Publications

4606

Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid, and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific loop-mediated isothermal amplification (LAMP) assay for detection of a distinct strain of grapevine ‘Candidatus Phytoplasma asteris’ that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted the ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South Africa, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

AbstractThe mutualistic interactions between Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone (short Endoriftia) have been extensively researched. However, the closed Endoriftia genome is still lacking. Here, by employing single‐molecule real‐time sequencing we present the closed chromosomal sequence of Endoriftia. In contrast to theoretical predictions of enlarged and mobile genetic element‐rich genomes related to facultative endosymbionts, the closed Endoriftia genome is streamlined with fewer than expected coding sequence regions, insertion‐, prophage‐sequences and transposase‐coding sequences. Automated and manually curated functional analyses indicated that Endoriftia is more versatile regarding sulphur metabolism than previously reported. We identified the presence of two identical rRNA operons and two long CRISPR regions in the closed genome. Additionally, pangenome analyses revealed the presence of three types of secretion systems (II, IV and VI) in the different Endoriftia populations indicating lineage‐specific adaptations. The in depth mobilome characterization identified the presence of shared genomic islands in the different Endoriftia drafts and in the closed genome, suggesting that the acquisition of foreign DNA predates the geographical dispersal of the different endosymbiont populations. Finally, we found no evidence of epigenetic regulation in Endoriftia, as revealed by gene screenings and absence of methylated modified base motifs in the genome. As a matter of fact, the restriction‐modification system seems to be dysfunctional in Endoriftia, pointing to a higher importance of molecular memory‐based immunity against phages via spacer incorporation into CRISPR system. The Endoriftia genome is the first closed tubeworm endosymbiont to date and will be valuable for future gene oriented and evolutionary comparative studies.

AbstractCable bacteria of theDesulfobulbaceaefamily are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera:CandidatusElectronema, typically found in freshwater environments, andCandidatusElectrothrix, typically found in saltwater environments. This taxonomic framework is based on both 16S rRNA gene sequences and metagenome-assembled genome (MAG) phylogenies. However, most of the currently available MAGs are highly fragmented, incomplete, and thus likely miss key genes essential for deciphering the physiology of cable bacteria. To address this, we performed Nanopore long read (total 162.4 Gbp) and Illumina short read (total 148.3 Gbp) shotgun sequencing of selected environmental samples and a single-strain enrichment ofCa. Electronema aureum. We recovered multiple cable bacteria MAGs, including two circular and one single-contig. Phylogenomic analysis, also confirmed by 16S rRNA gene-based phylogeny, classified one circular MAG and the single-contig MAG as novel species of cable bacteria, which we propose to nameCa. Electronema halotolerans andCa. Electrothrix laxa, respectively. TheCa. Electronema halotolerans, despite belonging to the previously recognized freshwater genus of cable bacteria, was retrieved from brackish-water sediment. Metabolic predictions showed several adaptations to a high salinity environment, similar to the “saltwater”Ca. Electrothrix species, indicating howCa. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.

One highly diverse phylogenetic group of Bacteria, Ca . Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya.

An outbreak of citrus greening or Huanglongbing disease bacteria occurs in many areas. We sampled and identified an ongoing ~year 2020 orange tree endemic in northern Thailand as Candidatus Liberibacter asiaticus. We thereby developed a plant greening disease (C. Liberibacter asiaticus) detection assay using simple alkaline heat DNA lysis and loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB), and evaluated the developed assay for its feasibility as point-of-care detection on 65 plant leaf samples with 100–1×104 copies of C. Liberibacter asiaticus or mocked injection compared with commercial DNA lysis kit and PCR-GE. Our assay is sensitive to 5–8.9 copies of omp (equaling 0.0056–0.01 fg) compatible with PCR-GE limit of detection. This ultra sensitive limit of detection could allow the disease detection before clinical apparent state of disease when C. Liberibacter asiaticus infection number is few, i.e. fewer than 100 copies of C. Liberibacter asiaticus. The assay is also specific with 6 degenerate primers targeting every strain of C. Liberibacter asiaticus omp from GenBank database, rapid (40 min total assay time), inexpensive (~2–3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay accuracy (93.85–100% accuracy, 100% specificity, and 89.74–100% sensitivity) to bacterial DNA extraction by a commercial kit followed by PCR and gel electrophoresis (92.31% accuracy, 100% specificity, and 87.18% sensitivity) based on the real sample tests. Hence, the technique could be used in local or laboratory resource-restricted settings. The test result could be read by naked eyes through the color change from violet (negative) to sky blue (positive) for a C. Liberibacter asiaticus-infected specimen. Furthermore, this assay uses safe chemical reagents and, thus, is safe for the users.