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4474

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with “ Candidatus Mycoplasma haemominutum” (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

Witches'-broom disease of lime (WBDL) caused by ‘Candidatus Phytoplasma aurantifolia’ is a devastating disease in the Sultanate of Oman, United Arab Emirates, and southern Iran. The disease primarily affects lime (Citrus aurantifolia), but in Iran, it is also found in bakraee, a natural C. reticulata hybrid. The disease has been experimentally transmitted from lime to several citrus cultivars by grafting and to a number of herbaceous hosts by dodder. However, the natural vector of ‘Ca. P. aurantifolia’ has not been determined. The most common phloem-feeding insect associated with lime trees in the area is the leafhopper Hishimonus phycitis. The WBDL phytoplasma has been detected in the body of this leafhopper by ELISA and PCR (1), but previous attempts to establish its vector status have failed. It was recently reported that the leafhopper can release the phytoplasma into a sugar solution by feeding through a Parafilm membrane (4). Here we report successful transmission of WBDL phytoplasma to bakraee seedlings by H. phycitis. The leafhopper nymphs and adults were collected in a WBDL-infected lime orchard in Minab (Hormozgan Province) in May of 2006. Of more than 100 leafhopper samples tested, at least 70% were positive for the phytoplasma by PCR using P1/P7 primer pair (3). Additional field-collected leafhoppers were caged (five per plant) on bakraee seedlings at the two-leaf stage in pots in the greenhouse in Zarghan (Fars Province). After 8 weeks, the remaining leafhoppers were killed with an insecticide. Six months after inoculation, 3 of 10 inoculated plants showed typical symptoms of WBDL, including bud proliferation, general chlorosis, and stunting. Symptomatic plants were strongly positive in PCR assays using primer pair P1/P7. No amplification was obtained with healthy control lime or nonsymptomatic bakraee seedlings. Amplified P1/P7 primed PCR products (1,800 bp) from experimentally vector-challenged bakraee seedlings, captured H. phycitis, and a naturally infected lime tree from Minab were subjected to restriction fragment length polymorphism (RFLP) analysis using AluI, HhaI, HpaII, RsaI, and TaqI enzymes. RFLP patterns from these sources were identical and similar to those reported earlier (2). These analyses verified the identity of WBDL phytoplasma in experimentally infected bakraee seedlings. To our knowledge, this is the first report of natural transmission of ‘Ca. P. aurantifolia’ by H. phycitis. References: (1) J. M. Bové et al. Proc. Conf. IOCV 12:342. 1993. (2) A. J. Khan et al. Phytopathology 92:1038, 2002. (3) B. Schneider et al. Pages 369–380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Vol. 2. S. Razin and J. G. Tully, eds. Academic Press, New York, 1995. (4) M. Siampour et al. Iran. J. Plant Pathol. 41:139 (Farsi) and 35 (English), 2006.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a ‘Candidatus Phytoplasma asteris’-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

A Gram-negative, chemoheterotrophic, facultatively anaerobic coccus, designated IMCC1545T, was isolated from the digestive tract of a marine clamworm, Periserrula leucophryna, inhabiting a tidal flat of the Yellow Sea. Cells of strain IMCC1545T are non-motile, dividing by binary fission. The predominant fatty acids are anteiso-C15 : 0 and C18 : 0. The respiratory quinone is menaquinone-7 and the DNA G+C content is 52.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences using three treeing algorithms revealed that the strain formed a novel genus-level lineage within the phylum ‘Verrucomicrobia’. The most closely related named organisms to strain IMCC1545T are ‘Fucophilus fucoidanolyticus’ SI-1234 (86.5 % 16S rRNA gene sequence similarity), Alterococcus agarolyticus ADT3T (81.8 %) and Opitutus terrae PB90-1T (80.3 %), which belong to subdivision 4 of the ‘Verrucomicrobia’. Subdivision 4 of the ‘Verrucomicrobia’ (here named Opitutae classis nov.) was divided into two clades, a clade containing strain IMCC1545T and a clade containing Opitutus terrae. From the taxonomic data obtained in this study, it is proposed that the new marine isolate be placed into a novel genus and species named Puniceicoccus vermicola gen. nov., sp. nov. (the type strain of Puniceicoccus vermicola is IMCC1545T=KCCM 42343T=NBRC 101964T) within Puniceicoccaceae fam. nov and Puniceicoccales ord. nov in the class Opitutae. The family Opitutaceae fam. nov. and order Opitutales ord. nov. are also formally proposed.