Schneider, Bernd

Pinus silvestris and Pinus halepensis trees grown in Germany and Spain, respectively, showing abnormal shoot branching, dwarfed needles and other symptoms were examined for the presence of plant-pathogenic mollicutes (phytoplasmas). While phytoplasmas could not be detected unambiguously with microscopical methods, PCR amplification using universal phytoplasma primers yielded positive results. Samples collected from symptomatic and non-symptomatic plant parts of both symptomatic Pinus silvestris and Pinus halepensis trees tested positive. Also, surrounding non-symptomatic trees proved to be phytoplasma-infected. Comparisons revealed that the 16S rRNA gene sequences of the phytoplasmas identified in Pinus silvestris and Pinus halepensis were nearly identical. However, the pine phytoplasma is only distantly related to other phytoplasmas. The closest relatives are members of the palm lethal yellowing and rice yellow dwarf groups and ‘Candidatus Phytoplasma castaneae’, which share between 94·5 and 96·6 % 16S rRNA gene sequence similarity. From these data it can be concluded that the phytoplasmas identified in the two Pinus species represent a coherent but discrete taxon; it is proposed that this taxon be distinguished at putative species level under the name ‘Candidatus Phytoplasma pini’.

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1·0 to 1·5 % of nucleotide positions and were thus below the recommended threshold of 2·5 % for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S–23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99·6 % 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of ‘Candidatus Phytoplasma pyri’.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.