz, resulted in systemic infection. Tomato plants then developed typical yellow leaf curl symptoms.

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

ABSTRACT A virus was isolated from soybean ( Glycine max ) plants with symptoms of dwarfing and bud blight in WenceslauBraz County, Parana, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purifiedvirus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related toTSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. TheCP gene shared 83% identity with the sequence of TSV CP from white clover ( Trifolium repens ) (GenBank CAA25133).This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed thatthis isolate be considered a strain of TSV named TSV-BR. Additional keywords : nucleotide sequence, Elisa, host range. RESUMOCaracterizacao biologica e molecular de um isolado de

Control of Russet Crack Disease in Sweetpotato Plants Using a Protective Mild Strain of Sweet potato feathery mottle virus

A mild isolate (designated as 10-O) of Sweet potato feathery mottle virus (SPFMV) was obtained from Ipomoea batatas. This isolate rarely caused skin discoloration and did not cause russet crack disease on the storage roots of I. batatas that are typical of infection with the severe strain of SPFMV (SPFMV-S). The yield of the 10-O-infected I. batatas ranged from 92 to 105% of the yield of healthy I. batatas. The coat protein gene of 10-O encodes 315 amino acids and has sequence identities of 91.1% at the nucleotide level and 95.6% at the amino acid level with the corresponding region of SPFMV-S. When I. batatas cuttings were inoculated with 10-O and later challenged with SPFMV-S, russet crack symptoms were much reduced and SPFMV-S was not detected in the challenged plants. These results indicate that 10-O can be an effective biological control agent against russet crack disease.

In vitro messenger properties of a satellite RNA of cucumber mosaic virus

The nucleotide sequence of a naturally occurring satellite RNA (satRNA) of cucumber mosaic virus strain F (CMV-F) isolated from Petasites japonicum Miq. has been determined. F-satRNA is 338 nucleotides long and possesses a single open reading frame (ORF; residues 11 to 91) in its 5-terminal region. Analysis of in vitro translation products of the RNA revealed that the major peptide synthesized had an Mr of 2800, corresponding to the value (Mr 2874) calculated from the amino acid sequence predicted from the ORF. Furthermore, a ribosome-binding fragment of the RNA in the initiation step of an in vitro translation system was found to contain the first AUG codon (residues 11 to 13). These results suggest that of the F-satRNA sequence, the coding region for the peptide can be assigned to the region from AUG to GGU (residues 11 to 91), accompanied by a termination codon UGA.

Geminate particle morphology of sweet potato leaf curl virus in partially purified preparation and its serological relationship to two Begomoviruses by western blotting

Sweet potato leaf curl virus (SPLCV) is a possible member of the genus Begomovirus; however, the presence of typical geminate particles in sap has not been confirmed. We attempted to observe SPLCV virions by partially purifying the virus using an enzyme-assisted procedure. The observed virions in the partially purified preparations were typical geminate particles with a size of ca. 18×30nm. This virus preparation was subjected to western blot analysis using antisera against Bean golden mosaic virus (BGMV) and Mungbean yellow mosaic virus (MYMV). SPLCV reacted with both antisera. Specific bands appeared to be slightly larger than the 30-kDa marker protein and were considered to be SPLCV coat protein. This western blot analysis revealed for the first time a serelogical relationship between SPLCV and the two well-characterized begomoviruses.

Cycas necrotic stunt virus isolated from gladiolus plants in Japan

An undescribed spherical virus ca. 30 nm in diameter was isolated from gladiolus (Gladiolus spp.) plants in Japan. The virus had a moderate host range within eight families. Purified virus preparations contained two large RNA components and one coat protein with mobility similar to Cycas necrotic stunt virus (CNSV) from cycas (Cycas revolute). The virus was serologically closely related to CNSV. Its nucleotide sequence of the coat protein gene had 89% common identity with that of CNSV. These results indicated that the virus isolated from gladiolus is a new strain of CNSV.

Semipersistency of Myzus persicae Transmission of Cucumoviruses Systemically Infecting Leguminous Plants

Sequential transmission tests of Peanut stunt virus (PSV) and Cucumber mosaic virus (CMV) systemically infecting common bean, Phaseolus vulgaris, were conducted using Myzus persicae allowed to fast for 2 hr and then to acquisition feed on infected common bean plants or purified virus for 10 min. In the sequential transmission tests using either one or 10 aphids per assay plant, three isolates of PSV (J,S,Y5) and one of CMV (V) were transmitted from and to common bean up to a third or fourth inoculation access. Many aphids transmitted these viruses to two or three plants. Purified viruses of PSV-S and CMV-V were also transmitted up to a third or second inoculation access at low percentage. On tobacco, Nicotiana tabacum, aphids transmitted PSV-S and CMV-V only in the first inoculation access, although PSV-S was transmitted to only one plant in the fourth and fifth inoculation access. These viruses may be transmitted in two phases by aphids, depending on the plant species.

Genomic Structure of a Geminivirus in the Genus Begomovirus from Yellow Vein-affected Eupatorium makinor

A virus from yellow vein-affected Eupatorium makinoi was tentatively designated as Eupatorium-infecting geminivirus (EuGV) on the basis of whitefly transmissibility, electron microscopic observation of geminate particles in sap and symptomatology. EuGV-speciflc DNA fragments were obtained by polymerase chain reaction (PCR). Based on the restriction analysis of the PCR products, EuGV was suggested to have a monopartite genome. We determined the putative, complete nucleotide sequence of the EuGV genome which is comprised of 2766 nucleotides. The EuGV sequence had two virus sense open reading frames (ORF)(V1, V2), four complementary sense ORFs (C1–C4) and a non-coding region termed the intergenic region. This genome structure is quite similar to other monopartite begomoviruses already reported. The nucleotide and amino acid sequences were compared with other begomoviruses. The results supported the conclusion that EuGV is distinct and divergent from other begomoviruses, whereas potential sequence motifs reported in other geminiviruses are well conserved.

Cryptic Polyadenylation of Transcripts of an RNA Virus Gene Introduced into Tobacco Plants

We constructed an expression vector for the coat protein (CP) gene and the 3′ untranslated region (3′ UTR) of RNA virus (sweet potato feathery mottle virus severe strain (SPFMV-S)) lacking a foreign terminator. Out of seven transgenic tobacco plants, expression of the transgene was observed in six plants. RT-PCR analysis revealed that the transcripts had a poly(A) tail, and in most of them, polyadenylation occurred on the 5′ side of the 3′ UTR. These results suggest that the viral sequence contains a cryptic polyadenylation signal that permits 3′-end processing of the transcripts.