Publications

3843

Three strictly anaerobic, methanogenic strains JH1T, JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales , and Methanobrevibacter wolinii SHT was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1T, JH4 and JH8 and M. wolinii SHT were 96.2 and 89.0 % respectively, and DNA–DNA hybridization of the isolates and M. wolinii DSM 11976T showed a 20 % reassociation. Strain JH1T exhibited 92 % DNA–DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5–1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37–40 °C and pH 6.5–7.0. The DNA G+C content of strain JH1T was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter , for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1T ( = KCTC 4102T = JCM 18376T).

The objective of this work was to evaluate the reaction of four sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' (Las) infection, a bacterium associated to huanglongbing (HLB) disease. Transgenic sweet orange plants of Hamlin, Natal, Pêra, and Valência cultivars, as well as nontransgenic controls received inocula by grafting budwood sections of HLB-infected branches. Disease progression was evaluated through observations of leaf symptoms and by polymerase chain reaction (PCR) analysis, eight months after inoculation. A completely randomized design was used, with four experiments (one for each cultivar) performed simultaneously. Bacteria title was estimated by quantitative PCR (qPCR). HLB symptoms and Las titers were present in nontransgenic and transgenic plants expressing the attacin A gene of the four sweet orange cultivars, eight months after bacteria inoculation. Five transgenic lines (transformation events) of 'Pêra' sweet orange expressing the attacin A gene have significantly lower Las titers in comparison with nontransgenic plants of this cultivar.

During a survey carried out in Jordanian vineyards in August and October 2012, grapevine (Vitis vinifera L.) plants showing typical grapevine yellows (GY) disease symptoms, including leaf discoloration and curling, berry shriveling, and irregular maturation of wood, were observed. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing stunting and leaf chromatic alteration were found, suggesting the involvement of phytoplasmas in the disease etiology. Using a CTAB method, total DNA was extracted from leaf veins of 25 symptomatic and two asymptomatic grapevines, and from five symptomatic and two asymptomatic bindweeds for PCR analysis. DNAs from periwinkle (Catharanthus roseus (L.) G. Don) plants infected by ‘Ca. Phytoplasma asteris’ strain SAY (group 16SrI), ‘Ca. Phytoplasma solani’ strain STOL (group 16SrXII), and ‘Ca. Phytoplasma ulmi’ strain EY1 (group 16SrV), were used as positive controls. DNAs from healthy periwinkle and reactions without template DNA were employed as negative controls. 16S rDNA nested PCRs, carried out using the primer pairs P1/P7, followed by R16F2n/R16R2 (1), yielded an amplicon of the expected size (1,250-bp) in three grapevine and in five bindweed samples, and in positive controls. Amplicons were not produced with DNA from 22 symptomatic grapevines (probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform [2]); nor from asymptomatic plants and negative controls. PCR products were sequenced by commercial services in Italy (Primm, Milan) and Korea (Macrogen Inc., Soul). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank with accessions KC835139 (from grapevine) and KC835140 (from bindweed). The 16S rDNA nucleotide sequences of phytoplasmas identified in grapevine and bindweed in Jordan shared >99.5% sequence identity with ‘Ca. Phytoplasma solani’ reference strain STOL (AF248959), and carried identical STOL-unique signature sequences and distinguishing sequence blocks (3). Phylogenetic and in silico RFLP analyses confirmed the affiliation of phytoplasma strains identified in grapevine and bindweed in Jordan to the species ‘Ca. Phytoplasma solani’ (subgroup 16SrXII-A), opening an avenue to future studies on the dissemination and impact of Bois noir (BN) in Jordan. These studies may add new information about BN, previously reported in neighboring countries (4). Further studies will investigate the role of Hyalesthes obsoletus Signoret, a polyphagous Cixiidae responsible for the BN phytoplasma transmission in Europe, and other possible insect vector(s) in the BN spread in Jordan. References: (1) I.-M. Lee et al. Int. J. Syst. Bact. 48:1153, 1998. (2) F. E. Constable et al. Plant Pathol. 52:267, 2003. (3) F. Quaglino et al. Int. J. Syst. Evol. Microb. 63:2879. (4) E. Choueiri et al. Plant Dis. 86:697, 2002.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV ‘Candidatus Phytoplasma cynodontis’ group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

ABSTRACT A recently described clinical isolate, “ Candidatus Bartonella ancashi,” was obtained from a blood sample of a patient presenting with verruga peruana in the Ancash region of Peru. This sample and a second isolate obtained 60 days later from the same patient were molecularly typed using multilocus sequence typing (MLST) and multispacer sequence typing (MST). The isolates were 100% indistinguishable from each other but phylogenetically distant from Bartonella bacilliformis and considerably divergent from other known Bartonella species, confirming their novelty.