Plant Disease

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.

The phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb., and M. lucida [G.Forst.] Mabb.) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5′trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and quantitative real-time PCR (qPCR) on two to four occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded internal transcribed spacer (ITS) region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. To our knowledge, this is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine whether infections persist in the absence of D. citri.

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium “Candidatus Liberibacter spp.”, among which “Ca. Liberibacter africanus” (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent “Ca. Liberibacter asiaticus” (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of “Ca. Liberibacter solanacearum”, the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

Recurrent epiphytotics of X-disease, caused by ‘Candidatus Phytoplasma pruni,’ have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes and, more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multilocus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define “true” X-disease–inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extraorchard environment.

‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp. where it causes witches broom symptoms and papaya where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study two qPCR assays are developed, one utilizing high resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMana ssay successfully amplified isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI, 16SrIII and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting Tm products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C difference, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.