Publications

3514

The bacterial strain WB46 was isolated from the rhizosphere of willow plants (Salix purpurea L.) growing in soil contaminated with petroleum hydrocarbons. The strain was subjected to whole-genome shotgun sequencing using Illumina HiSeq. Its draft genome is 7.15 Mb, with a 69.55% GC content, containing 6387 protein-coding genes and 51 tRNA and 15 rRNA sequences. The quality and reliability of the genome were assessed using CheckM, attaining an estimated genome completeness of 98.75% and an estimated contamination of 1.68%. These results indicate a high-quality genome (>95%) and low contamination (<5%). Many of these genes are responsible for petroleum hydrocarbon degradation, such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo). 16S rRNA gene analysis, including in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI), showed that strain WB46 belongs to the genus Nocardia, and the most closely related species is Nocardia asteroides. The strain WB46 showed a distance of 63.4% and sequence identity of 88.63%, respectively. These values fall below the threshold levels of 70% and 95%, respectively, suggesting that the strain WB46 is a new species. We propose the name of Nocardia canadensis sp. nov. for this new species. Interestingly, the sequence divergence of the 16S rRNA gene showed that the divergence only occurred in the V2 region. Therefore, the conventional V3–V4, V5–V7, or V8–V9 targeting metabarcoding, among others, would not be able to assess the diversity related to this new species.

The digestive organs of terrestrial isopods harbor bacteria of the recently proposed mollicute family Hepatoplasmataceae. The only complete genome available so far for Hepatoplasmataceae is that of “Candidatus Hepatoplasma crinochetorum”. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepoatoplamsataceae members: “Candidatus Tyloplasma littoralis” identified from the semiterrestrial isopod Tylos granuliferus, “Candidatus Hepatoplasma vulgare” identified from the common pill bug Armadillidium vulgare, and “Candidatus Hepatoplasma scaber” identified from the common rough woodlouse Porcellio scaber. Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. 16S ribosomal RNA gene sequences and phylogenomic analysis showed that “Candidatus Tyloplasma littoralis” and other semiterrestrial isopod-associated mollicutes represent the placeholder genus “g_Bg2” in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harboring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.

As hemp ( Cannabis sativa) emerges as a commercial crop in Nevada, mild to severe abnormal growth has been observed from many plants in commercial fields throughout the growing season. Affected plants exhibited a group of symptoms typically including stunting, leaf yellowing, excessive apical branching, clusters of witches’ broom, leaf rolling upwards, and leaf mottling and mosaic. These symptoms mostly showed up together in a plant or a crop and are defined here as abnormal growth syndrome (AGS). Between 2017 and 2019, the Nevada State Plant Pathology and Molecular Diagnostic Lab received 67 symptomatic hemp samples from Nevada for diagnosis, and ‘ Candidatus Phytoplasma trifolii’ was detected in 14 samples (21%). To investigate additional biotic agents associated with AGS, PCR products generated by primers P1/P7 were cloned into a pGEM-T vector and sequenced, and Spiroplasma citri DNA was found in two samples (3%). All 67 DNA samples were further tested for beet curly top virus (BCTV), and 57 samples (85%) were found to be infected by BCTV. Twelve samples (18%) were coinfected by both ‘ Ca. Phytoplasma trifolii’ and BCTV, and two (3%) were coinfected by S. citri, ‘ Ca. Phytoplasma trifolii’, and BCTV. The findings suggest that BCTV is the most prevalent pathogen causing the hemp abnormal growth in Nevada, but ‘ Ca. Phytoplasma trifolii’ and S. citri may also contribute to the severity and complexity of symptoms. Thus, hemp abnormal growth can be attributed to single, dual, or triple infections of these three leafhopper-vectored mollicutes and virus. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Phosphatidylcholine (PtdCho) is an unusual membrane phospholipid present in some endosymbiotic and intracellular pathogenic prokaryotes. ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem-limited, uncultured, fastidious α-Proteobacterium associated with the devastating citrus “greening” disease (huanglongbing). Phylogenetically related but nonpathogenic Liberibacter crescens (Lcr) was used as a culturable surrogate to examine PtdCho biosynthesis in pathogenic CLas. Genes encoding key enzymes for two alternative PtdCho biosynthetic routes are present in the Lcr genome: the one-step cytidine diphosphate (CDP)-choline ( pcs-encoding phosphatidylcholine synthase) and the three-step methyl-transferase pathway ( pmt-encoding phospholipid N-methyltransferase). However, only the CDP-choline pathway genes for incorporating exogenous Cho were identified in the CLas genome. Exogenous Cho enhanced growth and alleviated osmotic stress in wild-type Lcr and in the pmt insertion mutant strains when cultured in a sugar-rich medium. Quantitative RT-PCR analyses confirmed active uptake and condensation of nutritional Cho into PtdCho by CLas in both its plant host and psyllid vector. CLas-infected grapefruit leaves showed transcriptional activation of Cho biosynthesis genes and 2.8-fold higher levels of Cho. In plant cells, the compatible osmolyte glycine-betaine (GlyBet) is also derived from Cho. Expression of GlyBet biosynthesis genes and the GlyBet content were similar in both CLas-infected and healthy leaf tissue. The data presented here suggest that CLas likely exploits the Cho biosynthetic pathway in citrus hosts to expand the nutritional Cho pool. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

Two phloem-limited pathogens, ‘Candidatus Arsenophonus phytopathogenicus’ and ‘Candidatus Phytoplasma solani’, threaten sugar beet production in France, Switzerland, and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to ‘Ca. P. solani’ is found predominant in Saxony-Anhalt, unlike in France, where ‘Ca. P. solani’ has a minor role compared with ‘Ca. A. phytopathogenicus’. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The multilocus sequence analysis (MLSA) of nonribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported ‘Ca. P. solani’ strains including the strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020 and also in Bavaria in southern Germany. Based on 16S rDNA analysis, ‘Ca. A. phytopathogenicus’ in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany suggest that more attention should be directed toward understanding phytoplasma infection in sugar beet in this country.