Publications

4366

Strain IT6T, a thermoacidophilic and facultative methane-oxidizing bacterium, was isolated from a mud–water mixture collected from Pisciarelli hot spring in Pozzuoli, Italy. The novel strain is white when grown in liquid or solid media and forms Gram-negative rod-shaped, non-flagellated, non-motile cells. It conserves energy by aerobically oxidizing methane and hydrogen while deriving carbon from carbon dioxide fixation. Strain IT6T had three complete pmoCAB operons encoding particulate methane monooxygenase and genes encoding group 1d and 3b [NiFe] hydrogenases. Simple carbon–carbon substrates such as ethanol, 2-propanol, acetone, acetol and propane-1,2-diol were used as alternative electron donors and carbon sources. Optimal growth occurred at 50–55°C and between pH 2.0–3.0. The major fatty acids were C18 : 0, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 and C14 : 0, and the main polar lipids were phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, diphosphatidylglycerol, some unidentified phospholipids and glycolipids, and other unknown polar lipids. Strain IT6T has a genome size of 2.19 Mbp and a G+C content of 40.70 mol%. Relative evolutionary divergence using 120 conserved single-copy marker genes (bac120) and phylogenetic analyses based on bac120 and 16S rRNA gene sequences showed that strain IT6T is affiliated with members of the proposed order ‘Methylacidiphilales’ of the class Verrucomicrobiia in the phylum Verrucomicrobiota . It shared a 16S rRNA gene sequence identity of >96 % with cultivated isolates in the genus ' Methylacidiphilum ' of the family 'Methylacidiphilaceae’, which are thermoacidophilic methane-oxidizing bacteria. ‘ Methylacidiphilum sp.’ Phi (100 %), ‘Methylacidiphilum infernorum’ V4 (99.02 %) and ‘ Methylacidiphilum sp.’ RTK17.1 (99.02 %) were its closest relatives. Its physiological and genomic properties were consistent with those of other isolated ‘ Methylacidiphilum ’ species. Based on these results, we propose the name Methylacidiphilum caldifontis gen. nov., sp. nov. to accommodate strain IT6T (=KCTC 92103T=JCM 39288T). We also formally propose that the names Methylacidiphilaceae fam. nov. and Methylacidiphilales ord. nov. to accommodate the genus Methylacidiphilum gen. nov.

Phytoplasmas are phloem-limited plant pathogens, such as sugarcane white leaf (SCWL) phytoplasma, which are responsible for heavy economic losses to the sugarcane industry. Characterization of phytoplasmas has been limited because they cannot be cultured in vitro. However, with the advent of genome sequencing, different aspects of phytoplasmas are being investigated. In this study, we developed a DNA enrichment method for sugarcane white leaf (SCWL) phytoplasma, evaluated the effect of DNA enrichment via Illumina sequencing technologies, and utilized Illumina and Nanopore sequencing technologies to obtain the complete genome sequence of the “Candidatus Phytoplasma sacchari” isolate SCWL1 that is associated with sugarcane white leaf in China. Illumina sequencing analysis elucidated that only 1.21% of the sequencing reads from total leaf DNA were mapped to the SCWL1 genome, whereas 40.97% of the sequencing reads from the enriched DNA were mapped to the SCWL1 genome. The genome of isolate SCWL1 consists of a 538,951 bp and 2976 bp long circular chromosome and plasmid, respectively. We identified 459 protein-encoding genes, 2 complete 5S-23S-16S rRNA gene operons, 27 tRNA genes, and an incomplete potential mobile unit (PMU) in the circular chromosome. Phylogenetic analyses and average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values based on the sequenced genome revealed that SCWL phytoplasma and sugarcane grassy shoot (SCGS) phytoplasma belonged to the same phytoplasma species. This study provides a genomic DNA enrichment method for phytoplasma sequencing. Moreover, we report the first complete genome of a “Ca. Phytoplasma sacchari” isolate, thus contributing to future studies on the evolutionary relationships and pathogenic mechanisms of “Ca. Phytoplasma sacchari” isolates.

AbstractRecent outbreaks of ‘Candidatus Phytoplasma solani’ resulted in severe losses in potatoes, vegetable crops and grapevines in certain regions of Austria and constituted a major challenge for seed potato production. Therefore, the effects of various insecticides and insect deterrents on pathogen spread were studied both in laboratory and field experiments from 2018 to 2021. In laboratory transmission experiments, field captured Hyalesthes obsoletus were caged on differently treated Catharanthus roseus for five days. The insecticides lambda-cyhalothrin, deltamethrin, esfenvalerate, acetamiprid and chlorpyriphos showed the most rapid impact on insect survival and fully prevented phytoplasma transmission. The particle film forming products kaolin and diatomaceous earth had some effect. A transfer of the promising laboratory results to potato fields, however, was achieved to a limited extent only. Treatments with pyrethroids and acetamiprid every 8–10 days over the flight period of H. obsoletus roughly halved the number of symptomatic plants and tubers in case of moderately susceptible varieties and moderate infection pressure. In the event of susceptible varieties and high disease pressure, treatment effects were hardy discernible. In practical terms, the experiments indicate that insecticide applications alone are not sufficient to mitigate the disease. Spraying of diatomaceous earth and mineral oil did not affect disease incidence in the field.

AbstractAnaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by ‘Candidatus Methanoperedens nitroreducens’, an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that ‘Ca. M. nitroreducens’ uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.