The genus ‘Candidatus Phytoplasma’ was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus ‘Ca. Phytoplasma’, the proposed guidelines were revised and clarified to accommodate those ‘Ca. Phytoplasma’ species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing 98.65 % with other strain(s) within the same ‘Ca. Phytoplasma’ species should be considered related strains to that ‘Ca. Phytoplasma’ species. The guidelines herein, keep the original published reference strains. However, to improve ‘Ca. Phytoplasma’ species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of ‘Ca. Phytoplasma’ species and alternative reference strains described are reported. For new ‘Ca. Phytoplasma’ species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published ‘Candidatus Phytoplasma’ species, including ‘Ca. P. cocostanzaniae’ and ‘Ca. P. palmae’ described in this manuscript.

Quantitative PCR (qPCR), loop-mediated amplification (LAMP), and lateral flow strip-based recombinase polymerase amplification (RPA-LFS) assays were assessed for early detection of Phytophthora infestans, the global causal agent of potato and tomato late blight, on passive wind-powered spore traps known as Spornados. Spore traps were deployed in potato and tomato fields during the 2018, 2019, and 2020 growing seasons in the provinces of Alberta, British Columbia, Manitoba, Prince Edward Island, and Ontario. All assays used DNA extracts from Spornado cassette membranes targeting the P. infestans nuclear ribosomal internal transcribed spacer. A total of 1,003 Spornado samples were qPCR tested, yielding 115 positive samples for P. infestans spores. In further assessment of these samples, LAMP detected P. infestans in 108 (93.9%) of 115 qPCR positive samples, and RPA-LFS detected it in 103 (89.6%). None of the assays showed cross-reaction with other Phytophthora species or pathogenic fungi known to infect potato and tomato. The qPCR detected ≤1 fg of P. infestans DNA, and LAMP and RPA-LFS amplified 10 fg in as little as 10 min. All assays detected P. infestans before the first report of late blight symptoms in commercial potato or tomato fields within each region or province. The combination of Spornado passive samplers with qPCR, LAMP, or RPA-LFS proved a valuable spore trapping system for early surveillance of late blight in potato and tomato. Both LAMP and RPA-LFS showed potential as alternative approaches to qPCR for in-field monitoring of P. infestans.

Peach trees exhibiting peach rosette-like disease symptoms and infected by a phytoplasma of group 16SrI ‘Candidatus Phytoplasma asteris’ at the Canadian Clonal Genebank were further tested for the pathogen characterization based on the 16S rRNA gene. Nested PCR with phytoplasma universal primers R16mF2/R1 and R16F2n/R2 resulted in amplification of products of approximately 1.25 kb from all four symptomatic trees tested. Virtual RFLP of the R16F2n/R2 sequenced amplicons with selected restriction endonucleases showed unique RFLP patterns when compared to the described 16SrI phytoplasma subgroups; these data were confirmed by phylogenetic analyses. The phytoplasma was therefore assigned as a member of a new 16SrI subgroup (16SrI-W). Results represent the first report of a new phytoplasma 16SrI subgroup infecting peach in Canada, and provide a valuable tool for further epidemiological studies on this phytoplasma in peach.

Coconut farms located in the southern coast of Grand-Lahou in Côte d’Ivoire are severely affected by a lethal yellowing disease (CILY) associated with the group 16SrXXII-B, ‘Candidatus Phytoplasma palmicola’-related strains. Given the high prevalence of weed species on most of the farms, plants growing within and in the periphery of five selected coconut farms were assessed for the presence of the CILY phytoplasma to identify potential alternative hosts. A total of 396 plant samples belonging to 84 plant species and 35 botanical families were collected. Total DNA was extracted and tested by nested PCR with primers targeting the 16S rRNA and the translocation protein (secA) phytoplasma genes, and sequenced. Twenty samples from six plant species and five botanical families yielded PCR amplicons of the expected size, and both the secA and 16S rDNA sequences showed over 99% similarity with that of the Côte d’Ivoire lethal yellowing phytoplasma previously identified from coconut palms grown in Grand-Lahou coconut farms. Plant species from the families Poaceae (Paspalum vaginatum, Pennisetum pedicillatum), Verbenaceae (Stachytarpheta indica), Plantaginaceae (Scoparia dulcis), Phyllanthaceae (Phyllantus muellerianus) and Cyperacea (Diplacrum capitatum) were positive for the presence of the CILY phytoplasma, suggesting they may have epidemiological implications for disease spread in coconut farms in Grand-Lahou.