Publications

4938

AbstractCandidatus Liberibacter asiaticus (CLas) bacterium has been strongly implicated as the causative agent of huanglongbing (HLB), or citrus greening, which is currently the most devastating citrus disease worldwide. HLB is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in a persistent manner. We used quantitative-polymerase chain reaction (PCR) to detect CLas in dissected organs of individual D. citri adults infected with HLB in the laboratory or collected from field-infected citrus trees in South Florida. The proportion of infected (CLas-positive) dissected organs was 47–70% for the salivary glands, 72–80% for the alimentary canal, and 79–97.5% for the rest of the insect body. Statistical analysis indicated that, in both field- and laboratory-infected D. citri, the proportion of infected salivary glands was significantly lower than that of other parts in the insect body. With field-collected psyllids, the relative copy number of CLas genomes, compared with psyllid genomic DNA in each sample, was significantly higher in both the salivary gland and alimentary canal compared with that in the rest of the insect body for both males and females. These results provide the first PCR confirmation of CLas in the alimentary canal and salivary glands of D. citri and strongly suggest that the salivary glands constitute an important transmission barrier to CLas in the psyllid vector. Our results also suggest that CLas may replicate or accumulate in both the alimentary canal and salivary glands of D. citri.

ABSTRACT Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This “mycetocytic belt” might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria , and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya . The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, “ Candidatus Rohrkolberia cinguli,” is proposed for this newly characterized clade of symbiotic bacteria.

Five new species of cyanobacteria, Scytonematopsis maxima, Tolypothrix pseudorexia, Hormoscilla irregularis, Phormidium arthurensis, and Godleya alpina, the last belonging to a new genus, are described from cultured material isolated from the alpine zone of Mt Philistine, New Zealand. Tolypothrix pseudorexia resembles Rexia erecta in formation of upright trichomes on agar, but clearly lacks the ability to divide in two planes that is characteristic of the latter species. Godleya displays characteristics of the Scytonemataceae-isopolar hormogone development and binary branching-but groups with a species of Coleodesmium (Microchaetaceae) in analyses of 16S rDNA, despite its morphological differences, thus conflicting with the current classification system. We also show that material referable to the widely reported species Phormidium autumnale is found in the New Zealand alpine zone; molecular data indicate that this is likely the same organism reported recently from the Hutt River, Wellington, New Zealand, a quite different habitat. In this site, a lowland river, the species may form extensive mats. These have not been seen on Mt Philistine.

Elm yellows phytoplasmas (EY) belonging to the 16SrV‐A subgroup were recently proposed as a new candidate species ‘ Candidatus Phytoplasma ulmi’. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22 ‐ rps3 , secY and map . In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1 T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1 T and the new ‘ Ca . Phytoplasma ulmi’ strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1 T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22 ‐ rps3 and secY ) used for the characterization and assignment of 16SrV‐A phytoplasmas to the ‘ Ca.  Phytoplasma ulmi’ species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of ‘ Ca.  Phytoplasma ulmi’ (EY‐S1, EY‐S2, EY‐S3 and EY‐S4). New diagnostic enzymes for practical use in ‘ Ca.  Phytoplasma ulmi’ identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of ‘ Ca . Phytoplasma’ species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.

Prophages are important genetic elements of bacterial genomes and are involved in lateral gene transfer, pathogenicity, environmental adaptations, and interstrain genetic variability. In this study, the sequence of a prophage terminase gene of ‘Candidatus Liberibacter asiaticus’, a bacterium associated with citrus Huanglongbing (HLB), was selected as a molecular marker to assess the genetic variation in two ‘Ca. L. asiaticus’ populations from geographically distinct provinces (Guangdong and Yunnan) in China. The frequency of the prophage terminase gene was 15.8% (19/120) in Guangdong (altitude <500 m) and 97.4% (38/39) in Yunnan (altitude >2,000 m). The difference was highly significant (P < 0.0001) based on χ2 analysis. However, the partial prophage terminase gene sequences obtained from 10 Guangdong strains and 6 Yunnan strains were identical or highly similar, suggesting that at least some bacterial strains in the two locations shared a common recent origin. This is the first report on population variation of ‘Ca. L. asiaticus’ in China, where HLB was first described. The population variation of ‘Ca. L. asiaticus’ in the two geographical regions and the related HLB epidemiology were discussed.