Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.

Evaluating Nicotinamide Adenine Dinucleotide for Its Effects on Halo Blight of Snap Bean.

Halo blight, caused by Pseudomonas syringae pv. phaseolicola, is one of the major bacterial diseases on snap bean in Florida, and the outbreaks of this disease have occurred more often in recent years. Current management of this disease primarily depends on application of fixed copper-based bactericides but climate change and resistance development in the pathogen populations still cause hardship for management of this disease, especially in south Florida. In this study, nicotinamide adenine dinucleotide (NAD+) was evaluated in the greenhouse for its potential to reduce halo blight on snap bean. When NAD+ at 5 mM was applied by soil drench, foliar spray, or leaf infiltration, NAD+ significantly (P < 0.05) reduced disease severity of halo blight on snap bean compared with the untreated control. When NAD+ was applied by leaf infiltration, among the tested concentrations, NAD+ at 0.5 to 1.0 mM was most effective in decreasing halo blight disease. NAD+ at 2.5 mM applied as a foliar spray in rotation with Kocide 3000 (copper hydroxide) at 0.5 mg/ml further reduced disease severity compared with Kocide 3000 alone. In the in vitro study, no inhibitory effects of NAD+ were detected on the bacterial pathogen P. syringae pv. phaseolicola. Results of real-time PCR showed that the defense-related genes PR1, AZI1, EDS1, SARD1, PDF1.2, and PAL1 were upregulated in the NAD+ treatment. Taken together, these data indicated that NAD+ significantly suppressed halo blight on snap bean, and application of NAD+ has the potential in management of this important disease.

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.

Genomic Inference of Recombination-Mediated Evolution in Xanthomonas euvesicatoria and X. perforans.

Recombination is a major driver of evolution in bacterial populations, because it can spread and combine independently evolved beneficial mutations. Recombinant lineages of bacterial pathogens of plants are typically associated with the colonization of novel hosts and the emergence of new diseases. Here we show that recombination between evolutionarily and phenotypically distinct plant-pathogenic lineages generated recombinant lineages with unique combinations of pathogenicity and virulence factors. Xanthomonas euvesicatoria and Xanthomonas perforans are two closely related lineages causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of atypical strains collected from tomato in Nigeria and observed recombination in the type III secretion system and effector genes, which showed alleles from both X. euvesicatoria and X. perforans Wider horizontal gene transfer was indicated by the fact that the lipopolysaccharide cluster of one strain was most similar to that of a distantly related Xanthomonas pathogen of barley. This strain and others have experienced extensive genomewide homologous recombination, and both species exhibited dynamic open pangenomes. Variation in effector gene repertoires within and between species must be taken into consideration when one is breeding tomatoes for disease resistance. Resistance breeding strategies that target specific effectors must consider possibly dramatic variation in bacterial spot populations across global production regions, as illustrated by the recombinant strains observed here.IMPORTANCE The pathogens that cause bacterial spot of tomato and pepper are extensively studied models of plant-microbe interactions and cause problematic disease worldwide. Atypical bacterial spot strains collected from tomato in Nigeria, and other strains from Italy, India, and Florida, showed evidence of genomewide recombination that generated genetically distinct pathogenic lineages. The strains from Nigeria and Italy were found to have a mix of type III secretion system genes from X. perforans and X. euvesicatoria, as well as effectors from Xanthomonas gardneri These genes and effectors are important in the establishment of disease, and effectors are common targets of resistance breeding. Our findings point to global diversity in the genomes of bacterial spot pathogens, which is likely to affect the host-pathogen interaction and influence management decisions.

Multilocus sequence analysis of xanthomonads causing bacterial spot of tomato and pepper plants reveals strains generated by recombination among species and recent global spread of Xanthomonas gardneri.

Four Xanthomonas species are known to cause bacterial spot of tomato and pepper, but the global distribution and genetic diversity of these species are not well understood. A collection of bacterial spot-causing strains from the Americas, Africa, Southeast Asia, and New Zealand were characterized for genetic diversity and phylogenetic relationships using multilocus sequence analysis of six housekeeping genes. By examining strains from different continents, we found unexpected phylogeographic patterns, including the global distribution of a single multilocus haplotype of X. gardneri, possible regional differentiation in X. vesicatoria, and high species diversity on tomato in Africa. In addition, we found evidence of multiple recombination events between X. euvesicatoria and X. perforans. Our results indicate that there have been shifts in the species composition of bacterial spot pathogen populations due to the global spread of dominant genotypes and that recombination between species has generated genetic diversity in these populations.