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.

Potential and Metabolic Pathways of Eugenol in the Management of Xanthomonas perforans, a Pathogen of Bacterial Spot of Tomato.

Bacterial spot of tomato continues to pose a significant problem to tomato production worldwide. In Florida, bacterial spot of tomato caused by Xanthomonas perforans is one of the most important diseases responsible for tomato yield loss. This disease is difficult to control, and new strategies are continually being investigated to combat the devastating effect of this disease. Recent efforts focusing on essential oils based on small molecules have spurred interests in the utilization of this class of chemicals for disease management. In this study, we evaluated the efficacy of eugenol for the management of bacterial spot of tomato caused by X. perforans. In the greenhouse experiments, eugenol applied as a foliar spray significantly (p < 0.5) reduced bacterial spot disease compared to the untreated control. In the field experiments, the area under the disease progress curve (AUDPC) was significantly (p < 0.5) lower in the plots treated with eugenol or eugenol combined with the surfactant Cohere than in the untreated control plots, and it was comparable to the copper-based treatments. To provide additional insights into the possible pathways of eugenol activities, we applied a liquid chromatography mass spectrometry (LC-MS)-based metabolomic study using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) on X. perforans strain 91−118 treated with eugenol. Our results showed that eugenol affected metabolite production in multiple pathways critical to bacterial survival. For example, treatment of cells with eugenol resulted in the downregulation of the glutathione metabolism pathway and associated metabolites, except for 5-oxoproline, which accumulation is known to be toxic to living cells. While the peaks corresponding to the putatively identified sarmentosin showed the most significant impact and reduced in response to eugenol treatment, branched-chain amino acids, such as L-isoleucine, increased in production, suggesting that eugenol may not negatively affect the protein biosynthesis pathways. The results from our study demonstrated the efficacy of eugenol in the management of bacterial spot of tomato under greenhouse and field conditions and identified multiple pathways that are targeted.

Lasiodiplodia iraniensis, a New Causal Agent of Tuber Rot on Yam (Dioscorea Species) Imported into the United States and Implications for Quarantine Decisions.

One negative consequence of international trade of agricultural commodities is the inadvertent global spread of crop diseases. Yam (Dioscorea spp.) is a staple food crop in many countries and is traded globally. Most of the commercially traded yams in the United States are imported. In late 2020, samples of yam tubers from a commercial facility were submitted to the plant diagnostic clinic at the UF/IFAS Tropical Research and Education Center in Homestead, Florida. Samples showed rotten symptoms and were drawn from lots that were marked to be destroyed because the source of the rotting symptoms was unknown. Preliminary isolation showed that a fungus was consistently associated with the symptoms and was confirmed in the subsequent pathogenicity test as the causal agent. The fungus grew profusely on potato dextrose agar (PDA) with highly melanized hyphae. Matured conidia showed longitudinal striations. Based on its growth pattern and morphology, it was suspected that this fungus may be in the genus Lasiodiplodia. DNA-based identification using partial sequences of the internal transcribed spacer (ITS), ß-tubulin (TUB2), 28S rDNA (LSU), and elongation factor alpha (EF1-α) genes confirmed the identity of the isolates as Lasiodiplodia iraniensis Abdollahz., Zare & A.J.L. Phillips (synonym: L. iranensis). This is the first report of L. iraniensis affecting yam and has implications for international trade. This finding will provide an important foundation for making quarantine decisions to prevent spread of this disease.

Bacterial Spot of Tomato and Pepper in Africa: Diversity, Emergence of T5 Race, and Management.

Bacterial spot disease was first reported from South Africa by Ethel M. Doidge in 1920. In the ensuing century after the initial discovery, the pathogen has gained global attention in plant pathology research, providing insights into host-pathogen interactions, pathogen evolution, and effector discovery, such as the first discovery of transcription activation-like effectors, among many others. Four distinct genetic groups, including Xanthomonas euvesicatoria (proposed name: X. euvesicatoria pv. euvesicatoria), Xanthomonas perforans (proposed name: X. euvesicatoria pv. perforans), Xanthomonas gardneri (proposed name: Xanthomonas hortorum pv. gardneri), and Xanthomonas vesicatoria, are known to cause bacterial spot disease. Recently, a new race of a bacterial spot pathogen, race T5, which is a product of recombination between at least two Xanthomonas species, was reported in Nigeria. In this review, our focus is on the progress made on the African continent, vis-à-vis progress made in the global bacterial spot research community to provide a body of information useful for researchers in understanding the diversity, evolutionary changes, and management of the disease in Africa.

Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol.

Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; ß-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems.