First Report of Candidatus phytoplasma asteris-related strains (subgroup 16SrI-A) Associated with Aster Yellows on Chrysanthemums in Oklahoma.

In November and December of 2023, samples of cultivars of chrysanthemums (Chrysanthemum morifolium) from a commercial greenhouse facility within the state were submitted to the Oklahoma State University (OSU) Plant Disease and Insect Diagnostic Laboratory (Stillwater, OK). More than 300 symptomatic and asymptomatic stems with flowers and flower buds were submitted. Symptomatic samples were like those reported for aster yellows on multiple plant species, with visible phyllody and virescence. A total of 238 cultivars were processed for phytoplasma detection. Initially, samples were processed in batches based on cultivars. Each batch was made up of 3-9 flowers and/or flower buds from plants belonging to the same cultivar. Genomic DNA was extracted from batched samples using Qiagen DNeasy Plant Mini Kit (Qiagen Inc., MD) following the manufacturer's instruction. A qPCR assay, using 18S gene as internal control and 23S gene for phytoplasma detection was used to confirm the presence of phytoplasma (Oberhansli et al. 2011). The number of composite samples that tested positive was 67. A nested PCR assay using P1/P7 followed by R16F2n/R16R2 primers (Gundersen and Lee 1996; Smart et al. 1996; Lee et al. 1993; Lee et al. 1998) was used to retest and confirm batched samples that tested positive. PCR products were purified and sequenced at the OSU DNA Core Facility using Sanger Sequencing. A BLAST search of generated sequences on NCBI database confirmed the sequences as aster yellows phytoplasma sequences. To confirm individual plant materials from batches that were positive for aster yellows, DNA extracted from 12 samples representing different cultivars. PCR was carried out using the P1/P7 primers, followed by R16F2n/R16R2 primers as described previously. Another PCR using primer pair L15F1/MapR1 was used to amplify the partial spc operon that includes the complete secY gene (Lee et al. 2010). All PCR products were purified and sequenced at the OSU DNA Core Facility using Sanger Sequencing. BioEdit (https://thalljiscience.github.io/ ) was used to trim and generate consensus sequences of the forward and reverse sequences of all sequenced strains. All sequences have been deposited on the NCBI database (Accession numbers: PP539932-PP539940 and PQ249170-PQ249174 for 16SrRNA and secY genes respectively). The 16S rRNA sequences of strains share between 99.71-99.88% (query coverage= 99-100%, E-value= 0.0) while the secY gene share 98.33-99.49% (query coverage=100%, E-value=0.0) similarity to the 16S rRNA and secY genes of Aster yellows witches'-broom phytoplasma (CP000061.1; Bai et al. 2006). In silico iPhyClassifier 16Sr group/subgroup analyses (Wei et al. 2007; Zhao et al. 2009) showed that these strains belonged to the Candidatus phytoplasma asteris- related strains (subgroup 16SrI-A). Although aster yellows have been reported on other crops in Oklahoma, to our knowledge, this is the first report and confirmation of Candidatus phytoplasma asteris- related strains (subgroup 16SrI-A) causing aster yellows on chrysanthemums in Oklahoma. Considering that this was recovered in one of the major cut flowers growing facilities serving Oklahoma and other states, a concerted effort is required for improved surveillance to mitigate spread of this pathogen.