Rapid detection of viral, bacterial, fungal, and oomycete pathogens on tomato with microneedles, LAMP on a microfluidic chip, and smartphone device.

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles (MN) and combined these with LAMP assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated and disease severity was measured. Extractions were performed using MN and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, while P. infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid MN extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.

Reconstructing historic and modern potato late blight outbreaks using text analytics.

In 1843, a hitherto unknown plant pathogen entered the US and spread to potato fields in the northeast. By 1845, the pathogen had reached Ireland leading to devastating famine. Questions arose immediately about the source of the outbreaks and how the disease should be managed. The pathogen, now known as Phytophthora infestans, still continues to threaten food security globally. A wealth of untapped knowledge exists in both archival and modern documents, but is not readily available because the details are hidden in descriptive text. In this work, we (1) used text analytics of unstructured historical reports (1843-1845) to map US late blight outbreaks; (2) characterized theories on the source of the pathogen and remedies for control; and (3) created modern late blight intensity maps using Twitter feeds. The disease spread from 5 to 17 states and provinces in the US and Canada between 1843 and 1845. Crop losses, Andean sources of the pathogen, possible causes and potential treatments were discussed. Modern disease discussion on Twitter included near-global coverage and local disease observations. Topic modeling revealed general disease information, published research, and outbreak locations. The tools described will help researchers explore and map unstructured text to track and visualize pandemics.

Evaluation of a Formulation of Bacillus subtilis for Control of Phytophthora Blight of Bell Pepper.

Phytophthora blight, caused by Phytophthora capsici, is one of the most economically significant diseases of bell pepper in the United States. Over the past several decades, isolates of P. capsici exhibiting resistance to mefenoxam and other fungicides have been reported. Fungicide resistance coupled with an increased market for organically grown crops has led to interest in biological control as a disease management option. In this work, an isolate of Bacillus subtilis (AFS032321) was evaluated for control of Phytophthora blight of bell pepper in the greenhouse and field. A 28% active ingredient wettable powder formulation of the strain was applied as a soil drench at transplanting prior to inoculation. Treatment with this formulation of B. subtilis significantly reduced the area under the disease progress curve (AUDPC) by up to 52% compared to untreated control plants in greenhouse tests. Comparisons between applying the biocontrol weekly after seeding for 5 weeks versus a single application at transplanting (5 weeks) indicated no significant benefits of additional applications. The formulation of B. subtilis reduced disease caused by a mefenoxam-resistant isolate of P. capsici, while mefenoxam failed. The biocontrol efficacy of formulated strains was not affected in different soil types or potting media. However, disease was more severe in sandy soils. In field experiments that were conducted with a mefenoxam-sensitive isolate, disease incidence and severity of Phytophthora blight were significantly reduced at all rates of B. subtilis in 2019 except the 16.8 kg ha-1 rate. In both years, mefenoxam was more effective than B. subtilis in controlling disease in the field. B. subtilis did not affect the spatial dynamics of pathogen spread within rows. While the precise mechanism(s) of action is unclear, in vitro dual-culture tests suggest direct antagonism, as B. subtilis significantly inhibited colony growth of P. capsici. AgBiome has recently released a new formulation of the AFS032321 strain named Theia, with higher active ingredients for commercial applications and biocontrol of P. capsici.

Abaxial leaf surface-mounted multimodal wearable sensor for continuous plant physiology monitoring.

Wearable plant sensors hold tremendous potential for smart agriculture. We report a lower leaf surface-attached multimodal wearable sensor for continuous monitoring of plant physiology by tracking both biochemical and biophysical signals of the plant and its microenvironment. Sensors for detecting volatile organic compounds (VOCs), temperature, and humidity are integrated into a single platform. The abaxial leaf attachment position is selected on the basis of the stomata density to improve the sensor signal strength. This versatile platform enables various stress monitoring applications, ranging from tracking plant water loss to early detection of plant pathogens. A machine learning model was also developed to analyze multichannel sensor data for quantitative detection of tomato spotted wilt virus as early as 4 days after inoculation. The model also evaluates different sensor combinations for early disease detection and predicts that minimally three sensors are required including the VOC sensors.

An open-access T-BAS phylogeny for emerging Phytophthora species.

Phytophthora species cause severe diseases on food, forest, and ornamental crops. Since the genus was described in 1876, it has expanded to comprise over 190 formally described species. There is a need for an open access phylogenetic tool that centralizes diverse streams of sequence data and metadata to facilitate research and identification of Phytophthora species. We used the Tree-Based Alignment Selector Toolkit (T-BAS) to develop a phylogeny of 192 formally described species and 33 informal taxa in the genus Phytophthora using sequences of eight nuclear genes. The phylogenetic tree was inferred using the RAxML maximum likelihood program. A search engine was also developed to identify microsatellite genotypes of P. infestans based on genetic distance to known lineages. The T-BAS tool provides a visualization framework allowing users to place unknown isolates on a curated phylogeny of all Phytophthora species. Critically, the tree can be updated in real-time as new species are described. The tool contains metadata including clade, host species, substrate, sexual characteristics, distribution, and reference literature, which can be visualized on the tree and downloaded for other uses. This phylogenetic resource will allow data sharing among research groups and the database will enable the global Phytophthora community to upload sequences and determine the phylogenetic placement of an isolate within the larger phylogeny and to download sequence data and metadata. The database will be curated by a community of Phytophthora researchers and housed on the T-BAS web portal in the Center for Integrated Fungal Research at NC State. The T-BAS web tool can be leveraged to create similar metadata enhanced phylogenies for other Oomycete, bacterial or fungal pathogens.

Understanding the Genotypic and Phenotypic Structure and Impact of Climate on Phytophthora nicotianae Outbreaks on Potato and Tomato in the Eastern United States.

Samples from potato fields with lesions with late blight-like symptoms were collected from eastern North Carolina in 2017 and the causal agent was identified as Phytophthora nicotianae. We have identified P. nicotianae in potato and tomato samples from North Carolina, Virginia, Maryland, Pennsylvania, and New York. Ninety-two field samples were collected from 46 fields and characterized for mefenoxam sensitivity, mating type, and simple sequence repeat genotype using microsatellites. Thirty-two percent of the isolates were the A1 mating type, while 53% were the A2 mating type. In six cases, both A1 and A2 mating types were detected in the same field in the same year. All isolates tested were sensitive to mefenoxam. Two genetic groups were discerned based on STRUCTURE analysis: one included samples from North Carolina and Maryland, and one included samples from all five states. The data suggest two different sources of inoculum from the field sites sampled. Multiple haplotypes within a field and the detection of both mating types in close proximity suggests that P. nicotianae may be reproducing sexually in North Carolina. There was a decrease in the average number of days with weather suitable for late blight, from 2012 to 2016 and 2017 to 2021 in all of the North Carolina counties where P. nicotianae was reported. P. nicotianae is more thermotolerant than P. infestans and grows at higher temperatures (25 to 35°C) than P. infestans (18 to 22°C). Late blight outbreaks have decreased in recent years and first reports of disease are later, suggesting that the thermotolerant P. nicotianae may cause more disease as temperatures rise due to climate change.

Metagenomic study reveals hidden relationships among fungal diversity, variation of plant disease, and genetic distance in Cornus florida (Cornaceae).

Introduction: Understanding patterns of plant-microbe interactions across plant species and populations is a critical yet poorly characterized aspect in the field of plant pathology. Microbial DNA sequences present as contaminants in omics data of plants obtained using next-generation sequencing methods provide a valuable source to explore the relationships among endophytic microbial diversity, disease and genetic differentiation of host plants, and environmental variation, but few such studies have been conducted. The flowering dogwood tree (Cornus florida L.), an ecologically important species in North America, is threatened by powdery mildew and dogwood anthracnose diseases, and knowledge of the microbial diversity harbored within genetically and environmental distinct populations of this species remains largely unknown. Methods: We conducted a metagenomics study utilizing the sequences of RAD-tag/genotype-by-sequence libraries from leaf tissues of C. florida to examine such host-fungus interactions across the dogwood's US range. We performed various combinations of alignments to both host and pathogen genomes to obtain filtered sets sequences for metagenomics analysis. Taxonomic assignments were determined on each filtered set of sequences, followed by estimation of microbial diversity and correlation to environment and host-genetic variation. Results: Our data showed that microbial community composition significantly differed between visually healthy and diseased sites. Several microbial taxa known to interact with dogwood were identified from these sequences. We found no correlation between microbial diversity and relative abundances of sequences aligning to draft genomes of either pathogen causing powdery mildew or dogwood anthracnose. We found a significant relationship between differences of fungal communities and geographic distances of plant populations, suggesting roles of environments in shaping fungal communities in leaf tissues. Significant correlations between the genetic differentiation of plant samples and fungal community dissimilarity (beta diversity) were also observed in certain sets of our analyses-suggesting the possibility of a relationship between microbial community composition and plant genetic distance. This relationship persisted in significance even after controlling for significant effects of geographic-bioclimatic variation of microbial diversity. Discussion: Our results suggest that both genetics and the environment play a significant role in shaping foliar fungal communities. Our findings underscore the power of leveraging hidden microbial sequences within datasets originally collected for plant genetic studies to understand plant-pathogen interactions.

Microsatellite Markers from Peronospora tabacina, the Cause of Blue Mold of Tobacco, Reveal Species Origin, Population Structure, and High Gene Flow.

Peronospora tabacina is an obligate parasite that causes blue mold of tobacco. The pathogen reproduces primarily by sporangia, whereas the sexual oospores are rarely observed. A collection of 122 isolates of P. tabacina was genotyped using nine microsatellites to assess the population structure of individuals from subpopulations collected from central, southern, and western Europe; the Middle East; Central America; North America; and Australia. Genetic variations among the six subpopulations accounted for ∼8% of the total variation, including moderate levels of genetic differentiation, high gene flow among these subpopulations, and a positive correlation between geographic and genetic distance (r = 0.225; P < 0.001). Evidence of linkage disequilibrium (P < 0.001) showed that populations contained partially clonal subpopulations but that subpopulations from Australia and Mediterranean Europe did not. High genetic variation and population structure among samples could be explained by continuous gene flow across continents via infected transplant exchange and/or long-distance dispersal of sporangia via wind currents. This study analyzed the most numerous P. tabacina collection and allowed conclusions regarding the migration, mutation, and evolutionary history of this obligate biotrophic oomycete. The evidence pointed to the species origin in Australia and identified intracontinental and intercontinental migration patterns of this important pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Global historic pandemics caused by the FAM-1 genotype of Phytophthora infestans on six continents.

The FAM-1 genotype of Phytophthora infestans caused late blight in the 1840s in the US and Europe and was responsible for the Irish famine. We sampled 140 herbarium specimens collected between 1845 and 1991 from six continents and used 12-plex microsatellite genotyping (SSR) to identify FAM-1 and the mtDNA lineage (Herb-1/Ia) present in historic samples. FAM-1 was detected in approximately 73% of the historic specimens and was found on six continents. The US-1 genotype was found later than FAM-1 on all continents except Australia/Oceania and in only 27% of the samples. FAM-1 was the first genotype detected in almost all the former British colonies from which samples were available. The data from historic outbreak samples suggest the FAM-1 genotype was widespread, diverse, and spread to Asia and Africa from European sources. The famine lineage spread to six continents over 144 years, remained widespread and likely spread during global colonization from Europe. In contrast, modern lineages of P. infestans are rapidly displaced and sexual recombination occurs in some regions.

The persistent threat of emerging plant disease pandemics to global food security.

Plant disease outbreaks are increasing and threaten food security for the vulnerable in many areas of the world. Now a global human pandemic is threatening the health of millions on our planet. A stable, nutritious food supply will be needed to lift people out of poverty and improve health outcomes. Plant diseases, both endemic and recently emerging, are spreading and exacerbated by climate change, transmission with global food trade networks, pathogen spillover, and evolution of new pathogen lineages. In order to tackle these grand challenges, a new set of tools that include disease surveillance and improved detection technologies including pathogen sensors and predictive modeling and data analytics are needed to prevent future outbreaks. Herein, we describe an integrated research agenda that could help mitigate future plant disease pandemics.

Integrated microneedle-smartphone nucleic acid amplification platform for in-field diagnosis of plant diseases.

We demonstrate an integrated microneedle (MN)-smartphone nucleic acid amplification platform for "sample-to-answer" diagnosis of multiplexed plant pathogens within 30 min. This portable system consists of a polymeric MN patch for rapid nucleic acid extraction within a minute and a 3D-printed smartphone imaging device for loop-mediated isothermal amplification (LAMP) reaction and detection. We expanded the extraction of the MN technology for DNA targets as in the previous study (ACS Nano, 2019, 13, 6540-6549) to more fragile RNA biomarkers, evaluated the storability of the extracted nucleic acid samples on MN surfaces, and developed a smartphone-based LAMP amplification and fluorescent reader device that can quantify four LAMP reactions on the same chip. In addition, we have found that the MN patch containing as few as a single needle tip successfully extracted enough RNA for RT-PCR or RT-LAMP analysis. Moreover, MN-extracted RNA samples remained stable on MN surfaces for up to three days. The MN-smartphone platform has been used to detect both Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA down to 1 pg, comparable to the results from a benchtop thermal cycler. Finally, multiplexed detection of P. infestans and TSWV through a single extraction from infected tomato leaves and amplification on the smartphone without benchtop equipment was demonstrated.

Protective plant immune responses are elicited by bacterial outer membrane vesicles.

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.

DNA Extraction from Plant Leaves Using a Microneedle Patch.

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.

Detection of Phytophthora infestans by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR.

Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.

Non-invasive plant disease diagnostics enabled by smartphone-based fingerprinting of leaf volatiles.

Plant pathogen detection conventionally relies on molecular technology that is complicated, time-consuming and constrained to centralized laboratories. We developed a cost-effective smartphone-based volatile organic compound (VOC) fingerprinting platform that allows non-invasive diagnosis of late blight caused by Phytophthora infestans by monitoring characteristic leaf volatile emissions in the field. This handheld device integrates a disposable colourimetric sensor array consisting of plasmonic nanocolorants and chemo-responsive organic dyes to detect key plant volatiles at the ppm level within 1 min of reaction. We demonstrate the multiplexed detection and classification of ten individual plant volatiles with this field-portable VOC-sensing platform, which allows for early detection of tomato late blight 2 d after inoculation, and differentiation from other pathogens of tomato that lead to similar symptoms on tomato foliage. Furthermore, we demonstrate a detection accuracy of ≥95% in diagnosis of P. infestans in both laboratory-inoculated and field-collected tomato leaves in blind pilot tests. Finally, the sensor platform has been beta-tested for detection of P. infestans in symptomless tomato plants in the greenhouse setting.

Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases.

In-field molecular diagnosis of plant diseases via nucleic acid amplification is currently limited by cumbersome protocols for extracting and isolating pathogenic DNA from plant tissues. To address this challenge, a rapid plant DNA extraction method was developed using a disposable polymeric microneedle (MN) patch. By applying MN patches on plant leaves, amplification-assay-ready DNA can be extracted within a minute from different plant species. MN-extracted DNA was used for direct polymerase chain reaction amplification of plant plastid DNA without purification. Furthermore, using this patch device, extraction of plant pathogen DNA ( Phytophthora infestans) from both laboratory-inoculated and field-infected leaf samples was performed for detection of late blight disease in tomato. MN extraction achieved 100% detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional gold standard cetyltrimethylammonium bromide (CTAB)-based DNA extraction method and 100% detection rate for all blind field samples tested. This simple, cell-lysis-free, and purification-free DNA extraction method could be a transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field.

Genetic Structure and Subclonal Variation of Extant and Recent U.S. Lineages of Phytophthora infestans.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

Phytophthora acaciae sp. nov., a new species causing gummosis of black wattle in Brazil.

A new Phytophthora species was found associated with gummosis in black wattle plantations in the subtropical, humid, south of Brazil. The new species Phytophthora acaciae is formally named herein based on phylogenetic and morphological analyses. This is the fourth Phytophthora species found from this pathogen complex in black wattle plantations causing gummosis in Brazil. The other three species are P. nicotianae, P. boehmeriae, and P. frigida. Phytophthora acaciae is heterothallic with amphigynous antheridia, noncaducous, papillate sporangia and is placed in the Phytophthora clade 2 based on nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequences. Maximum parsimony and maximum likelihood phylogenetic analyses of P. acaciae isolates based on multigene sequences, including partial DNA sequences of three nuclear protein-coding genes (ß-tubulin, translation elongation factor-1α, and ras-related protein), two mitochondrial protein-coding genes (cytochrome c oxidase subunits I and II), in addition to ITS sequence data, support the delimitation of this new species on Acacia mearnsii from the other previously described clade 2 Phytophthora species. Pathogenicity trial confirmed that the new species causes necrotic lesions on the plant stem, with either the presence or absence of gum.

Large sub-clonal variation in Phytophthora infestans from recent severe late blight epidemics in India.

The population structure of the Phytophthora infestans populations that caused the recent 2013-14 late blight epidemic in eastern India (EI) and northeastern India (NEI) was examined. The data provide new baseline information for populations of P. infestans in India. A migrant European 13_A2 genotype was responsible for the 2013-14 epidemic, replacing the existing populations. Mutations have generated substantial sub-clonal variation with 24 multi-locus genotypes (MLGs) found, of which 19 were unique variants not yet reported elsewhere globally. Samples from West Bengal were the most diverse and grouped alongside MLGs found in Europe, the UK and from neighbouring Bangladesh but were not linked directly to most samples from south India. The pathogen population was broadly more aggressive on potato than on tomato and resistant to the fungicide metalaxyl. Pathogen population diversity was higher in regions around the international borders with Bangladesh and Nepal. Overall, the multiple shared MLGs suggested genetic contributions from UK and Europe in addition to a sub-structure based on the geographical location within India. Our data indicate the need for improved phytosanitary procedures and continuous surveillance to prevent the further introduction of aggressive lineages of P. infestans into the country.

Population structure and migration of the Tobacco Blue Mold Pathogen, Peronospora tabacina, into North America and Europe.

Tobacco blue mold, caused by Peronospora tabacina, is an oomycete plant pathogen that causes yearly epidemics in tobacco (Nicotiana tabacum) in the United States and Europe. The genetic structure of P. tabacina was examined to understand genetic diversity, population structure and patterns of migration. Two nuclear loci, Igs2 and Ypt1, and one mitochondrial locus, cox2, were amplified, cloned and sequenced from fifty-four isolates of P. tabacina from the United States, Central America-Caribbean-Mexico (CCAM), Europe and the Middle East (EULE). Cloned sequences from the three genes showed high genetic variability across all populations. Nucleotide diversity and the population mean mutation parameter per site (Watterson's theta) were higher in EULE and CCAM and lower in U.S. POPULATIONS: Neutrality tests were significant and the equilibrium model of neutral evolution was rejected, indicating an excess of recent mutations or rare alleles. Hudson's Snn tests were performed to examine population subdivision and gene flow among populations. An isolation-with-migration analysis (IM) supported the hypothesis of long-distance migration of P. tabacina from the Caribbean region, Florida and Texas into other states in the United States. Within the European populations, the model documented migration from North Central Europe into western Europe and Lebanon, and migration from western Europe into Lebanon. The migration patterns observed support historical observations about the first disease introductions and movement in Europe. The models developed are applicable to other aerial dispersed emerging pathogens and document that high-evolutionary-risk plant pathogens can move over long distances to cause disease due to their large effective population size, population expansion and dispersal.