Detection and molecular characterization of alfalfa witches'-broom phytoplasma and its leafhopper vector in Riyadh region of Saudi Arabia

This study was conducted to detect for the first time the occurrence of phytoplasma in symptomatic alfalfa plants as well as in its insect vectors in Riyadh region of Saudi Arabia together with its characterization by comparing it with isolates detected elsewhere in the world. Disease symptoms similar to those described for phytoplasma diseases were observed on alfalfa plants growing in the Riyadh region (Wadi aldawasir, Sagir and AlZulfi), Saudi Arabia. Three representative alfalfa samples and two groups of leafhopper samples out of the total samples collected from the fields in the visited areas tested positive for plant pathogenic phytoplasmas using P1/P7 primer pair in the first round of PCR test. An additional PCR was conducted using the nested R16F2n/R16R2 primer pair, which yielded fragments of approximately 1.2 kb. The rest of the collected samples were tested using a synthesized cDNA probe for phytoplasma detection by dot blot hybridization. Fifty four out of 84 alfalfa samples and 65 out of 94 leafhopper samples collected from the above mentioned locations hybridized with the cDNA probe, representing 64.3% and 69.1%, respectively. The nucleotide sequences for the five positive samples were determined and were found to have 99.3%-100% nucleotide sequences identity among them, and share 97.3-98.8% sequence similarity with seven phytoplasma isolates belonging to the 16S rII (peanut witches-broom group) obtained from the GenBank database. The nucleotide sequences of the five isolates detected in this study were published in the GenBank with the following accession numbers: JQ808130, JQ818819, JQ818820, JX646694 and JX646695.