Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

The Oman strain of Tomato yellow leaf curl virus (TYLCV-OM) and its associated betasatellite, an isolate of Tomato leaf curl betasatellite (ToLCB), were previously reported from Oman. Here we report the isolation of a second, previously undescribed, begomovirus [Tomato leaf curl Oman virus (ToLCOMV)] and an alphasatellite from that same plant sample. This alphasatellite is closely related (90 % shared nucleotide identity) to an unusual DNA-2-type Ageratum yellow vein Singapore alphasatellite (AYVSGA), thus far identified only in Singapore. ToLCOMV was found to have a recombinant genome comprising sequences derived from two extant parents, TYLCV-OM, which is indigenous to Oman, and Papaya leaf curl virus from the Indian subcontinent. All possible combinations of ToLCOMV, TYLCV-OM, ToLCB and AYVSGA were used to agro-inoculate tomato and Nicotiana benthamiana. Infection with ToLCOMV yielded mild leaf-curl symptoms in both hosts; however, plants inoculated with TYLCV-OM developed more severe symptoms. Plants infected with ToLCB in the presence of either helper begomovirus resulted in more severe symptoms. Surprisingly, symptoms in N. benthamiana infected with the alphasatellite together with either of the helper viruses and the betasatellite were attenuated and betasatellite DNA accumulation was substantially reduced. However, in the latter plants no concomitant reduction in the accumulation of helper virus DNA was observed. This is the first example of an attenuation of begomovirus-betasatellite symptoms by this unusual class of alphasatellites. This observation suggests that some DNA-2 alphasatellites encode a pathogenicity determinant that may modulate begomovirus-betasatellite infection by reducing betasatellite DNA accumulation.

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Tomato is cultivated in the coastal region of Al-Batinah, in the Sultanate of Oman, during the winter season, to meet the high demand for fresh produce in the domestic market. In order to identify the causal agent of a widespread disease associated with infestations of the whitefly Bemisia tabaci (Genn.) leaves were collected from tomato plants showing symptoms characteristic of the disease in Al-Batinah during 2004 and 2005. Total nucleic acids were isolated from the tomato leaves and used as the template for Phi29 DNA polymerase amplification of begomoviral circular DNA. Putative full unit length begomoviral DNA multimers were digested with Nco I and cloned into the plasmid vector pGEM7Zf+. The complete nucleotide (nt) sequence was determined as 2,765 bases, indicative of a monopartite begomoviral genome. A comparison of the genome sequence for the seven field isolates examined, indicated that they shared 99% nt identity. The virus from Oman was most closely related to TYLCV-IR at 91% nt identity, a monopartite begomoviral species described previously from Iran. Based on the guidelines of the ICTV the Oman isolate has been designated TYLCV-Om and is considered an isolate of TYLCV-IR. A satellite DNA (satDNA beta), was amplified by polymerase chain reaction using degenerate primers and cloned, and the DNA sequence was determined. Analysis of the complete nt sequence of 1,371 bases indicated that the satDNA shared 88.5% similarity with its closest relatives, which are DNA beta molecules from tomato in Pakistan. This is the first report of a satDNA beta associated with the TYLCV species. The TYLCV-Om and associated satDNA, thus represent a begomovirus-complex at the Asian-Middle East crossroads that quiet uniquely share geographical and genetic hallmarks of both.