Publications

4452

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.

The anaerobic nitrite-reducing methanotroph ‘CandidatusMethylomirabilis oxyfera’ (‘Ca.M. oxyfera’) produces oxygen from nitrite by a novel pathway. The major part of the O2is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of ‘Ca.M. oxyfera’ harbours four sets of genes encoding terminal respiratory oxidases: two cytochromecoxidases, a third putativebo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV–visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by ‘Ca.M. oxyfera’, notably the membrane-boundbo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.

Abstract Candidatus Liberibacter asiaticus (Las) has been reported to increase the susceptibility of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), to selected insecticides. Reduced general esterase activity in Las-infected, compared with uninfected, D. citri has been proposed as a possible explanation for this difference in insecticide susceptibility. The current study was conducted to quantify glutathione transferase (GST) and cytochrome P450 (general oxidase) activities in Las-infected D. citri to further explain the possible mechanisms for altered susceptibility to insecticides due to Las infection. GST and cytochrome P450 activities (indirectly through general oxidase levels) were quantified in Las-infected and uninfected D. citri nymphs and adults. Mean (±SEM) GST activity was significantly lower in Las-infected (468.23 ± 26.87 /µmol/min/mg protein) than uninfected (757.63 ± 59.46 µmol/min/mg protein) D. citri adults. Likewise, mean cytochrome P450 activity was significantly lower in Las-infected (0.23 ± 0.02 equivalent units [EU] cytochrome P450/mg protein) than uninfected (0.49 ± 0.05 EU cytochrome P450/mg protein) D. citri adults. Immature stages (second and fifth instars) were characterized by significantly lower GST activity than adults for uninfected D. citri. However, cytochrome P450 activity was significantly higher in second instar nymphs than adults and fifth-instar nymphs for uninfected D. citri. Lower activities of GST and general oxidase in Las-infected D. citri indicate that infection with Las alters D. citri physiology in a manner that could increase insecticide susceptibility. The reduced activities of these detoxifying enzymes due to Las infection may be explained by examining expression levels of associated genes in Las-infected and uninfected D. citri.

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated “Candidatus Phytoplasma brasiliense” (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). “Ca. Phytoplasma brasiliense” has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.