The diversity of cytochrome b sequences of Carasobarbus species from Iran and adjacent areas was investigated. Two divergent haplotype groups were found for C. luteus. The first one is widespread and congruent with the biogeographical hypothesis of a recent isolation among the various populations as a consequence of rising sea levels following the last Pleistocene glaciation. The second one is restricted to the Khabur River in Syria. The possibility that one of these groups corresponds to C. albus is discussed, but we conclude that it is more likely that C. luteus is a single species that retained two divergent mitochondrial lineages. The mitochondrial sequence diversity found for C. kosswigi and C. sublimus is high, possibly due to small population size and consequent genetic drift. Carasobarbus kosswigi is paraphyletic with respect to C. sublimus, indicating a recent speciation event of these taxa.
Background:The witches' broom disease of lime caused by Candidatus Phytoplasma aurantifolia, is the most devastating disease of acidian lime in the southern parts of Iran. Objectives: At present, no efficient method has been developed for controlling the disease, therefore quarantine approaches such as early detection and subsequent eradication of infected trees is very important. Toward this aim, developing a reliable and sensitive detection method would be the first step to prevent transportation of infected plant materials to other places. Materials and Methods: In this study, Immunodominant membrane protein (IMP) of the pathogen was selected as a target for detection and preparation of polyclonal antibody. The IMP is the major protein present on the surface of phytoplasma cells. For this purpose, the DNA region encoding IMP gene was isolated and cloned into pET28a bacterial expression vector. The recombinant protein was expressed in a large scale in Escherichia coli. Purification was performed under native conditions and the purity and integrity of produced recombinant protein were confirmed by western immuno blot analysis using anti His-tag and anti-IMP polyclonal antibodies. The purified recombinant IMP was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with the alkaline phosphatase enzyme. Results: The purified antibodies and conjugates were applied for efficient detection of infected plants in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and dot immunosorbent assay (DIBA). Conclusions: These antibodies were proven to be very powerful tools to detect the Candidatus Phytoplasma aurantifolia in plants.
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