Publications

4368

AbstractIsothermal recombinase polymerase amplification (RPA) assays for the specific detection of “Candidatus Phytoplasma mali (Ca. P. mali),” the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real‐time mode (TwistAmp®exo assay) using a fluorophore‐labelled probe or by direct visualization employing a lateral flow device (TwistAmp®nfo assay/Milenia®HybriDetect). The RPA assays specifically amplified DNA from “Ca. P. mali” strains, and cross‐reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real‐time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster. Simplified nucleic acid extraction procedures from plant tissue with Tris‐ and CTAB‐based buffers revealed that crude Tris–DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA‐based assays for the detection of “Ca. P. mali”. The assays are suitable for high‐throughput screening of plant material and point‐of‐care diagnostic and can be potentially combined with a simplified DNA extraction procedure.

Abstract Asian citrus psyllid D iaphorina citri is the vector of the citrus H uanglongbing ( HLB ) associated bacterial agent ‘ C andidatus L iberibacter asiaticus’ ( C L as). The molecular interactions between C L as and D . citri remain unclear. In the present study, protein profiles of mitochondrial, microsomal and cytosolic fractions from uninfected and C Las‐infected adult D . citri are investigated using two‐dimensional gel electrophoresis. The comparative analysis reveals a total of 18, 24 and 20 protein spots that are unique or differentially expressed in mitochondrial, microsomal and cytosolic proteins fractions respectively. These proteins are successfully identified by mass spectrometry. Among the 62 identified proteins, 30 are up‐regulated, whereas 32 are down‐regulated. These proteins include important components in energy metabolism such as ATP synthase, ATP ase, ATP / ADP carrier protein, etc.; host stress responses such as heat shock proteins; host detoxification processes (i.e., cytochrome P 450 and glutathione S ‐transferase); and the cytoskeleton (such as actin, tubulin, myosin and tropomyosin). These data suggest that, after C L as infection, several proteins of D . citri , especially energy metabolism and protein biosynthesis, are altered, and extensive host defence responses are induced. In conclusion, the present study reports proteomic information that is helpful in understanding the vector–pathogen relationship between C L as and D . citri , and could be used to identify potential targets for limiting the spread of C L as, as well as to provide new insights into HLB management.

ABSTRACT The high-quality draft genome of “ Candidatus Methanoperedens sp.” strain BLZ2, a nitrate-reducing archaeon anaerobically oxidizing methane, is presented. The genome was obtained from an enrichment culture and measures 3.74 Mb. It harbors two nitrate reductase gene clusters, an ammonium-forming nitrite reductase, and the complete reverse methanogenesis pathway. Methane that escapes to the atmosphere acts as a potent greenhouse gas. Global methane emissions are mitigated by methanotrophs, which oxidize methane to CO 2 . “ Candidatus Methanoperedens spp.” are unique methanotrophic archaea that can perform nitrate-dependent anaerobic oxidation of methane. A high-quality draft genome sequence of only 85 contigs from this archaeon is presented here.