2.2.11
ASH YELLOWS PHYTOPLASMAS, A COHERENT, WIDELY DISTRIBUTED GROUP IN NORTH AMERICA

HM GRIFFITHS, GT HILL, and WA SINCLAIR

Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA

Background and objectives

Ash yellows (AshY) phytoplasmas parasitize Fraxinus spp. (ash) and Syringa spp. (lilac) [1] and comprise a phylogenetic subclade of the phytoplasmas based on analyses of 16S rRNA and ribosomal protein genes [2]. The subclade was based on data from only the type strain AshY1 but is supported by DNA hybridization data from several strains. The objectives of research summarized here were to determine identity and similarity of phytoplasma strains from ash and lilac across the known range of AshY (71–113° W longitude), evaluate differences in strains’ aggressiveness, and identify potential vectors of AshY.

Materials and methods
DNA samples from plants and insects served as templates in PCRs using phytoplasma-universal primer pairs R16F2/R2 (1.2 kb product) and/or P1/P7 (1.8 kb product). Amplimers of rDNA thus obtained were digested with restriction endonucleases and/or sequenced. Immunofluorescence microscopy was performed with a monoclonal antibody raised against strain AshY2. The aggressiveness of 13 strains associated with AshY or lilac witches broom was evaluated in terms of growth suppression and chlorosis in graft-inoculated F. pennsylvanica (green ash) and Catharanthus roseus (periwinkle). Relative rates of multiplication of strains AshY1 and AshY3, which differed in aggressiveness, were assessed in co-inoculated periwinkle plants by means of PCR-RFLP analysis after 3 and 5 months of incubation. Airborne insects collected from Malaise traps at two sites of high AshY incidence in New York State, USA, were assayed for phytoplasmal DNA using PCR-RFLP analysis.

Results and conclusions
All phytoplasmas detected in ash or lilac appeared to represent one coherent group. The amplimers from 14 strains, including AshY1, obtained with primer pair R16F2/R2 and digested with AluI, had identical RFLP profiles. A total of four RFLP profiles were obtained with AluI, TaqI, and HhaI from P1/P7 amplimers, and the corresponding restriction sites were located in sequence data. Sequence similarity of P1/P7 amplimers from strains representing all RFLP patterns was >99%. Thus, at least four rDNA genotypes of AshY phytoplasmas exist. One strain, AshY3 from Utah,USA, did not react with the monoclonal antibody. Strains varied significantly (P< 0.01) in growth suppression and chlorosis induced in either ash or periwinkle. AshY3 was the most aggressive and AshY4 from New York State the least. Only AshY3 could be detected in periwinkle plants 3 months after co-inoculation with AshY1 and AshY3; 2 months later, AshY1 was also detected. Thus, AshY3 may multiply more rapidly than AshY1, which could explain its aggressiveness. Seventeen genera of Homoptera, including 10 with known phytoplasma vectors, were sampled by PCR-RFLP analysis. AshY phytoplasmas were detected in two genera of leafhoppers, Scaphoideus and Colladonus (10 out of 460 and one out of 68 specimens, respectively). The phytoplasmas associated with AshY and lilac witches broom appear to be a discrete and coherent, but inhomogeneous, leafhopper-borne taxon.

References
1. Sinclair WA, Griffiths HM, Davis RE, 1996. Plant Disease 80, 6468-75.
2. Gundersen DE, Lee IM, Rehner SA, Davis RE, Kingsbury DT, 1994. Journal of Bacteriology 176, 5244-54.