Caicedo-Chávez, Jorge D.

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    Identification and molecular characterization of Pigeon pea witches’- broom phytoplasma in plants and its potential vectors in Puerto Rico
    (2014) Caicedo-Chávez, Jorge D.; Rivera-Vargas, Lydia I.; College of Agricultural Sciences; Davis, Robert E.; Irish, Brian M.; Department of Crops and Agro-Environmental Sciences; Kolterman, Duane A.
    Few studies have determined the presence of phytoplasma from important crops in Puerto Rico. Disease symptoms resembling those caused by phytoplasma were observed in different plant species such as pigeon pea (Cajanus cajan), periwinkle (Catharanthus roseus), tabebuia (Tabebuia heterophylla), Spanish lime (Melicoccus bijugatus), ixora (Ixora coccinea), mango (Mangifera indica), cactus (Opuntia spp.), citrus trees (Citrus spp.), and coffee (Coffea arabica). Sixty-two plant samples from these species were tested using end point PCR with universal and specific primers (i.e., nested PCR) that prime amplification of the 16S rDNA and ribosomal protein genes (rpIV-rpsC). Fifty-one percent of the samples tested corresponding to periwinkle, pigeon pea, citrus, coffee and tabebuia were positive for phytoplasmas with amplicons of 0.8 and 1.2kb, respectively, depending upon the primers used in PCRs. In both cases the DNA sequences showed 99% of identity with pigeon pea witches’-broom phytoplasma (PPWB) and by restriction patterns (RLFP) obtained from these samples belonged to group 16SrIX. Due to the lack of studies of potential insect vectors, common auchenorrhyncha species were sweep-collected from pigeon pea and citrus and tested for phytoplasma. Of nine insect genera collected, Empoasca kraemeri, Melornemis antillarum and Colpoptera maculifrons were positive for PPWB phytoplasma based on results from conventional PCR and DNA sequence analysis. These findings indicate that these insects fed upon the aforementioned plant species, and may act as potential phytoplasma vectors in the field. Finally, specific primers were designed for qPCR assay to amplify a 102-bp region of the 16S rDNA gene from samples with low level infections of phytoplasma. By the SYBR® Green method, the melting temperature (Tm) recorded in positive samples was 82.3oC. These primers amplified and identified DNA of phytoplasma belonging to the groups and subgroups 16SrV-A, 16SrIII- H, 16SrII-D, 16SrV-C, 16SrII-C, 16SrVI-A, 16SrXII-A and 16SrIX-C.