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In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region. References: (1) R. E. Davis et al. New Dis. Rep. 20:35, 2010. (2) M. Salehi et al. Plant Pathol. 56:669, 2007. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) M. G. Zamharir. Afr. J. Microbiol. Res. 5:6013, 2011.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including ‘CandidatusPhytoplasma australiense’, ‘CandidatusPhytoplasma japonicum’ and ‘CandidatusPhytoplasma fragariae’. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis viniferaL.). Such latter strains have been informally proposed to represent a separate species, ‘CandidatusPhytoplasma solani’, but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the ‘CandidatusPhytoplasma’ genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated (‘Ca.Phytoplasma solani’) strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. ‘Ca.Phytoplasma solani’ is the only phytoplasma known to be transmitted byHyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse ‘Ca.Phytoplasma solani’ strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA,tuf,secYandrplV–rpsCgene sequences supported this view and yielded congruent trees in which ‘Ca.Phytoplasma solani’ strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of ‘Ca.Phytoplasma australiense’-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, ‘Ca.Phytoplasma solani’; STOL11 is designated the reference strain.

Summary Knowledge of intraspecific variation in symbioses may aid in understanding the ecology of widespread insects in different parts of their range. We investigated bacterial symbionts of A delges tsugae , a pest of hemlocks in eastern N orth A merica introduced from A sia. Amplification, cloning, and sequencing of bacterial 16 S rDNA , in situ hybridizations, and electron microscopy revealed that A . tsugae harbours up to five bacterial phylotypes, according to population. Three G ammaproteobacteria species are maternally transmitted. The first, designated ‘ C a . P seudomonas adelgestsugas’ resides in the haemocoel, and was detected in all populations except T aiwan. The second phylotype, ‘ C a . S erratia symbiotica’, resides in bacteriocytes of populations on T suga sieboldii in J apan and in E . N orth A merica. The third phylotype, designated ‘ C a . A nnandia adelgestsuga’, clustered within a lineage of several insect endosymbionts that included B uchnera aphidicola . It was detected in bacteriocytes in all populations, and in salivary glands of first instars. Two B etaproteobacteria phylotypes were detected in some J apanese T . sieboldii and eastern N orth A merica populations, and were observed only in salivary glands with no evidence of maternal transmission. Our results support the ideas that symbiont gain and loss has been volatile in adelgids, and that symbionts may help to trace the source of invasive species.