Molecular Characterization of the 16S rRNA Gene of Phytoplasmas Detected in Two Leafhopper Species Associated with Alfalfa Plants Infected with Witches' Broom in Oman

Abstract

Two leafhopper species, Austroagallia avicula and Empoasca sp., were consistently found in alfalfa fields
infected with witches’ broom phytoplasma (OmanAlfWB) in the Al-Batinah, Dakhliya, North and South Sharqiya,
Muscat, and Al-Bureimi regions of the Sultanate of Oman. Phytoplasmas from both leafhoppers were detected by
specific polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the spacer region in direct PCR
using P1/P7 primer pairs. Comparative RFLP profiles of the amplified rRNA gene and the spacer region from leafhopper
phytoplasmas and from 20 phytoplasma controls yielded patterns referable to phytoplasmas belonging to the peanut
witches’ broom group (16SrII group). In particular, extensive RFLP analyses with the endonucleases HpaII, Tru9I,
Tsp509I, and RsaI indicated that the phytoplasmas in A. avicula and Empoasca sp. were identical but showed some
differences from OmanAlfWB; however, RFLP patterns obtained with TaqI showed the OmanAlfWB and the
phytoplasmas from the two leafhoppers to be identical. Direct PCR products amplified from phytoplasma leafhopper
DNA using the P1/P7 primer pair were cloned and sequenced yielding 1758 bp and 1755 bp products from A. avicula
and Empoasca sp. respectively; the homology of these sequences with OmanAlfWB and papaya yellow crinkle
phytoplasmas was more than 98%. A phylogenetic tree based on the 16S rRNA gene and spacer region sequences
from 44 phytoplasmas revealed that the phytoplasmas from the leafhoppers clustered with OmanAlfWB, papaya
yellow crinkle, and gerbera phyllody phytoplasmas, all belonging to 16SrII group, but were distinct from lime witches’
broom phytoplasma, the most commonly found phytoplasma in the Sultanate of Oman.