Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/15/2002
Publication Date: 10/1/2002
Citation: Interpretive Summary: Alfalfa plants showing witches' broom symptoms typical of plant pathogenic bacteria called phytoplasmas were observed in the Sultanate of Oman. By molecular methods, it was identified that the phytoplasma associated with this diseased alfalfa (AlfWB) was similar to the known phytoplasma, peanut witches'-broom phytoplasma (subgroup 16SrII). However, this phytoplasma was slightly different from all the other phytoplasma strains employed belonging to this subgroup. A phylogenetic analysis based on sequence and molecular data of 35 phytoplasma strains, mainly from isolates from the Southern hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to papaya yellow crinkle from New Zealand and Chinese pigeon pea witches' broom from Taiwan. This information is important because it identifies the specific phytoplasma responsible for alfalfa witches'-broom disease and gives us detection methods that may be used to monitor spread and identify disease vectors. This information will benefi scientists and those involved in the identification of and monitor of spread of phytoplasma-associated diseases among agricultural crops.
Technical Abstract: Alfalfa plants showing witches' broom symptoms typical of phytoplasmas were observed in regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths, when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism (RFLP) profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches' broom group (16SrII). Restriction enzyme profiles showed identity of phytoplasmas in all the 300 infected samples. Comparative analyses on P1/P7 amplimers of 20 strains with Tru9I, Tsp509I, HpaII, TaqI and RsaI indicated that this phytoplasma is different from all other strains employed belonging to subgroup 16SrII, except tomato big bud from Australia, and could be therefore classified in subgroup 16SrII-D. The P1/P7 PCR product was sequenced directly after cloning. The alfalfa witches' broom (AlfWB) phytoplasma 16S-23S rRNA gene yielded a 1690 bp product. The homology search using BLAST showed 99% similarity with papaya yellow crinkle phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasma strains showed that AlfWB is a new phytoplasma species, with closest relationships to papaya yellow crinkle (PapayaYC) from New Zealand and Chinese pigeon pea witches' broom (CPPWB) from Taiwan.