I.-M. Lee

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Detection and characterization of a phytoplasma associated with annual blue grass (Poa annua) white leaf disease in southern Italy

A phytoplasma was detected in annual blue grass (Poa annua L. Fienardo), exhibiting white leaf symptoms, that was grown in the fields near Caserta in southern Italy. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with annual blue grass white leaf disease was identified as a new member of phytoplasma 16S rRNA group XI (16SrXI) (type strain, rice yellow dwarf phytoplasma). The annual blue grass white leaf phytoplasma is most closely related to Bermuda grass white leaf phytoplasma found in Asia. Annul blue grass white leaf and Bermuda grass white leaf phytoplasmas were designated as the third subgroup (16SrXI-C) of group XI. This is the first report that a plant pathogenic phytoplasma belonging to group 16SrXI is present on the European continent.

Russian Isolates of Potato spindle tuber viroid Exhibit Low Sequence Diversity

Potato spindle tuber viroid (PSTVd) is currently widespread in seed potatoes grown in Russia. Characterization of 39 PSTVd isolates collected over a 15-year period from widely separated areas in Russia revealed the presence of 17 different sequence variants, all but one of which were previously unknown. Most variants were recovered only once, but two were more widely distributed; one of these was a mild variant previously isolated in Germany, the second was a novel variant inducing symptoms similar to those of the type strain in tomato. Despite this apparent lack of population diversity, several informative PSTVd variants were recovered. Sequence changes in the pathogenicity and variable domains were particularly common, but previously unknown changes were also detected within the loop E motif in the central domain, a structural motif known to play a key role in PSTVd replication and host range determination.

New subgroup 16SrIII-Y phytoplasmas associated with false-blossom diseased cranberry ( Vaccinium macrocarpon ) plants and with known and potential insect vectors in New Jersey

The identity of the presumed phytoplasmal pathogen associated with cranberry false-blossom disease has never been fully clarified. In the present study a molecular-based procedure was employed to determine the identity of the phytoplasma. Tissues of cranberry plants exhibiting cranberry false-blossom symptoms were collected from multiple bogs on each of three randomly selected commercial cranberry farms in New Jersey. Leafhoppers, including the known vector Limotettix vaccinii (Van Duzee) (=Scleroracus vaccinii, Euscellis striatulus) and the sharp-nosed leafhopper Scaphytopius magdalensis (Provancher), a known vector of blueberry stunt disease, were collected from two different farms in New Jersey. Nested PCR assays and RFLP analysis of 16S rRNA gene sequences were employed for the detection and identification of the associated phytoplasmas. All of 20 cranberry plants sampled and five out of 14 batches of leafhoppers tested positive for phytoplasma. Virtual RFLP and sequence analyses revealed that all the associated phytoplasmas were members or variants of a new subgroup, 16SrIII-Y. Phylogenetic analysis of 16S rRNA sequences indicated that cranberry false-blossom phytoplasma strains represented a lineage distinct from other 16SrIII subgroups. This is the first report confirming that a new phytoplasma (designated as a new subgroup 16SrIII-Y) is associated with cranberry false-blossom disease and associated with both leafhopper species in New Jersey.

First report of new phytoplasma diseases associated with soybean, sweet pepper, and passion fruit in Costa Rica.

A new soybean disease outbreak occurred in 2002 in a soybean (Glycine max) plantation in Alajuela Province, Costa Rica. Symptoms on the affected plants included general stunting, small leaves, formation of excessive buds, and aborted seed pods. In the same region, two other diseases, one in sweet pepper (Capsicum annuum) fields and another affecting passion fruit (Passiflora edulis) vines, were also found. Symptoms on sweet pepper plants included unusually dark green leaves, some of which exhibited a rugose symptom with a zigzag pattern to the midvein, and purple vein discoloration. Passion fruit vines exhibited bud proliferation. Collectively, symptoms resembled those commonly attributed to phytoplasmal infections. Total nucleic acid was extracted from veinal tissues of leaves or buds (soybean). A nested PCR assay using primer pair P1/P7 followed by R16F2n/R16R2 (1) was employed for the detection of putative phytoplasmas that might be associated within symptomatic plants. All seven symptomatic plants (three soybean, three sweet pepper, and one passion fruit) tested, but not healthy controls, yielded positive results. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products using restriction enzymes AluI, BfaI, HhaI, MseI, and RsaI indicated that the three diseases were associated with a very similar or identical phytoplasma. RFLP patterns and sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. FJ226068-FJ226073) revealed that the phytoplasma shared less than 97.5% sequence homology with all previously classified phytoplasmas, and, as such, represents a new taxon most closely related to 16SrXII group (1) strains. To our knowledge, this is the first report of a new phytoplasma associated with diseases of soybean, sweet pepper, and passion fruit in Costa Rica. Reference: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.