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Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.

Anaerobic methane metabolism is among the hallmarks of Archaea, originating very early in their evolution. Here, we show that the ancestor of methane metabolizers was an autotrophic CO 2 -reducing hydrogenotrophic methanogen that possessed the two main complexes, methyl-CoM reductase (Mcr) and tetrahydromethanopterin-CoM methyltransferase (Mtr), the anaplerotic hydrogenases Eha and Ehb, and a set of other genes collectively called “methanogenesis markers” but could not oxidize alkanes. Overturning recent inferences, we demonstrate that methyl-dependent hydrogenotrophic methanogenesis has emerged multiple times independently, either due to a loss of Mtr while Mcr is inherited vertically or from an ancient lateral acquisition of Mcr. Even if Mcr is lost, Mtr, Eha, Ehb, and the markers can persist, resulting in mixotrophic metabolisms centered around the Wood-Ljungdahl pathway. Through their methanogenesis remnants, Thorarchaeia and two newly reconstructed order-level lineages in Archaeoglobi and Bathyarchaeia act as metabolically versatile players in carbon cycling of anoxic environments across the globe.

The Asian citrus psyllid, Diaphorina citri, is an invasive insect and a vector of ‘ Candidatus Liberibacter asiaticus’ ( CLas), a bacterium whose growth in Citrus species results in huanglongbing (HLB), also known as citrus greening disease. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification and nevertheless contain significant quantities of host DNA. To overcome these hurdles, we developed a simple pretreatment DNase and filtration (PDF) protocol to remove host DNA and directly sequence CLas and the complete, primarily uncultivable microbiome from D. citri adults. The PDF protocol yielded CLas abundances upward of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids. The PDF protocol confirmed our lab strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this method for bacterial strain tracking. CLas genetic polymorphisms arising in lab-reared psyllid populations included prophage-encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest that laboratory propagation of CLas could result in different phenotypic trajectories among laboratories and could confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can directly inform agricultural management strategies related to bacterial strain tracking, insect microbiome surveillance, and antibiotic resistance screening.

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 .