Publications

4470

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of ‘Candidatus Liberibacter solanacearum’, the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of ‘Ca. Liberibacter solanacearum’ was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected ‘Ca. Liberibacter solanacearum’ and the closely related species ‘Ca. Liberibacter asiaticus’, the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting ‘Ca. Liberibacter’ pathogens in psyllids and field-grown potato plants and tubers.

Abstract. This study investigated the bacterial diversity associated with microbial mats of polar deep-sea cold seeps. The mats were associated with high upward fluxes of sulfide produced by anaerobic oxidation of methane, and grew at temperatures close to the freezing point of seawater. They ranged from small patches of 0.2–5 m in diameter (gray mats) to extensive fields covering up to 850 m2 of seafloor (white mats) and were formed by diverse sulfide-oxidizing bacteria differing in color and size. Overall, both the dominant mat-forming thiotrophs as well as the associated bacterial communities inhabiting the mats differed in composition for each mat type as determined by microscopy, 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis. While the smaller gray mats were associated with a highly diverse composition of sulfide oxidizers, the larger white mats were composed of only 1–2 types of gliding Beggiatoa filaments. Molecular analyses showed that most of the dominant mat-forming sulfide oxidizers were phylogenetically different from, but still closely related to, thiotrophs known from warmer ocean realms. The psychrophilic nature of the polar mat-forming thiotrophs was tested by visual observation of active mats at in situ temperature compared to their warming to >4 °C. The temperature range of mat habitats and the variation of sulfide and oxygen fluxes appear to be the main factors supporting the diversity of mat-forming thiotrophs in cold seeps at continental margins.

Summary Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout ( Salmo trutta ) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site‐specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected ‘ Candidatus Piscichlamydia salmonis’ and ‘ Candidatus Clavochlamydia salmonicola’ infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘ Candidatus Liberibacter asiaticus’ and ‘ Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘ Ca.  L. asiaticus’, which has taken over from ‘ Ca.  L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘ Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘ Ca.  L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘ Ca.  L. asiaticus’ was distributed throughout the inoculated plants, with mean C t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘ Ca.  L. asiaticus’ reached the highest titres in mature leaves (mean C t value = 26·7) of citrus plants maintained at 22/27°C. ‘ Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C t value in mature leaves = 33·6).