Publications

4475

A survey carried out in Georgian vineyards, located in the Khaketi region, in September 2013, showed the presence of vines of the cultivar Chardonnay with typical grapevine yellows (GY) symptoms including leaf discoloration and curling, berry shriveling, and irregular maturation of wood. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing shoot proliferation and leaf yellowing were found, suggesting the involvement of phytoplasmas in the disease etiology. Total DNA was extracted by a CTAB method from leaf veins of 18 symptomatic and two asymptomatic grapevines, and from four symptomatic and two asymptomatic bindweeds, and analyzed by PCR assays. Moreover, DNA extracted from ‘Candidatus Phytoplasma asteris’ strain SAY (group 16SrI), ‘Ca. P. solani’ strain STOL (group 16SrXII), and ‘Ca. P. ulmi’ strain EY1 (group 16SrV) were used as positive controls. DNA extracted from healthy periwinkle and a reaction mixture without template were employed as negative controls. Nested PCRs targeting the 16S rDNA, carried out using the primer pairs P1/P7 followed by R16F2n/R16R2 (1), produced a band of the expected size (1,250 nt) in all the symptomatic grapevine and bindweed plants, and in the positive controls. No amplification was observed with DNA from asymptomatic plants nor the negative controls. PCR products were sequenced by a commercial sequencing service (Primm, Milan, Italy). The 16S rDNA nucleotide sequences of phytoplasmas identified in all grapevines and in two bindweed samples shared >99.5% sequence identity with ‘Ca. P. solani’ reference strain STOL (GenBank Accession No. AF248959), and carried identical STOL-unique signature sequence and distinguishing sequence blocks (3). Moreover, nucleotide sequences of phytoplasmas identified in the other two bindweed samples shared >99.6% sequence identity with ‘Ca. P. convolvuli’ reference strain BY-S57/11 (JN833705) (2). RFLP and phylogenetic analyses confirmed the affiliation of the phytoplasma strains identified in grapevine and bindweed plants in Georgia to the species ‘Ca. P. solani’ (subgroup 16SrXII-A) and ‘Ca. P. convolvuli’ (subgroup 16SrXII-H). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank website with accession nos. KF996535 and KF996536 (‘Ca. P. solani’ from grapevine and bindweed, respectively), and KF996537 (‘Ca. P. convolvuli’). Future studies will focus on investigating the spread and impact of ‘Ca. P. solani’-associated bois noir (BN) in Georgia. In particular, the identification of ‘Ca. P. solani’ in bindweeds suggested the presence of the insect Hyalesthes obsoletus, a polyphagous cixiidae responsible for BN phytoplasma transmission in vineyards in Europe. Accurate surveys and molecular analyses will be performed for identifying the insect vector(s) of the BN associated phytoplasma strains in Georgia. Additional studies will be performed to study the spread and impact of ‘Ca. P. convolvuli,’ identified only in Italy, Germany, Serbia, and Bosnia and Herzegovina (2), throughout the Caucasian countries. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) M. Martini et al. Int. J. Syst. Evol. Microbiol. 62:2910, 2013. (3) F. Quaglino et al. Int. J. Syst. Evol. Microbiol. 63:2879, 2013.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with ‘Candidatus Liberibacter solanacearum’ and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect ‘Ca. L. solanacearum’. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of ‘Ca. Liberibacter’ were observed using electron microscopy in celery plant tissues. A fifth haplotype of ‘Ca. L. solanacearum’, named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

Abstract Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila’, is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila’ traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.

Abstract In two of Iran's central provinces, several herbaceous plants showing phytoplasma disease symptoms were collected to detect 'Canididatus Phytoplasma asteris'-related phytoplasmas. Confirmation of an association of phytoplasmas with diseased plants was done using polymerase chain reaction (PCR) assays having the phytoplasma universal primer pairs P1/P7 followed by R16F2n/ R16R2 in nested PCR. Then, for detection of 'Ca. P. asteris', DNA samples were subjected to amplification of rp and tuf genes using specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy, respectively. Restriction fragment length polymorphism or RFLP analyses of rp gene fragments using Tsp509I restriction enzyme as well as sequence analyses indicated that 'Ca. P. asteris'-related phytoplasmas associated with carrot, niger seed and scallion plants in these regions, belong to the rpI-L subgroup. This research is the first report of carrot, niger seed, and scallion infection with phytoplasmas belonging to the rpI-L subgroup.