First Report of Alfalfa Witches Broom Disease in Oman Caused by a Phytoplasma of the 16Sr II Group.

Abstract

Alfalfa (Medicago sativa L.) is a primary forage crop in the Sultanate of Oman. A new disease of alfalfa in Oman is characterized by proliferation of shoots and yellowing of leaves in 1- to 2-year-old plants and tillering of stems in 4- to 5-year-old plants. Annual losses due to this disease are estimated at more than US$ 23 million. Samples of healthy and infected alfalfa plants were collected from different regions. Total DNA was extracted according to Khadhair et al. (1), with minor modifications. Amplification of 16S rDNA was done using a nested polymerase chain reaction (PCR) approach with primers P1/P7 and R16F2n/R16R2. DNA from healthy leaves and sterile water was used as a negative control, while DNA from periwinkle infected with faba bean phyllody (16SrII-C), aster yellows (16SrI), tomato big bud (16SrII-D), sweet potato little leaf (16SrII-D), catharanthus phyllody (16SrVI), and sesame phyllody (16SrII-A) were used as positive controls and for restriction fragment length polymorphism (RFLP) comparisons. Nested 1.25-kb PCR products from infected plant samples were subjected to RFLP analysis with restriction endonucleases RsaI, AluI, HaeIII, HhaI, EcoRI, TaqI, Tru9I, and Sau3AI. The analysis showed that the alfalfa witches' broom phytoplasma (AWBP) belonged to the 16SrII group (peanut witches' broom) and that the AWBP was most similar to sweet potato little leaf (16SrII-D) but distinct from "Candidatus Phytoplasma aurantifolia," the cause of lime witches' broom in Oman. Other phytoplasmas infecting alfalfa have been reported from Europe and North America (1,3), but they belong to the 16SrVI (clover phyllody) and 16SrI (aster yellows) groups. An alfalfa witches' broom reported from Italy (2) forms a separate grouping (4). To our knowledge, this is the first report of a phytoplasma from the peanut witches' broom group infecting alfalfa in the Sultanate of Oman.