Wei, Wei

In 2024, an infestation of corn leafhoppers, Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae), was detected in several commercial corn fields across Oklahoma (OK) (Faris et al. 2024). Corn hybrids in the infested field showed symptoms resembling maize bushy stunt, including chlorotic striping, reddening of leaves, production of multiple ears, and stunting. Visual disease incidence ranged from 25% to 100% among affected fields. Leaf samples from symptomatic plants were collected from 15 commercial corn fields across seven counties in OK (Payne, Logan, Caddo, Kay, Texas, Pottawatomie, and Grady) during September, October, and November of 2024. Each sample consisted of 3-4 symptomatic corn leaves randomly collected in each field. In addition, 43 corn leafhopper samples were collected from the symptomatic fields using handheld vacuums. Each corn leafhopper sample consisted of a minimum of 10 corn leafhoppers per field. In the laboratory, the corn leafhoppers were identified as D. maidis based on morphological characteristics as described (Faris et al. 2024). DNA from the 15 leaf samples was extracted from the leaf midrib and blade tissues using the DNeasy® Plant Mini Kit. Insect DNA was extracted using a CTAB-based method (Gorayeb et al. 2023), with each sample consisting of five pooled insects per field. DNA samples from plants and insects were initially screened using the R16F2n/R16R2 primer pair (Gundersen and Lee, 1996) for the presence of phytoplasmas. Seventeen insect and five plant samples tested positive for phytoplasma. Subsequent phytoplasma screening was performed on insect and plant samples collected from the same fields on the same dates, comprising a total of four insect and five plant samples. To further characterize the phytoplasma, a semi-nested PCR targeting the 16S rRNA gene was performed on these samples (four insect and five plant samples) using primer pairs P1/16S-SR followed by P1A/16S-SR (Wei et al. 2021), yielding an expected amplicon of approximately 1.5 Kb. An additional gene marker, the ribosomal protein (rp) gene, was also amplified using universal primers rpF1/rpR1 (Lee et al. 1998), producing 1.2 Kb PCR amplicons. PCR products were subjected to Sanger sequencing, and 16S rRNA and rp gene sequences were deposited in GenBank under accession numbers PX927497–PX927505 and PX993703-PX993711, respectively. BLASTn analysis of the 16S rRNA sequences showed 99.14–99.93% nucleotide identity with reference strains of ‘Candidatus Phytoplasma asteris’ associated with maize bushy stunt (accessions MW578284 and CP015149). Similarly, the rp gene sequences shared 99.91–100% nucleotide identity with the same reference strains. Using the iPhyClassifier tool (Zhao et al. 2013), the 16S rRNA gene sequences were classified within 16Sr group I, subgroup B, with a similarity coefficient of 1.0 (AP006628). The pairwise comparison and subsequent phylogenetic analysis showed that the OK isolates clustered within the 16SrI-B clade. This finding represents the first report of a 16SrI-B phytoplasma (‘Candidatus Phytoplasma asteris’) associated with maize bushy stunt in OK. Although currently classified as ‘Candidatus Phytoplasma asteris’, a proposal to establish a novel phytoplasma species, ‘Candidatus Phytoplasma zeae’, has been made based on ecological distinctiveness, vector specificity, whole-genome comparisons, and community consensus (Pellegrinetti et al. 2025).

‘Candidatus Phytoplasma trifolii’ is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches’ broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of ‘Candidatus Phytoplasma trifolii’-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.

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 % sequence identity with the reference strain but >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.

Abstract The reference strain of 'Ca. Phytoplasma trifolii' is the causative agent of clover proliferation (CP) disease of alsike clover (Trifolium hybridum). The CP disease was first reported in Canada in the early 1960s when the aetiological agent was mistakenly presumed to be a yellows-type virus (Chiykowski, 1965). Subsequent investigations revealed that the disease was associated with infection by a mycoplasma-like organism (Chen and Hiruki, 1975>; Hiruki and Chen, 1984), now termed phytoplasma, strain CPR (Hiruki and Wang, 2004). Later, phytoplasmas of the same lineage (subgroup 16SrVI-A) were found in the USA, Mexico, and many countries in Europe and Asia, causing diseases in diverse leguminous and vegetable crops, responsible for significant yield losses and quality reductions. Phytoplasmas of the same lineage also caused disease in elm trees in the USA. Phytoplasmas of closely-related lineages (various subgroups of group 16SrVI) also have wide distributions around the world.

Abstract Elm yellows develops in sporadic epidemics after introduction of 'Ca. Phytoplasma ulmi' to areas where vectors are abundant.