Core-Genome Multilocus Sequence Typing for Epidemiological and Evolutionary Analyses of Phytopathogenic Xanthomonas citri.

Xanthomonas citri subsp. citri is the cause of bacterial citrus canker, responsible for major economic losses to the citrus industry. X. citri subspecies and pathovars are responsible for diseases in soybean, common bean, mango, pomegranate, and cashew. X. citri disease has been tracked using several typing methods, but recent studies using genomic sequencing have been key to understanding the evolutionary relationships within the species, including fundamental differences among X. citri subsp. citri pathotypes. Here, we describe a core-genome multilocus sequence typing (cgMLST) scheme for X. citri based on 250 genomes comprising multiple examples of X. citri subsp. citri pathotypes A, A*, and Aw; X. citri subsp. malvacearum; X. citri pv. aurantifolii, pv. fuscans, pv. glycines, pv. mangiferaeindicae, pv. viticola, and pv. vignicola; and single isolates of X. citri pv. dieffenbachiae and pv. punicae. This data set included genomic sequencing of 100 novel X. citri subsp. citri isolates. cgMLST, based on 1,618 core genes across 250 genomes, is implemented at PubMLST (https://pubmlst.org/organisms/xanthomonas-citri/). GrapeTree minimum-spanning tree and Interactive Tree of Life (iTOL) neighbor-joining phylogenies generated from the cgMLST data resolved almost identical groupings of isolates to a core-genome single nucleotide polymorphism (SNP)-based neighbor-joining phylogeny. These resolved identical groupings of X. citri subsp. citri pathotypes and X. citri subspecies and pathovars. X. citri cgMLST should prove to be an increasingly valuable resource for the study of this key species of plant-pathogenic bacteria. Users can submit genomic data and associated metadata for comparison with previously characterized isolates at PubMLST to allow the rapid characterization of the local, national, and global epidemiology of these pathogens and examine evolutionary relationships. IMPORTANCE Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others. Recent genomic studies have shown that host-adapted X. citri subspecies and pathovars and X. citri subsp. citri pathotypes form distinct clades. In this study, we describe a core-genome multilocus sequence typing (cgMLST) scheme for this species that can rapidly and robustly discriminate among these ecologically distinct, host-adapted clades. We have established this scheme and associated databases containing genomic sequences and metadata at PubMLST, which users can interrogate with their own genome sequences to determine X. citri subspecies, pathovars, and pathotypes. X. citri cgMLST should prove to be an invaluable tool for the study of the epidemiology and evolution of this major plant pathogen.

An electronic, unsupervised patient-reported Expanded Disability Status Scale for multiple sclerosis.

BACKGROUND: In persons with multiple sclerosis (MS), the Expanded Disability Status Scale (EDSS) is the criterion standard for assessing disability, but its in-person nature constrains patient participation in research and clinical assessments. OBJECTIVE: The aim of this study was to develop and validate a scalable, electronic, unsupervised patient-reported EDSS (ePR-EDSS) that would capture MS-related disability across the spectrum of severity. METHODS: We enrolled 136 adult MS patients, split into a preliminary testing Cohort 1 (n = 50), and a validation Cohort 2 (n = 86), which was evenly distributed across EDSS groups. Each patient completed an ePR-EDSS either immediately before or after a MS clinician's Neurostatus EDSS evaluation. RESULTS: In Cohort 2, mean age was 50.6 years (range = 26-80) and median EDSS was 3.5 (interquartile range (IQR) = [1.5, 5.5]). The ePR-EDSS and EDSS agreed within 1-point for 86% of examinations; kappa for agreement within 1-point was 0.85 (p < 0.001). The correlation coefficient between the two measures was 0.91 (<0.001). DISCUSSION: The ePR-EDSS was highly correlated with EDSS, with good agreement even at lower EDSS levels. For clinical care, the ePR-EDSS could enable the longitudinal monitoring of a patient's disability. For research, it provides a valid and rapid measure across the entire spectrum of disability and permits broader participation with fewer in-person assessments.

Using the PARAFAC2 tensor factorization on EHR audit data to understand PCP desktop work.

BACKGROUND: Activity or audit log data are required for EHR privacy and security management but may also be useful for understanding desktop workflow. OBJECTIVE: We determined if the EHR audit log file, a rich source of complex time-stamped data on desktop activities, could be processed to derive primary care provider (PCP) level workflow measures. METHODS: We analyzed audit log data on 876 PCPs across 17,455 ambulatory care encounters that generated 578,394 time-stamped records. Each individual record represents a user interaction (e.g., point and click) that reflects all or part of a specific activity (e.g., order entry access). No dictionary exists to define how to combine clusters of sequential audit log records to represent identifiable PCP tasks. We determined if PARAFAC2 tensor factorization could: (1) learn to identify audit log record clusters that specifically represent defined PCP tasks; and (2) identify variation in how tasks are completed without the need for ground-truth labels. To interpret the result, we used the following PARAFAC2 factors: a matrix representing the task definitions and a matrix containing the frequency measure of each task for each encounter. RESULTS: PARAFAC2 automatically identified 4 clusters of audit log records that represent 4 common clinical encounter tasks: (1) medications' access, (2) notes' access, (3) order entry access, and (4) diagnosis modification. PARAFAC2 also identified the most common variants in how PCPs accomplish these tasks. It discovered variation in how the notes' access task was done, including identification of 9 distinct variants of notes access that explained 77% of the input data variation for notes. The discovered variants mapped to two known workflows for notes' access and to two distinct PCP user groups who accessed notes by either using the Visit Navigator or the Wrap-Up option. CONCLUSIONS: Our results demonstrate that EHR audit log data can be rapidly processed to create higher-level constructed features that represent time-stamped PCP tasks.

Improved deferred antagonism technique for detecting antibiosis.

The deferred antagonism technique has been utilized for several decades for detecting antibiosis activity. Most protocols require the elimination of antibiotic-producing cells by exposing them to chloroform vapour, UV radiation or filter sterilizing the filtrate steps that require additional time and expense to complete. We provide a modified approach to current soft agar overlay practices, which involves addition of antibiotics to the soft agar overlay to inhibit growth of the producer but not the indicator strain. This technique can be used to reproducibly and efficiently screen for antibiotic production with ease. We demonstrate the effectiveness of this technique with three bacterial systems: inhibition of the bacterial spot of tomato pathogen, Xanthomonas euvesicatoria, by its pathogenic competitor Xanthomonas perforans; and inhibition of the fire blight pathogen, Erwinia amylovora, by Pantoea vagans C9-1 or Pseudomonas fluorescens A506. SIGNIFICANCE AND IMPACT OF THE STUDY: Deferred antagonism assays are used commonly to observe antibiotic production by micro-organisms. Killing or removing the producer cells prior to introduction of the indicator strain is a standard practice but requires additional time and special handling procedures. We evaluated a modification of the assay, where the overlay medium is amended with an antibiotic to which the indicator strain is resistant and the producer strain is sensitive. This modification obviates extra steps to kill the producer strain prior to overlaying with the indicator strain and provides a rapid, consistent and cost-effective method to detect antibiosis.

Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas perforans.

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologous-recombination events derived from X. euvesicatoria In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation.IMPORTANCE Bacterial leaf spot of tomato and pepper is an endemic plant disease with a global distribution. In this study, we investigated the evolutionary processes leading to the emergence of novel X. perforans lineages identified in Alabama. While one lineage was isolated from symptomatic tomato and pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, here designated PthXp1. Functional analysis of PthXp1 indicated that it does not induce Bs4-mediated resistance in tomato and contributes to virulence, providing an adaptive advantage to strains on tomato. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts that results in the emergence of novel pathogen lineages and associated phenotypes, including host range.

Reclassification of Xanthomonas gardneri (ex Sutic 1957) Jones et al. 2006 as a later heterotypic synonym of Xanthomonas cynarae Trébaol et al. 2000 and description of X. cynarae pv. cynarae and X. cynarae pv. gardneri based on whole genome analyses.

Multilocus sequence analysis of Xanthomonas species revealed a very close relationship between Xanthomonas cynarae, an artichoke pathogen and Xanthomonas gardneri, a tomato and pepper pathogen. Results of whole genome sequence comparisons using average nucleotide identity between representative strains of X. gardneri and X. cynarae were well above the threshold of 95-96 %. Inoculations of X. gardneri strains in artichoke leaves caused mild disease symptoms, but only weak symptoms were observed in the bracts. Both X. cynarae and X. gardneri grew equally and caused typical bacterial spot symptoms in pepper after artificial inoculation. However, X. cynarae induced a hypersensitive reaction in tomato, while X. gardneri strains were virulent. Pathogenicity-associated gene clusters, including the protein secretion systems, type III effector profiles, and lipopolysaccharide cluster were nearly identical between the two species. Based on our results from whole genome sequence comparison, X. gardneri and X. cynarae belong to the same species. The name X. cynarae has priority and X. gardneri should be considered as a later heterotypic synonym. An emended description of X. cynarae (type strain=CFBP 4188T, =DSM 16794T) is given. However, due to the host specificity in artichoke and tomato, two pathovars, X. cynarae pv. cynarae and X. cynarae pv. gardneri, are proposed.

Recombinase Polymerase Amplification Assay for Field Detection of Tomato Bacterial Spot Pathogens.

Bacterial spot of tomato is caused by Xanthomonas gardneri, X. euvesicatoria, X. perforans, and X. vesicatoria. Current diagnostic methods for the pathogens are not in-field assays. Recombinase polymerase amplification (RPA) is ideal for in-field detection assays, because it is an isothermal technique that is rapid and more tolerant to inhibitors compared with polymerase chain reaction. Hence, novel RPA probes and primers were designed to amplify regions of the hrcN gene of X. gardneri, X. euvesicatoria, and X. perforans. The X. gardneri RPA is specific to X. gardneri with a detection limit of 106 CFU/ml and detected X. gardneri in lesions from naturally (n = 6) or artificially (n = 18) infected plants. The X. euvesicatoria RPA detects both X. euvesicatoria and X. perforans with a detection limit of 106 CFU/ml and detected both pathogens in plants artificially infected (n = 36) or naturally infected (n = 85) with either X. euvesicatoria or X. perforans. The X. perforans RPA is specific to X. perforans with a detection limit of 107 CFU/ml. Although the X. perforans RPA assay was unable to detect X. perforans from lesions, the X. euvesicatoria RPA was successfully used in field to detect X. perforans from symptomatic field samples (n = 31). The X. perforans RPA was then used to confirm the pathogen in the laboratory. The X. euvesicatoria and X. gardneri RPA is promising for rapid, real-time in-field detection of bacterial spot and one of the first developed among plant pathogenic bacteria.

In-Grove Spatiotemporal Spread of Citrus Huanglongbing and Its Psyllid Vector in Relation to Weather.

Reports of spatial patterns of 'Candidatus Liberibacter asiaticus'-infected asymptomatic citrus trees and 'Ca. L. asiaticus'-positive Asian citrus psyllids (ACP) are rare, as are published relationships between huanglongbing (HLB), ACP, and weather. Here, spatial patterns of 'Ca. L. asiaticus'-positive asymptomatic and symptomatic trees were determined every half year in a small grove over 2.5 years, and of HLB-symptomatic trees and ('Ca. L. asiaticus'-positive) ACP populations every month in two commercial groves for 1 year. Spread of symptomatic trees followed that of asymptomatic 'Ca. L. asiaticus'-positive trees with <6 months' delay. 'Ca. L. asiaticus'-positive asymptomatic and symptomatic fronts moved at 2.5 to 3.6 m month-1. No spatial relationship was detected between ACP populations and HLB-infected trees. HLB incidence and 'Ca. L. asiaticus'-positive ACP dynamics were tentatively positively correlated with monthly rainfall data and, to a lesser extent, with average minimum temperature.

Nano-Magnesium Oxide: A Novel Bactericide Against Copper-Tolerant Xanthomonas perforans Causing Tomato Bacterial Spot.

Bacterial spot caused by Xanthomonas perforans causes significant damage on tomato in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, Cu bactericides are not effective in disease management. Hence, there is a critical need to find alternatives for Cu. Antibacterial activity of magnesium oxide (Nano-MgO), and other metal oxide nanoparticles, were evaluated against a Cu-tolerant and -sensitive X. perforans strain. In vitro experiments demonstrated high antibacterial activity of Nano-MgO against both strains compared with the commercial Cu. The minimum inhibitory concentration of Nano-MgO is 25 µg/ml and the minimum bactericidal concentration is 100 µg/ml against a Cu-tolerant X. perforans strain after 4 h of exposure. Structural changes in the bacterial membrane following exposure to Nano-MgO treatments compared with the controls were observed using transmission electron microscopy. In two greenhouse experiments with a Cu-tolerant strain, bacterial spot severity was significantly reduced by Nano-MgO at 200 µg/ml compared with Cu-ethylene bis-dithiocarbamate (grower standard), and the untreated control (P = 0.05). In three field experiments, Nano-MgO at 200 µg/ml significantly reduced disease severity with no negative impact on yield compared with the untreated control. Inductively coupled plasma mass spectrometric analysis of the fruit confirmed that Nano-MgO application did not lead to the accumulation of Mg, Cu, Ca, K, Mn, P, and S. This study is the first to demonstrate the potential of Nano-MgO against bacterial spot of tomato.

Pathogenicity of the bacterium New Zealand rickettsia-like organism (NZ-RLO2) in Chinook salmon Oncorhynchus tshawytscha smolt.

Farmed New Zealand Chinook salmon Oncorhynchus tshawytscha Walbaum have been found to be infected by rickettsia-like organisms (NZ-RLO). While these Gram-negative intra-cellular bacteria are closely related to Piscirickettsia salmonis, a significant pathogen for farmed salmon globally, the pathogenicity of NZ-RLO is unknown. The aim of the present study was to determine if one strain, NZ-RLO2, causes disease in Chinook salmon. Post-smolt salmon were inoculated with NZ-RLO2 by intraperitoneal injection at high, medium and low doses and observed for 30 d. All fish in the high and medium dosed groups died by the end of the study and 63% of the low dose group died within 30 d of inoculation. Necropsy revealed the fish inoculated with NZ-RLO2 had internal multifocal haemorrhages. The most consistent histological finding in fish inoculated with NZ-RLO2 was neutrophilic and necrotizing pancreatitis and steatitis with intra-cytoplasmic organisms often visible within areas of inflammation. Other histological lesions included multifocal hepatic necrosis, haematopoietic cell necrosis and splenic and renal lymphoid depletion. The presence of NZ-RLO2 within the inoculated fish was confirmed by replication in cell culture and qPCR. The results suggest NZ-RLO2 can cause disease in Chinook salmon and therefore could be a significant pathogen in farmed Chinook salmon.

Impacts of epizootic ulcerative syndrome on subsistence fisheries and wildlife.

Epizootic ulcerative syndrome (EUS), caused by the water mould (Oomycota) Aphanomyces invadans, has spread throughout the world's major continents over the last 50 years, with the apparent exception of South and Central America. With over 160 susceptible fish species representing 54 families and 16 orders recorded to date, EUS is of international concern and infection with A. invadans is a World Organisation for Animal Health (OIE) listed disease. This paper examines what little has been reported on the impacts of EUS on subsistence fisheries and wildlife, or what can be deduced about those impacts, and concludes that there is a need for systematic data collection on the size and socio-economic importance of subsistence fisheries. Such fisheries are often relied upon by the poorest communities, thus food and nutrition security impacts can be significant. Similarly, impacts on wildlife are poorly documented, emphasising the lack of, and the need for, research on and modelling of the ecosystem-level impacts of EUS and other aquatic animal diseases. The history of EUS and several other aquatic animal diseases also brings into question the effectiveness of current measures for controlling the international spread of aquatic animal diseases and calls for a re-think on how best to meet this ongoing challenge.

En l'espace d'un demi-siècle, le syndrome ulcératif épizootique dû à l'oomycète Aphanomyces invadans s'est propagé dans toutes les régions du monde, à l'exception, semble-t-il, de l'Amérique du Sud et Centrale. Avec plus de 160 espèces de poissons répertoriées comme sensibles, réparties en 54 familles et 16 ordres, le syndrome ulcératif épizootique est une maladie préoccupante à l'échelle internationale et figure parmi les maladies listées par l'Organisation mondiale de la santé animale (OIE) sous le nom d'infection à A. invadans. Les auteurs font état des très rares signalements concernant l'impact du syndrome ulcératif épizootique sur la pêche de subsistance et sur la faune sauvage et tentent d'en tirer quelques conclusions, en insistant sur la nécessité de procéder à une collecte systématique de données afin de déterminer l'envergure et l'importance socioéconomique de la pêche de subsistance. Les communautés les plus pauvres étant souvent celles qui dépendent le plus de cette activité, la maladie a sans doute un impact majeur sur la sécurité alimentaire et nutritionnelle de ces populations. De même, l'impact sur la faune sauvage n'a pas vraiment été étudié jusqu'à présent, d'où la nécessité de conduire des travaux de recherche et de modélisation sur l'impact du syndrome ulcératif épizootique (et d'autres maladies des animaux aquatiques) à l'échelle des écosystèmes. L'histoire du syndrome ulcératif épizootique et d'autres maladies des animaux aquatiques pose également la question de l'efficacité des mesures appliquées actuellement pour maîtriser la propagation internationale de ces maladies et invite à repenser la réponse à apporter à ce défi toujours présent.

En los últimos 50 años, el síndrome ulcerante epizoótico, causado por el hongo acuático (oomiceto) Aphanomyces invadans, se ha diseminado por casi todos los continentes del planeta, con la aparente salvedad de Sudamérica y Centroamérica. Se trata de una enfermedad de importancia internacional que, hasta donde consta a día de hoy, afecta a más de 160 especies piscícolas de 54 familias y 16 órdenes. De ahí que la infección por A. invadans sea una patología inscrita en la lista de la Organización Mundial de Sanidad Animal (OIE). Los autores, tras exponer lo poco que hasta ahora se ha descrito de los efectos de la enfermedad sobre la pesca de subsistencia y la fauna silvestre o lo que es posible inferir acerca de esos efectos, llegan a la conclusión de que se requiere una labor sistemática de obtención de datos sobre la magnitud e importancia socioeconómica de la actividad pesquera de subsistencia, de la que dependen a menudo las comunidades más pobres, por lo que las consecuencias para la seguridad nutricional y alimentaria pueden ser de calado. Tampoco están bien descritas las repercusiones de la enfermedad en los animales silvestres, lo que pone de relieve la ausencia, y por ende la necesidad, de investigaciones y de modelos sobre los efectos ecosistémicos del síndrome ulcerante epizoótico y otras varias enfermedades de los animales acuáticos. La historia de estas patologías también arroja dudas sobre la eficacia de las medidas aplicadas actualmente para controlar la propagación internacional de las enfermedades de los animales acuáticos y exige replantearse cuál es la respuesta idónea a este problema que no cesa.

Advanced Copper Composites Against Copper-Tolerant Xanthomonas perforans and Tomato Bacterial Spot.

Bacterial spot, caused by Xanthomonas spp., is a widespread and damaging bacterial disease of tomato (Solanum lycopersicum). For disease management, growers rely on copper bactericides, which are often ineffective due to the presence of copper-tolerant Xanthomonas strains. This study evaluated the antibacterial activity of the new copper composites core-shell copper (CS-Cu), multivalent copper (MV-Cu), and fixed quaternary ammonium copper (FQ-Cu) as potential alternatives to commercially available micron-sized copper bactericides for controlling copper-tolerant Xanthomonas perforans. In vitro, metallic copper from CS-Cu and FQ-Cu at 100 µg/ml killed the copper-tolerant X. perforans strain within 1 h of exposure. In contrast, none of the micron-sized copper rates (100 to 1,000 µg/ml) from Kocide 3000 significantly reduced copper-tolerant X. perforans populations after 48 h of exposure compared with the water control (P < 0.05). All copper-based treatments killed the copper-sensitive X. perforans strain within 1 h. Greenhouse studies demonstrated that all copper composites significantly reduced bacterial spot disease severity when compared with copper-mancozeb and water controls (P < 0.05). Although there was no significant impact on yield, copper composites significantly reduced disease severity when compared with water controls, using 80% less metallic copper in comparison with copper-mancozeb in field studies (P < 0.05). This study highlights the discovery that copper composites have the potential to manage copper-tolerant X. perforans and tomato bacterial spot.

Regional Spatial-Temporal Spread of Citrus Huanglongbing Is Affected by Rain in Florida.

Citrus huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus' (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month-1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year-1) than in counties with smaller groves in hotspot areas (0.67 year-1), confirming reports that large-scale HLB management may slow epidemic progress.

Molecular Epidemiology of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spot of Watermelon and Squash in Florida.

From 2013 to 2014, bacterial leaf spot epidemics incited by Pseudomonas syringae pv. syringae affected an estimated 3,000 ha of watermelon and squash in Florida, and caused foliar blighting and transplant losses in severely affected fields. To investigate the diversity of the causal agent, we isolated 28 P. syringae strains from diseased plants grown in 10 Florida and Georgia counties over the course of 2 years. Strains were confirmed as P. syringae through sequence analysis of the 16S ribosomal RNA, phenotypic, and biochemical profiling; however, 20 displayed an atypical phenotype by exhibiting nonfluorescent activity on King's medium B agar and being negative for ice-nucleating activity. Multilocus sequence analysis and BOX polymerase chain reaction revealed the presence of two haplotypes among the collected strains that grouped into two distinct clades within P. syringae phylogroup 2. Pathogenicity testing showed that watermelon, cantaloupe, and squash seedlings were susceptible to a majority of these strains. Although both haplotypes were equally virulent on cantaloupe, they differed in virulence on watermelon and squash. The distribution of one haplotype in 9 of 10 Florida and Georgia counties sampled indicated that these epidemics were associated with the recent introduction of a novel clonal P. syringae lineage throughout major watermelon production areas in Florida.

Epidemiology and management of bacterial leaf spot on watermelon caused by Pseudomonas syringae.

Bacterial leaf spot of watermelon caused by Pseudomonas syringae has been an emerging disease in the southeastern United States in recent years. Disease outbreaks in Florida were widespread from 2013 to 2014 and resulted in foliar blighting at the early stages of the crop and transplant losses. We conducted a series of field trials at two locations over the course of two years to examine the chemical control options that may be effective in management of this disease, and to investigate the environmental conditions conducive for bacterial leaf spot development. Weekly applications of acibenzolar-S-methyl (ASM) foliar, ASM drip, or copper hydroxide mixed with ethylene bis-dithiocarbamate were effective in reducing the standardized area under the disease progress curve (P < 0.05). Pearson's correlation test demonstrated a negative relationship between the average weekly temperature and disease severity (-0.77, P = 0.0002). When incorporated into a multiple regression model with the square root transformed average weekly rainfall, these two variables accounted for 71% of the variability observed in the weekly disease severity (P < 0.0001). This information should be considered when choosing the planting date for watermelon seedlings as the cool conditions often encountered early in the spring season are conducive for bacterial leaf spot development.

Nocardiosis in freshwater reared Chinook salmon (Oncorhynchus tshawytscha).

CASE HISTORY: An investigation was conducted to identify the cause of mortalities in freshwater reared Chinook salmon (Oncorhynchus tshawytscha). Mortalities occurred in juvenile salmon, at a salmon rearing facility in the South Island of New Zealand. The affected fish were from a pen inside the facility with no surrounding pens or other year classes affected. CLINICAL FINDINGS: Clinically affected fish presented with skin lesions. The majority of skin lesions were unruptured, boil-like, raised circular masses up to 4 cm in diameter, particularly on the dorsolateral aspects and the flank. A number of fish presented with large ulcers resulting from rupturing of the raised lesions described above. This clinical presentation showed similarities to that of furunculosis caused by typical Aeromonas salmonicida, a bacterium exotic to New Zealand. LABORATORY FINDINGS: Samples were taken from two representative fish in the field for histopathology, bacterial culture and molecular testing. Histopathological findings included granulomatous lesions in the kidney, liver, spleen and muscle. When stained with Fite-Faraco modified acid fast stain filamentous branching rods were identified within these granulomas. Following bacterial culture of kidney swabs pure growth of small white matt adherent colonies was observed. This isolate was identified as a Nocardia species by biochemical testing and nucleotide sequencing of the partial 16S rRNA gene. All samples were negative for A. salmonicida based on bacterial culture and PCR testing. DIAGNOSIS: Nocardiosis caused by a Nocardia species. CLINICAL RELEVANCE: Nocardiosis in these fish was caused by a previously undescribed Nocardia species that differs from the species known to be pathogenic to fish: N. asteroides, N. salmonicida and N. seriole. This bacterium is likely to be a new or unnamed environmental species of Nocardia that has the potential to cause disease in Chinook salmon under certain conditions. The clinical presentation of this Nocardia species manifested as raised, boil-like skin lesions which has similarities to the presentation of furunculosis caused by the bacterium typical A. salmonicida, a species exotic to New Zealand.

Low Concentrations of a Silver-Based Nanocomposite to Manage Bacterial Spot of Tomato in the Greenhouse.

Bacterial spot, caused by four Xanthomonas spp., is one of the most damaging diseases of tomato worldwide. Due to limited disease management options, growers rely heavily on copper-based bactericides, which are often ineffective due to the presence of copper-resistant Xanthomonas strains. This study was undertaken to characterize the antibacterial activity of a silver-based nanocomposite, Ag-dsDNA-GO, and its potential as an alternative to copper. Ag-dsDNA-GO at rates as low as 10 µg/ml killed all bacterial cells of copper-tolerant and -sensitive Xanthomonas perforans strains in suspensions containing approximately 103 CFU/ml within 15 min of exposure in vitro, whereas equivalent rates of copper (10, 25, and 50 µg/ml) were unable to significantly reduce populations compared with the untreated control after 24 h of exposure (P = 0.05). All copper concentrations killed the copper-sensitive X. perforans strain but required exposure for ≥1 h. Ag-dsDNA-GO also exhibited antibacterial activity against copper-tolerant X. vesicatoria, X. euvesicatoria, and X. gardneri strains. In greenhouse studies, tomato plants treated with Ag-dsDNA-GO at either 75 or 100 µg/ml prior to artificial inoculation significantly reduced disease severity when compared with copper-mancozeb and negative controls (P = 0.05). This study highlights the potential of Ag-dsDNA-GO as an alternative to copper in tomato transplant production.

A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato.

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

Angular Leaf Spot of Cucurbits is Associated With Genetically Diverse Pseudomonas syringae Strains.

Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on King's medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.