Molecular analysis of new isolates of Tomato leaf curl Philippines virus and an associated betasatellite occurring in the Philippines.

Three new begomovirus isolates and one betasatellite were obtained from a tomato plant exhibiting leaf curl symptom in Laguna, the Philippines. Typical begomovirus DNA components representing the three isolates (PH01, PH02 and PH03) were cloned, and their full-length sequences were determined to be 2754 to 2746 nucleotides. The genome organizations of these isolates were similar to those of other Old World monopartite begomoviruses. The sequence data indicated that PH01 and PH02 were variants of strain B of the species Tomato leaf curl Philippines virus, while PH03 was a variant of strain A of the species Tomato leaf curl Philippines virus. These isolates were designated ToLCPV-B[PH:Lag1:06], ToLCPV-B[PH:Lag2:06], and ToLCPV-A[PH:Lag3:06], respectively. Phylogenetic analysis revealed that the present isolates form a separate monophyletic cluster with indigenous begomoviruses reported earlier in the Philippines. A betasatellite isolated from same sample belongs to the betasatellite species Tomato leaf curl Philippines betasatellite and designated Tomato leaf curl Philippines betasatellite-[Philippines:Laguna1:2006], ToLCPHB-[PH:Lag1:06]. When co-inoculated with this betasatellite, tomato leaf curl Philippines virus induced severe symptoms in N. benthamiana and Solanum lycopersicum plants. Using a PVX-mediated transient assay, we found that the C4 and C2 proteins of tomato leaf curl Philippines virus and the ßC1 protein of ToLCPHB-[PH:Lag1:06] function as a suppressor of RNA silencing.

Occurrence of Two Little Cherry Viruses in Sweet Cherry in Washington State.

Little cherry disease, one of the major viral diseases of sweet cherry (Prunus avium) worldwide, is associated with either of two closteroviruses, Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2). Two sets of primers corresponding to a portion of the replicase gene of LChV-1 and LChV-2 were used in one-tube reverse-transcription polymerase chain reactions to detect these viruses in total RNA extracts of field-collected sweet cherry tissues. LChV-1 and LChV-2 were detected both alone and in combination in five sweet cherry orchards in Washington State. Sequence analysis of a 240-nucleotide (nt) fragment of the replicase open reading frame (ORF)1b and a 232-nt fragment from a portion of ORF8 and the 3' untranslated region (UTR) of LChV-1 indicated that North American (NA) isolates shared 90 to 99% nucleotide identity in both genome segments analyzed. In contrast, comparisons of NA isolates to two Eurasian isolates of LChV-1 indicated shared nucleotide identities of 79 to 82% in the replicase fragment and 89 to 90% in the ORF8/3'UTR fragment. Sequence variation in the replicase region did not affect detection of LChV-1 in 12 isolates using the replicase-specific primers reported here. This article represents the first report of LChV-1 and LChV-2 in sweet cherry in Washington.

Genetic variation of rice tungro bacilliform virus in the Philippines.

Restriction fragment length polymorphisms (RFLPs) were found in 27 full genome length clones from a glasshouse isolate of rice tungro bacilliform pararetrovirus (RTBV) from the International Rice Research Institute (IRRI), the Philippines and from clones from 5 field isolates from different parts of the Philippines. There was much less variation between the IRRI clones than between the field isolate clones. The RFLPs were due to single base changes and represented about 10% of the potential sites. Sequencing across the region between nt 7772 and 7989 confirmed that the field isolates differed from the published sequence more than did the IRRI clones. The most common substitutions were G > A, A > G and T > C. Sequence heterogeneity was also noted in PCR products from RTBV DNA from the isolates. These observations are discussed in relation to the quasispecies population concept of viruses.