Davis, Robert E.

Abstract 'Ca. Phytoplasma pini' is a member of phytoplasma 16S rRNA gene RFLP group XXI, subgroup XXI-A. It has been identified in a number of European countries, including Germany, Poland, Lithuania, Spain, Czech Republic and Croatia. Outside of Europe it has been identified in China and Mozambique. A related strain has also been identified in Maryland, USA. In the USA, favourable climatic conditions and wide availability of potential host plants of the phytoplasma, suggest that the potential for spread of 'Ca. Phytoplasma pini' could be significant. Host plants include Pinus sylvestris, P. halepensis, P. mugo, P. banksiana, P. nigra, P. tabuliformis, Abies procera and Tsuga canadensis. Symptoms include the formation of ball-like growths containing dwarfed needles, yellowed or reddish needles and the loss of needles. It is transmitted by insect vectors that are currently unknown.

Abstract 'Ca. Phytoplasma pini' is a member of phytoplasma 16S rRNA gene RFLP group XXI, subgroup XXI-A. It has been identified in a number of European countries, including Germany, Poland, Lithuania, Spain, Czech Republic and Croatia. Outside of Europe it has been identified in China and Mozambique. A related strain has also been identified in Maryland, USA. In the USA, favourable climatic conditions and wide availability of potential host plants of the phytoplasma, suggest that the potential for spread of 'Ca. Phytoplasma pini' could be significant. Host plants include Pinus sylvestris, P. halepensis, P. mugo, P. banksiana, P. nigra, P. tabuliformis, Abies procera and Tsuga canadensis. Symptoms include the formation of ball-like growths containing dwarfed needles, yellowed or reddish needles and the loss of needles. It is transmitted by insect vectors that are currently unknown.

Wheat blue dwarf (WBD) is one of the most economically damaging cereal crop diseases in northwestern PR China. The agent associated with the WBD disease is a phytoplasma affiliated with the aster yellows (AY) group, subgroup C (16SrI-C). Since phytoplasma strains within the AY group are ecologically and genetically diverse, it has been conceived that the AY phytoplasma group may consist of more than one species. This communication presents evidence to demonstrate that, while each of the two 16 rRNA genes of the WBD phytoplasma shares >97.5 % sequence similarity with that of the ‘CandidatusPhytoplasma asteris’ reference strain, the WBD phytoplasma clearly represents an ecologically separated lineage: the WBD phytoplasma not only has its unique transmitting vector (Psammotettix striatus) but also elicits a distinctive symptom in its predominant plant host (wheat). In addition, the WBD phytoplasma possesses molecular characteristics that further manifest its significant divergence from ‘Ca. P. asteris’. Such molecular characteristics include lineage-specific antigenic membrane proteins and a lower than 95 % genome-wide average nucleotide identity score with ‘Ca. P. asteris’. These ecological, molecular and genomic evidences justify the recognition of the WBD phytoplasma as a novel taxon, ‘CandidatusPhytoplasma tritici’.

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

‘Candidatus Phytoplasma pini’-related strain MDPP, the reference strain of subgroup 16SrXXI-B, is a pathogen associated with witches’ broom disease of Pinus spp. in North America. Here, we report the first draft genome sequence of ‘Ca. Phytoplasma pini’ strain MDPP, which consists of 474,136 bases, with a G + C content of 22.22%. This information will facilitate comparative genomics of gymnosperm-infecting phytoplasmas.

AbstractIn order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma.

Grapevine yellows diseases occur in cultivated grapevine (Vitis vinifera L.) on several continents, where the diseases are known by different names depending upon the identities of the causal phytoplasmas. In this study, phytoplasma strains associated with grapevine yellows disease (North American grapevine yellows [NAGY]) in vineyards of Pennsylvania were characterized as belonging to 16S ribosomal RNA (rRNA) gene restriction fragment length polymorphism group 16SrI (aster yellows phytoplasma group), subgroup 16SrI-B (I-B), and variant subgroup I-B*. The strains (NAGYI strains) were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico three-dimensional modeling of the SecY protein. Although the NAGYI strains are closely related to aster yellows (AY) phytoplasma strains and are classified like AY strains in subgroup I-B or in variant subgroup I-B*, the results from genotyping and protein modeling may signal ongoing evolutionary divergence of NAGYI strains from related strains in subgroup 16SrI-B.

Phy.to.plas'ma. Gr. neut. n.phyton, a plant; Gr. neut. n.plasmasomething formed or molded, a form; N.L. neut. n.Phytoplasma, a form from a plant.Tenericutes / Mollicutes / Acholeplasmatales / Incertae Sedis ‐ Family II /CandidatusPhytoplasmaThe generic name ‘CandidatusPhytoplasma’ has been adopted by specialists to refer to a monophyletic clade of wall‐less phytopathogenic bacteria affiliated with the orderAcholeplasmatalesin the classMollicutes. These small (<1 µm) pleomorphic cells occur within the nutritionally‐rich phloem sap and sieve elements of plants, and in the gut or hemolymph of insects that feed on plants. Sustained culture in cell‐free media has not yet been demonstrated for any phytoplasma, and a recent report of successful axenic culture remains to be independently replicated. Reference strains are maintained in plants by periodic graft inoculation. Sequence analysis of PCR‐amplified rDNA, plus considerations of the plant host range and vector species, provide a basis for phytoplasma strain identification and assignment to phylogenetic groups. Hundreds of plant species are susceptible to infection, with signs including discoloration, stunting, virescence, phyllody, proliferation of flowers or shoots, or sterility. The necessity for strict quarantine regulations to control the spread of phytoplasmas among important plants such as fruit trees, palms, and grapevines illustrates the importance of accurate identification and nomenclature for these organisms.