AbstractEchinorhynchus and Pomphorhynchus . The accuracy of the algorithm's formula for calculating hook cross-sectional area was validated by data collected using a digitizing tablet. Meristograms were created from data in public respositories for eight Echinorhynchus taxa: E.bothniensis, E. 'bothniensis', E.gadi spp. A, B and I, E.brayi, E.salmonis and E.truttae. In this preliminary analysis, the meristogram differentiated E.bothniensis, E.brayi, E.gadi sp. B, E.salmonis and E.truttae from each other, and from the remaining taxa in this study, but independent data will be required for validation. Sample sizes for E. 'bothniensis' and E.gadi spp. A and I were too small to identify diagnostic features with any degree of confidence. Meristogram differences among the sibling species of the E.gadi and E.bothniensis groups suggest that the 'intra-specfic' variation in meristogram previously reported for some Echinorhynchus taxa, may have actually represented morphological divergence between unrecognized cryptic species. Hierarchical clustering of taxa based on Meristogram data yielded dendrograms that were largely concordant with phylogenetic relationships inferred from DNA sequence data, indicating the presence of a strong phylogenetic signal.The Meristogram algorithm was implemented in the  Acanthocephalans are characterized by the presence of a retractable proboscis armed with recurved hooks which provides the means of attachment to the definitive host's gut wall. In many acanthocephalans the proboscis hooks are arranged in longitudinal rows and display positional variation in size and shape. The Meristogram was developed by Echinorhynchusgadi Zoega in Müller, 1776 from New England and discriminated two groups: one from Pseudopleuronectesamericanus  and Limandaferruginea  possessing an "arched" Meristogram, and another from Gadusmorhua L., Pollachiusvirens L., and P.americanus having a "looped" Meristogram. The latter group could be subdivided into two populations on the basis of differences in absolute blade length, although they possessed identical Meristograms. One population was found in P.americanus and the other in G.morhua and P.virens. Now recognised, all three populations can apparently be readily distinguished using conventional morphometrics, without the aid of Meristograms. Echinorhynchussalmonis Müller, 1784 and demonstrated differences between the Meristograms of three species of Pomphorhynchus Monticelli, 1905: P.bulbocolli Linkins in Van Cleave, 1919, P.sebastichthydis Yamaguti, 1939 and P.yamagutii Schmidt & Hugghins, 1973. In subsequent studies, Meristogram analysis was successfully applied to the problem of discriminating Pomphorhynchusrocci Cordonnier & Ward, 1967 from P.bulbocolli , E.brayi Wayland, Sommerville & Gibson, 1999, E.gadi sp. I  at each integer value in the range of percent-position values yielded by the moving average routine. This linear interpolation step often improves the appearance of the Meristogram, but more importantly, by providing common percent-position coordinates it facilitates comparison of Meristograms using multivariate statistics (please see next section).This new implementation of the Meristogram algorithm also enforces a minimum moving average segment size. It seems reasonable to argue that each proboscis in a collection should be represented in every moving average segment. The minimum moving average interval is thus defined as 100/(x+1), rounded up to the nearest integer, where n is the length of the shortest row of hooks in the collection.R . Experienced users of R can call the functions to generate and display Meristograms from the command line. The graphical user interface  minimum moving average interval (MMAI) for the taxon; (2) 1.5 × MMAI; (3) 2 × MMAI.To assess inter-taxon variation, hook measurement data for females and males were pooled and a Meristogram was generated for each taxon using a moving average segment of 17% and linear interpolation. For comparative purposes it was important to use the same moving average segment for all Meristograms and 17% was the MMAI which could be applied to all taxa.In addition to visual inspection of Meristograms, a more objective comparison of Meristograms was achieved with the aid of statistical pattern recognition methods. Meristogram data from all taxa were concatenated into a single matrix, where columns were hook variables at each integer percent-position and rows were taxa. Principal component analysis (PCA) was then used to reduce the number of dimensions in this data-set. Similarity of Meristograms was further assessed by hierarchical clustering of the scores for the first two principal components (accounting for most of the variation in the data-set) using the unweighted pair group method with arithmetic mean (UPGMA) algorithm and four different distance metrics .E.gadi spp.No new data are presented in this study. However, files of hook measurement data in the appropriate format for input to the Meristogram software have been prepared and these are available as supplementary material  were 0.887, 0.882 and 0.816 for E.gadi spp. A, B & I, respectively. The estimated hook areas were remarkably close to the measured values for all three taxa. Linear regression demonstrated that in E.gadi spp. A & B, the Meristogram tended to slightly over-estimate hook area  on the dorsal surface of the proximal end of the proboscis (positions > 79%), corresponding to a flattening of the tail of the Meristogram. Length and area of the last and smallest hook in dorsal rows of female worms ranged from 34.3-59.2µm and 137-477µmE.salmonis, but did not distinguish between dorsal and ventral hook rows; their Meristograms resemble those presented here.Potential diagnostic featuresRadial asymmetryHooks are markedly reduced in size at the proximal end of the proboscis, especially on the dorsal surface.E.truttaeDescriptionE.truttae from Drummore and the River Carron catchment display several loops; three prominent loops are shared by both collections  and base  are located around two-thirds of the way along a row from the distal end of the proboscis.The Drummore collection has peak values of L, B, A and R at positions 40%, 72%, 70% and 77%, respectively Fig. a. The lo2 in females and 626-1,096µm2 in males) are usually situated just distal to the middle of the row. The hooks with the broadest base  are found two-thirds of the way along the row from the distal end of the proboscis.In the collection from the River Carron catchment maximal values of L, B, A and R are found at positions 33%, 64%, 43% and 75%, respectively Fig. 5. The lonPotential diagnostic featuresE.truttae exhibits the least serial variation in hook size. The shortest hooks are 80% (River Carron catchment) - 82% (Drummore) of the length of the longest hooks in their respective rows. In the other taxa studied the shortest hooks are 42-71% of the length of the longest hooks. Similarly the smallest hooks of E.truttae have an area 62% (Drummore) - 66% (River Carron catchment) that of the largest hooks, whereas in the other taxa this value ranges from 17-51%.E.bothniensisDescriptionE.salmonis and E.truttae. A smaller loop occurs in the 60-75% position region of the proboscis, bordered above by B and below by A and L; this latter loop is also present in the Meristogram for E.salmonis. Peak values for L, B, A and R are found at positions 34%, 53%, 52% and 61%, respectively. The Meristogram of E.bothniensis resembles the "arched pattern" Meristogram of E.gadi from Pseudopleuronectesamericanus and Limandaferruginea described by The curves for B, A and R follow a similar trajectory in the distal half of the Meristogram, with a particularly strong monotonic relationship between B and A Fig. a. In thi2 in females and 393-547µm2 in males) are located in the middle of the row.The longest hooks  are located around a third of the way along a row from the distal end of the proboscis, and the shortest hooks  at the proximal end of a row. The stoutest hooks  are found just proximal to the middle of the row and the weakest  at the distal end of the row. The hooks with the broadest base  and greatest area  hooks are situated a third of the way along the row from the distal end of the proboscis, and the shortest (33.0-39.3µm) and smallest (135-187µm2) hooks at the proximal end of the row. The hooks with the broadest base (21.9-22.7µm) are also the stoutest , and are located in the proximal third of the row. The weakest hooks  are located at the distal end of the row.The Meristogram of is' Fig. b should Potential diagnostic featuresE. 'bothniensis' was created using data from just two specimens; additional data may well change the appearance of the Meristogram. For this reason diagnostic characters have not been proposed for this taxon.The Meristogram for E.gadi sp. ADescription2 in females and 628-761µm2 in males) are found just proximal to the middle of a row.Maximal values of L, B, A and R occur at positions: 27%, 67%, 57% and 67%  are situated just distal to the middle of the row and the largest hooks  are found a third of the way along the row from the distal end of the proboscis. The stoutest hooks  occur in the proximal third of the row.The Meristogram of d L Fig. d. MaximaE.gadi spp. in this study, broadly display what E.gadi sp. B appears to be closest to the exemplar of the "looped pattern" depicted in Fig. 11 of The Meristograms of all of the Potential diagnostic featuresE.gadi sp. B is the only taxon in this study in which the hook variable B peaks on the distal half of the proboscis.E.gadi sp. IDescriptionE.gadi sp. B, E. 'bothniensis' and E.salmonis. Peak values of L, B, A and R occur at positions: 18%, 68%, 24% and 69%. The longest hooks  are situated in the distal quarter of a row and the shortest hooks  at the proximal end of the row. The largest hooks  are situated in the distal third of the row. As in E. 'bothniensis' and E.gadi sp. A, the hooks with the broadest base  are also the stoutest . In E.gadi sp. I, these most robust hooks are located two thirds of the way along a row from the distal end of the proboscis. The weakest hooks  are found at the distal end of a row.In the 45-80% position region of the Meristogram are three obvious loops Fig. e. The loPotential diagnostic featuresE.gadi sp. I and A from the other taxa in this study have been described in the section on E.gadi sp. A (above).Meristogram characteristics which may differentiate E.salmonis by pooling hook measurement data from dorsal and ventral rows of hooks; the hook measurement data from the other taxa were assumed to comprise a mixture of dorsal and ventral rows of hooks. The two collections of E.truttae were treated as separate entities, because of the divergence observed between their Meristograms  revealed the major sources of variation in the Meristograms. Fig. 7E.salmonis and E.brayi. Concordance between dendrograms created using different distance metrics suggests that the clusters found are robust.Hierarchical clustering of the scores for the first two PCs depicts relationships between Meristograms as a dendrogram Fig. . Three d8E.truttae suggest intraspecific, geographical variation in hook patterns. However, given the existence of cryptic species in Echinorhynchus , the alternative explanation of interspecific variation should also be considered. Despite this subtle difference in Meristogram, the two populations of E.truttae displayed a much stronger affinity to each other, than to any of the other taxa in all cluster analyses  and so very unlikely to encompass the range of morphological variation potentially displayed by these taxa. Such variation might render some of the proposed diagnostics ineffectual, particularly for the discrimination of the closely related E.truttae, E.gadi spp, E.bothniensis and E. 'bothniensis'.None of the eight E.brayi, E.salmonis and E.truttae; these are also the taxa showing the greatest genetic divergence , then this represents the first morphological character to separate these allopatric sibling species. A previous attempt to discriminate these taxa using conventional morphometrics or the Proboscis Profiler tool was unsuccessful . In addition to facilitating further comparative analyses, these datasets are potentially useful in the testing and refinement of the Meristogram algorithm.Users of the Meristogram are encouraged to share their hook measurement data, either by including them as supplementary files to publications or submitting them to public repositories , etc." . The freely available source code for the current implementation of the Meristogram should provide a helpful starting point for anyone who would like to adapt the method for novel applications.The Meristogram is not limited to the analysis of the hooks of the acanthocephalan proboscis. It can potentially be applied to any organism in which there are patterns in the longitudinal variation of serially homologous structures, Supplementary material 1Data analysis scriptData type: R language scriptBrief description: The commands in this script will perform all of the data analyses described in this paper and generate all of the figures. The script is included here to enable readers to verify the findings of this study.File: oo_73745.RMatthew T WaylandSupplementary material 2Echinorhynchusgadi spp.Area of hooks from Data type: morphometricBrief description: Comma separated value file with the following nine columns: species, sex, specimen, hook, position, length, base, measured_area and estimated_area.E.gadi group species - species of the sex - sex of acanthocephalanhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)measured_area - cross-sectional area (square micrometres) of a hook viewed in profile and measured using a digitising tabletestimated_area - cross-sectional area (square micrometres) of hook estimated using the formula: area = length x base / 2http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63675.csvMatthew T WaylandSupplementary material 3EchinorhynchusbothniensisHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)Source: Wayland (2013)File: oo_63705.csvMatthew T WaylandSupplementary material 4EchinorhynchusbothniensisHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)Source: Wayland (2013)File: oo_63706.csvMatthew T WaylandSupplementary material 5Echinorhynchus 'bothniensis'Hook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)Source: Wayland (2013)File: oo_63707.csvMatthew T WaylandSupplementary material 6EchinorhynchusbrayiHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.62285Source: File: oo_63694.csvMatthew T WaylandSupplementary material 7EchinorhynchusbrayiHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.62285Source: File: oo_63695.csvMatthew T WaylandSupplementary material 8Echinorhynchusgadi sp. AHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63710.csvMatthew T WaylandSupplementary material 9Echinorhynchusgadi sp. AHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63711.csvMatthew T WaylandSupplementary material 10Echinorhynchusgadi sp. BHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63712.csvMatthew T WaylandSupplementary material 11Echinorhynchusgadi sp. BHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63713.csvMatthew T WaylandSupplementary material 12Echinorhynchusgadi sp. IHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63714.csvMatthew T WaylandSupplementary material 13Echinorhynchusgadi sp. IHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.hd5c7Source: File: oo_63715.csvMatthew T WaylandSupplementary material 14EchinorhynchussalmonisHook measurement data from dorsal rows of female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.v21k1Source: File: oo_63696.csvMatthew T WaylandSupplementary material 15EchinorhynchussalmonisHook measurement data from ventral rows of female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.v21k1Source: File: oo_63699.csvMatthew T WaylandSupplementary material 16EchinorhynchussalmonisHook measurement data from dorsal rows of male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.v21k1Source: File: oo_63701.csvMatthew T WaylandSupplementary material 17EchinorhynchussalmonisHook measurement data from ventral rows of male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)http://dx.doi.org/10.5061/dryad.v21k1Source: File: oo_63703.csvMatthew T WaylandSupplementary material 18Echinorhynchustruttae from Drummore, southwest ScotlandHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)Source: Wayland (2013)File: oo_68461.csvMatthew T WaylandSupplementary material 19Echinorhynchustruttae from Drummore, southwest ScotlandHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of hook blade (micrometres)base - width of hook base (micrometres)Source: Wayland (2013)File: oo_68463.csvMatthew T WaylandSupplementary material 20Echinorhynchustruttae from the River Carron catchment, central ScotlandHook measurement data from female Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of the hook blade (micrometres)base - width of the hook base (micrometres)Source: Wayland (2013)File: oo_68464.csvMatthew T WaylandSupplementary material 21Echinorhynchustruttae from the River Carron catchment, central ScotlandHook measurement data from male Data type: morphometricBrief description: The file is a comma separated value (CSV) format suitable for input to the Meristogram software. The file has four columns: specimen, hook, length, base.specimen - unique identifier for the specimenhook - numerical position of hook in longitudinal row as counted from the distal end of the proboscislength - length of the hook blade (micrometres)base - width of the hook base (micrometres)Source: Wayland (2013)File: oo_68466.csvMatthew T WaylandSupplementary material 22EchinorhynchusbothniensisMeristograms for female and male Data type: morphologicalEchinorhynchusbothniensis. For each sex Meristograms were generated with the following moving average intervals: 9, 14 and 18%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for five female and five male specimens of File: oo_64487.pdfMatthew T WaylandSupplementary material 23Echinorhynchus 'bothniensis'Meristograms for female Data type: morphologyEchinorhynchusbrayi, generated with the following moving average intervals: 8, 12 and 16%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for two female specimens of File: oo_64488.pdfMatthew T WaylandSupplementary material 24EchinorhynchusbrayiMeristograms for female and male Data type: morphologicalEchinorhynchusbrayi. For each sex Meristograms were generated with the following moving average intervals: 17, 26 and 34%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for four female and seven male specimens of File: oo_64449.pdfMatthew T WaylandSupplementary material 25Echinorhynchusgadi sp. AMeristograms for female and male Data type: morphologicalEchinorhynchusgadi sp. A. For each sex Meristograms were generated with the following moving average intervals: 9, 14 and 18%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for four female and two male specimens of File: oo_64463.pdfMatthew T WaylandSupplementary material 26Echinorhynchusgadi sp. BMeristograms for female and male Data type: morphologicalEchinorhynchusgadi sp. B. For each sex Meristograms were generated with the following moving average intervals: 8, 12 and 16%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for four female and four male specimens of File: oo_64464.pdfMatthew T WaylandSupplementary material 27Echinorhynchusgadi sp. IMeristograms for female and male Data type: morphologicalEchinorhynchusgadi sp. I. For each sex Meristograms were generated with the following moving average intervals: 8, 12 and 16%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for five female and one male specimens of File: oo_64466.pdfMatthew T WaylandSupplementary material 28EchinorhynchussalmonisMeristograms for female and male Data type: morphologicalEchinorhynchussalmonis. For each sex and proboscis surface  Meristograms were generated with the following moving average intervals: 10, 15 and 20%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for 36 female and 6 male specimens of File: oo_64457.pdfMatthew T WaylandSupplementary material 29EchinorhynchustruttaeMeristograms for female and male Data type: morphologicalEchinorhynchustruttae from two different geographical locations: Drummore, southwest Scotland  and the River Carron catchment, central Scotland . For each sex and locality Meristograms were generated with the following moving average intervals: 10, 15 and 20%. These intervals correspond to the minimum moving average interval (MMAI) for this collection of worms, 1.5 x MMAI and 2.0 x MMAI, respectively.Brief description: Meristograms for File: oo_68417.pdfMatthew T Wayland


Sporisorium scitamineum is the fungus that causes sugarcane smut. This fungus has the potential to infect all sugarcane species unless a species is resistant to biotrophic fungal pathogens. However, it remains unclear how the fungus breaks through the cell walls of sugarcane and causes the formation of black or gray whip-like structures on the sugarcane plants.Sugarcane smut can cause losses in cane yield and sugar content that range from 30% to total crop failure. Losses tend to increase with the passage of years. S. scitamineum assembled de novo with a contig N50 of 41 kb, a scaffold N50 of 884 kb and genome size 19.8 Mb, containing an estimated 6,636 genes. This phytopathogen can utilize a wide range of carbon and nitrogen sources. A reduced set of genes encoding plant cell wall hydrolytic enzymes leads to its biotrophic lifestyle, in which damage to the host should be minimized. As a bipolar mating fungus, a and b loci are linked and the mating-type locus segregates as a single locus. The S. scitamineum genome has only 6 G protein-coupled receptors (GPCRs) grouped into five classes, which are responsible for transducing extracellular signals into intracellular responses, however, the genome is without any PTH11-like GPCR. There are 192 virulence associated genes in the genome of S. scitamineum, among which 31 expressed in all the stages, which mainly encode for energy metabolism and redox of short-chain compound related enzymes. Sixty-eight candidates for secreted effector proteins (CSEPs) were found in the genome of S. scitamineum, and 32 of them expressed in the different stages of sugarcane infection, which are probably involved in infection and/or triggering defense responses. There are two non-ribosomal peptide synthetase (NRPS) gene clusters that are involved in the generation of ferrichrome and ferrichrome A, while the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes.Here, we report the first high-quality genome sequence of S. scitamineum genome will facilitate future research on the genomic basis and the pathogenic mechanisms of sugarcane smut.As a destructive pathogen to sugar industry, the The online version of this article (doi:10.1186/1471-2164-15-996) contains supplementary material, which is available to authorized users. Saccharum genus of the Poaceae family , an economically important seed plant family that includes maize, wheat, rice, sorghum, and many other forage crops. Sugarcane is the world’s largest crop. In 2012, the Food and Agriculture Organization estimates that it was cultivated in 101 countries, on approximately 26.1 million hectares, and with a worldwide harvest of 1.83 billion tons. The 10-year trend for sugarcane production has been upward. Sugarcane is not only a cash crop, but it can also be used as livestock fodder. The main product of sugarcane is sucrose, which accounts for 80% of all sugar produced in the world and 92% of that in China. It is also used as raw material in human food industries  and is fermented to produce sugarcane ethanol. Sugarcane ethanol represents 40% of the world’s total ethanol fuel.Sugarcane belongs to the Sporisorium scitamineum. Sugarcane smut has been an increasing large problem in almost all countries where sugarcane is grown. It was first reported in 1877 in the Natal region of South Africa. The genome version described in this paper is the first version and the accession number is [JFOL01000000].S. scitamineum genome assembly and scaffolding. Table S2. Gene prediction in the S. scitamineum genome. Table S3. Protein database annotation result for proteins of S. scitamineum. Table S4. Noncoding genes in the S. scitamineum genome. Table S5. Repeat elements in the S. scitamineum genome. Table S6. Expression of candidate genes for encoding pant cell wall degradation enzymes of S. scitamineum at six stages in the infection process. Table S7. Candidate virulence associated genes of S. scitamineum compared against pathogen-host interaction (PHI) database and their expression at six stages in the infection process. Table S8. Candidate genes for Secreted Effector Proteins in the S. scitamineum genome (aa length > 200 aa & Cys (%) > 1.5%) and their expression at six stages in the infection process. Table S9. Sixty-five compounds in the elicitor of S. scitamineum detected by GC-MS. Table S10. Primer information of Real-time quantitative PCR for candidate virulent factors. (XLSX 72 KB)Additional file 1: Table S1: Data used for (a) 15-mer depth distribution of 32 X data. Analysis X-coordinate is depth, and Y-coordinate is frequency. (b) GC content and depth correlative analysis. X-coordinate is GC content, and Y-coordinate is average depth. Through calculating GC content and average depth, we can analyze whether GC bias exists. If not seriously biased, this scatter diagram takes on the shape similar to Poisson distribution, that is there will be a peak near the GC content of the genome, and the more deviation from it, the lower the depth is. Figure S2. Pep1 is conserved among Sporisorium scitamineum and other sequenced smut fungi. (a) Sequence alignment of S. scitamineum Pep1 (Ss), S reilianum Pep1 (Sr), U. maydis Pep1 (Um), and U. hordei Pep1 (Uh). Identical amino acids are highlighted in green. Red boxes: conserved cysteine residues; black box: putative N-terminal secretion signal; blue box: poorly conserved glycine-rich C-terminal region. (b) Phylogenetic tree based on Pep1 multiple sequence alignment. (DOCX 844 KB)Additional file 2: Figure S1: The K-mer distribution and GC-depth analysis.


When planning clinical trials, it is a key element to choose appropriate outcomes that ensure the comparability of effects of interventions in ways that minimise bias. We hypothesise that outcome measures in cardiothoracic surgical trials are inconsistent and without standard. Therefore, comparing the relative effectiveness of interventions across studies is problematic. We surmise that cardiothoracic research has focused habitually on the identification of risk factors and on the reduction of adverse outcomes with less consideration of factors that contribute to well being and positive health outcomes .We conducted a systematic review of reviews to determine both the type and number of outcomes reported in current cardiothoracic surgery interventional research, in order to identify a list of potential outcomes for a minimum core outcome set (COS). Special focus was placed on outcomes that emphasise salutogenesis. We interpreted salutogenic outcomes as those relating to optimum and/or positive health and well being. We searched Issue 7 (July 2014) of the Cochrane Database of Systematic Reviews. Systematic reviews of randomised trials on non-minimal-invasive off- or on-pump cardiothoracic surgery  investigating pre-, intra- or postsurgical interventions related to the outcome of the procedure were eligible for inclusion. We excluded protocols and withdrawn systematic reviews. Two review authors extracted outcome data independently. Unique lists of salutogenically and non-salutogenically focused outcomes were established. 15 systematic reviews involving 371 randomized trials and 58,253 patients were included in this review. Applied definitions of single and composite endpoints varied significantly, and patient-centred, salutogenically focused outcomes were seldom reported. One third of included reviews did not assess patient-centred outcomes at all; all other reviews were unable to perform meta-analyses due to an absence of data or heterogeneity in outcome measures. This compares to 36 non-salutogenically focused outcome domains representing 121 individual non-salutogenically focused outcomes, whereof 50% were assessed only once. Measures of mortality, cerebrovascular complications and hospitalisation were reported most frequently. Two reviews chose a composite endpoint as primary outcome. Pooled analysis of composite endpoints was not possible, as the required data was not reported per patient in all components.In cardiothoracic surgical trials, choice and definition of non-salutogenically focused single and composite outcomes are inconsistent. There is an absence of patient centred, salutogenically focused outcome parameters in cardiac trials. We recommend the development of a core outcome set of salutogenically focused and non-salutogenically focused outcomes for cardiothoracic surgical research. When planning clinical trials, it is essential to choose appropriate outcomes to assure the comparability of effects of interventions in ways that minimise bias. However, there is a growing body of evidence indicating that inadequate attention has been paid to the outcomes measured in clinical trials; choice and definitions of outcome measures across trials vary considerably ,2. This Patients scheduled for cardiac surgery are at significant risk for the development of major adverse events during the postoperative course. Although surgical outcome has significantly improved over the last decade, cardiac surgery is generally associated with high incidences of myocardial, neurological and renal dysfunction, which account for up to 10% mortality, depending on the complexity of the surgical procedure, age, sex and co-morbidities –10. HencWith the major focus being the reduction of severe adverse events, it is evident that there is little focus of what contributes to, or enhances, health and the well being of cardiac patients or how salutogenically focused outcomes could complement cardiothoracic surgical research. The term salutogenesis describes an approach focussing on factors that promote health and personal well being rather than on factors that cause diseases. It is a stress resource orientated concept, first introduced by Aaron Antonovsky in the late 1990’s , which fthe process of enabling people to increase control over, and to improve, their health. To reach a state of complete physical, mental and social well being, an individual or group must be able to identify and to realize aspirations, to satisfy needs, and to change or cope with the environment. Health is, therefore, seen as a resource for everyday life, not the objective of living. Health is a positive concept emphasizing social and personal resources, as well as physical capacities. Therefore, health promotion is not just the responsibility of the health sector, but goes beyond healthy life-styles to well being“. However, we hypothesise that cardiothoracic research continues to focus on factors that cause diseases, how those factors could be influenced, and how the diseases can be treated.For nearly thirty years, the World Health Organisation (WHO)  has advohttp://www.comet-initiative.org. Minimum core outcome sets are well established in several clinical areas [Recent publications strongly recommend the development and use of agreed core outcome sets (COS), which should be measured and reported as a minimum in all trials for a specific clinical area ,14, alsoal areas –18, whichttp://www.omeract.org. The OMERACT Initiative is still very active today; focusing on different appearances of the disorder as well as patient reported outcomes (PRO’s) and quality of life assessment. The OMERACT Initiative is an impressive example of how patient stakeholders can influence research. After ten years, the OMERACT Initiative had to re-evaluate their core outcome set, despite of its success. The new core set omitted several outcomes of major importance to patients. One of these was fatigue, which is now included in the current version of the core outcome set on rheumatoid arthritis.In the early 1970’s, the WHO became aware of this enduring problem. Two meetings were held (1977 in Turin and 1979 in Brussels) in order to develop a “common language” to describe cancer treatment, and to agree upon internationally acceptable general principles for evaluating data ,17 that A minimum COS for cardiothoracic surgical research would address the problems depicted above. However, a recently published systematic review  on availhttp://www.comet-initiative.org/studies/details/630?result=true). The conduct and reporting of this review adheres to, as much as practicable, the standards of the Cochrane Collaboration as outlined in the Cochrane Handbook [In preparation of this systematic review, a protocol was composed . The revHandbook  and the Handbook , S1 ChecWe considered systematic reviews of randomised controlled trials for non-minimal-invasive off or on-pump cardiothoracic clinical trials  investigating any pre-, intra- or postsurgical interventions (participants > 18 years of age) for inclusion. Pre-, intra- and postsurgical interventions are defined as any intervention that occurred before, during or after cardiac surgery and were related to the outcome of the procedure. We excluded protocols for systematic reviews and systematic reviews that had been withdrawn.We searched Issue 7 (July 2014) of the Cochrane Database of Systematic Reviews for all reviews published by the Cochrane Heart Group, using the advanced search option limited to “all text” and reviews published by the Cochrane Heart Group using “*” in the search box. Citations and abstracts were exported to EndNote X4 . Each citation was then independently reviewed by two authors for inclusion via two complementary screening levels: level 1 = title or title and abstract screening, level 2 = full-text screening of citations judged relevant or considered relevant during screening level 1. Any disagreement of our judgements as to whether a review should be included was resolved through discussion and, if required, consultation with the team. Data was independently extracted from each included review by two review authors using a purposively pre-developed data extraction form. Any disagreement was resolved through discussion and, if required, consultation with the team for consensus. For the purpose of this review, we adopted the definition of a salutogenically focused outcome introduced by Smith and colleagues , which wWe identified 15 systematic reviews that matched our inclusion criteria, presenting data on 371 randomised controlled trials. The trials were performed between 1971 and 2013, comprising statistics on 58,253 participants. One third of the included systematic reviews evaluated a pre-surgical intervention (n = 5), and each of the four publications presented data for an intra- or postsurgical intervention. Two reviews were not limited to a pre-, intra- or postsurgical timeframe. Following this process, we identified two salutogenically focused outcomes , collaps“Mortality”, 13 different definitions and/or terms were used in 15 systematic reviews. This can be transferred to all other non-salutogenically focused outcome domains accordingly. Two of the included systematic reviews chose a composite outcome as a primary endpoint; both composite endpoints varied significantly. Both systematic reviews were unable to perform a pooled analysis since necessary data was not reported on patient level for all composites of the combined endpoint.In contrast, we were able to extract data of 121 individual non-salutogenically focused outcomes reported in the included reviews. Those 121 unique non-salutogenically focused outcomes collapsed into 36 outcome domains. Overall, 50% of reported outcomes were reported only once. To our knowledge, this is the first approach to determine the type and number of salutogenically focused and non-salutogenically focused outcomes reported in current cardiothoracic surgery interventional research. In the 371 randomised controlled trials  comprised in the 15 systematic reviews included in our study, we revealed that patient centred, salutogenically focused outcomes were seldom reported. Measures of quality of life (n = 9) and beneficial events  were intended, but analysed in none of the meta-analyses due to lack of data in included studies, heterogeneity in outcome measurement and inconsistency in reporting. The near complete absence of salutogenically focused outcomes contrasts to a unique list of 36 non-salutogenically focused outcome domains representing 121 individually reported outcomes across all reviews. Major outcome variables  were analysed and published most commonly. Utilised definitions of single and composite endpoints showed an alarming inconsistency for the “same” outcome. Overall, 50% of all outcomes had appeared only once in included reviews.The findings of our review support the hypothesis that the effectiveness of cardiothoracic surgery interventional research is measured against adverse effects, rather than increases in measures of health and well being. Patient-centred, salutogenically focused outcome parameters are not currently an inherent part of cardiothoracic clinical research. Avoidance of adverse events is a crucial element when exploring effectiveness of interventions for cardiothoracic surgical patients, and should remain an important component of cardiac surgical trials. However, the consistent, exclusive focus on risk reduction continues to form the basis for policy and practice development, without taking patients’ perception or evaluation of their condition, or quality of care and quality of life into consideration. When taking the contemporary curative nature of cardiothoracic surgery into account, it is vital that the perspective of all stakeholders—above all the patients’ perspective—is considered when deciding on outcome measurement. Here, the phrasing of clinical endpoints chosen by review authors to report on “any beneficial (…) events related to this review”  already At present, 3.127 clinical trials are registered at the “International Clinical Trials Registry Platform” (ICTRP) of the World Health Organisation , investiFurthermore, we were able to demonstrate a considerable inconsistency in choice and definition of single and composite outcomes in cardiothoracic interventional surgical trials. Even for major outcome variables , which are measured most frequently in cardiac trials, no approved definition is internationally recognised. The terminology used, while presumably aiming to characterise the same variable, is deceptive and misleading . This is further complicated by combination with a certain period of time . Again, the wording of systematic reviewers to analyse “mortality within the commonly accepted definitions of either prior to discharge or within thirty days”  as a priper se, but rather on reported single and composite endpoints and whether they were salutogenically focused or not. Although our search was limited to randomised controlled trials included in systematic reviews published by the Cochrane Heart Group , we were able to clearly demonstrate the necessity for the postulated minimum core outcome set, and we believe that all of our recommendations are justified on the basis of the evidence we have cited.Focusing so exclusively limits our review to a select patient group and to measurements of effectiveness against outcomes specific for a certain period. However, our review was not intentionally focused on a specific population, nor on any particular intervention There is an absence of salutogenically focused outcome parameters in cardiothoracic surgery interventional research. In the present review, we highlighted inconsistency and heterogeneity in reporting clinical endpoints of cardiac surgical studies. We recommend the development of a minimum core outcome set of salutogenically focused and non-salutogenically focused outcomes for intervention-based cardiothoracic surgical research.10.1002/14651858.CD003611.pub3.Arsenault KA, Yusuf AM, Crystal E, Healey JS, Morillo CA, Nair GM, Whitlock RP. Interventions for preventing postoperative atrial fibrillation in patients undergoing heart surgery. Cochrane Database of Systematic Reviews 2013, Issue 1. Art. No.: CD003611. DOI: 10.1002/14651858.CD004588.pub3.Bakhai A, Hill RA, Dundar Y, Dickson RC, Walley T. Percutaneous transluminal coronary angioplasty with stents versus coronary artery bypass grafting for people with stable angina or acute coronary syndromes. Cochrane Database of Systematic Reviews 2005, Issue 1. Art. No.: CD004588. DOI: 10.1002/14651858.CD007131.pub3.Karmali KN, Davies P, Taylor F, Beswick A, Martin N, Ebrahim S. Promoting patient uptake and adherence in cardiac rehabilitation.CochraneDatabase of SystematicReviews 2014, Issue 6.Art.No.:CD007131.DOI: 10.1002/14651858.CD005566.pub3.Dieleman JM, van Paassen J, van Dijk D, Arbous MS, Kalkman CJ, Vandenbroucke JP, van der Heijden GJ, Dekkers OM. Prophylactic corticosteroids for cardiopulmonary bypass in adults. Cochrane Database of Systematic Reviews 2011, Issue 5. Art. No.: CD005566. DOI: 10.1002/14651858.CD004466.pub3.Freitas ERFS, Soares BGO, Cardoso JR, Atallah ÁN. Incentive spirometry for preventing pulmonary complications after coronary artery bypass graft. Cochrane Database of Systematic Reviews 2012, Issue 9. Art. No.: CD004466. DOI: 10.1002/14651858.CD004815.pub3.Hoenig MR, Aroney CN, Scott IA. Early invasive versus conservative strategies for unstable angina and non-ST elevation myocardial infarction in the stent era. Cochrane Database of Systematic Reviews 2010, Issue 3. Art. No.: CD004815. DOI: 10.1002/14651858.CD010118.pub2.Hulzebos EHJ, Smit Y,Helders PPJM, vanMeeterenNLU. Preoperative physical therapy for elective cardiac surgery patients. Cochrane Database of Systematic Reviews 2012, Issue 11. Art. No.: CD010118. DOI: 10.1002/14651858.CD008493.pub2.Liakopoulos OJ, Kuhn EW, Slottosch I, Wassmer G,Wahlers T. Preoperative statin therapy for patients undergoing cardiac surgery. Cochrane Database of Systematic Reviews 2012, Issue 4. Art. No.: CD008493. DOI: 10.1002/14651858.CD003464.pub2.Massel DR, Little SH. Antiplatelet and anticoagulation for patients with prosthetic heart valves. Cochrane Database of Systematic Reviews 2013, Issue 7. Art. No.: CD003464. DOI: 10.1002/14651858.CD007224.pub2.Møller CH, Penninga L, Wetterslev J, Steinbrüchel DA, Gluud C. Off-pump versus on-pump coronary artery bypass grafting for ischaemic heart disease. Cochrane Database of Systematic Reviews 2012, Issue 3. Art. No.: CD007224. DOI: 10.1002/14651858.CD002138.Rees K, Beranek-Stanley M, Burke M, Ebrahim S. Hypothermia to reduce neurological damage following coronary artery bypass surgery. Cochrane Database of Systematic Reviews 2001, Issue 1. Art. No.: CD002138. DOI: 10.1002/14651858.CD009507.pub2.Spencer S, Tang A, Khoshbin E. Leukodepletion for patients undergoing heart valve surgery. Cochrane Database of Systematic Reviews 2013, Issue 7. Art. No.: CD009507. DOI: 10.1002/14651858.CD004472.pub3.Theologou T, Bashir M, Rengarajan A, Khan O, Spyt T, Richens D, Field M. Preoperative intra aortic balloon pumps in patients undergoing coronary artery bypass grafting. Cochrane Database of Systematic Reviews 2011, Issue 1. Art. No.: CD004472. DOI: 10.1002/14651858.CD007398.pub2.Unverzagt S, MachemerMT, Solms A, Thiele H, Burkhoff D, SeyfarthM, deWaha A, Ohman EM, Buerke M, Haerting J, Werdan K, Prondzinsky R. Intra-aortic balloon pump counterpulsation (IABP) for myocardial infarction complicated by cardiogenic shock. Cochrane Database of Systematic Reviews 2011, Issue 7. Art. No.: CD007398. DOI: 10.1002/14651858.CD003042.pub2.Wallen MA, Morrison AL, Gillies D, O’Riordan E, Bridge C, Stoddart F. Mediastinal chest drain clearance for cardiac surgery. Cochrane Database of Systematic Reviews 2002, Issue 2. Art. No.: CD003042. DOI: S1 Protocol(PDF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.


Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea  secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites.  Cladosporium fulvum [Zymoseptoria tritici [Magnaporthe oryzae [Stagnospora nodorum and Pyrenophora tritici-repentis, which cause disease on wheat, secrete the ToxA host-selective proteinaceous toxin that induces host cell death and susceptibility in wheat genotypes that harbour the corresponding Tsn1 toxin sensitivity gene [Phytopathogenic fungi form intimate associations with specific host plant species and cause disease. To be successful, each fungal pathogen, whether growing within or between living plant cells, or amongst dead plant tissue, must continually communicate with a susceptible host. Communication is achieved through the microbe secreting a cocktail of proteins, enzymes and metabolites which are either delivered into the plant cell or detected at the plant cell surface . Secretem fulvum ,3, Mg3Ly tritici  and Slp1e oryzae . Only inity gene ,7. Henceity gene .Sclerotinia sclerotiorum is an economically important fungal pathogen that causes annual losses in excess of $200 million in the United States. S. sclerotiorum forms melanised hyphal aggregates called sclerotia, from which apotheica or fruiting bodies germinate and release pathogenic ascospores. S. sclerotiorum causes disease on over 400 plant species, including the crop species; oilseed rape (Brassica napus), soybean (Glycine max), sunflower (Helianthus annuus) and many vegetable plants [S. sclerotiorum is considered to cause disease on all hosts by using a solely necrotrophic lifestyle [S. sclerotiorum is the secretion of oxalic acid during infection. This seemingly simple organic acid regulates a range of functions related to infection including, lowering the pH to increase the activity of polygalaturonases [S. sclerotiorum resistant crops, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), there are elevated levels of germin or oxalate oxidase, an enzyme capable of breaking down oxalic acid into CO2 and H2O2 [e plants . S. scleifestyle . Typicalifestyle . Centraluronases , suppresuronases  and alteuronases . By contand H2O2 . This laand H2O2  and Sspgand H2O2 , a fungaand H2O2  and an uand H2O2 .Botrytis cinerea is also an economically important plant necrotrophic pathogen of soft fruits, vegetables and flowers and is one of the most devastating plant pathogens of the grapevine. Its host range of over 200 plant species is comparatively smaller than S. sclerotiorum and disease symptoms are remarkably different. B. cinerea infection is characterised by grey masses of conidia which appear on infected fruit organs, including flowers, fruits, leaves, shoots and soil storage organs [B. cinerea also produce small sclerotia in necrotic tissues from which conidiophores and multinucleate conidia form [B. cinerea to infect dicotyledonous crop species is somewhat different from S. sclerotiorum and appears to involve the use of an arsenal of secreted cell wall degrading enzymes, the production of reactive oxygen species (ROS) via an NADPH oxidase [et al. [B. cinerea can secrete small RNAs that suppress plant immunity by hijacking specific host RNAs.e organs . B. cinedia form . The gro oxidase , the sec oxidase –23. Also [et al.  have demS. sclerotiorum and B. cinerea genomes revealed a high level of co-linearity and identity between these two closely related necrotrophs, in addition to a similar arsenal of genes associated with necrotrophic processes, such as plant cell wall degradation, in particular pectin, and oxalic acid production, plus the expansion B. cinerea-specific secondary metabolites [S. sclerotiorum—B. cinerea genome comparison, genes encoding for proteins with N-terminal signal peptides were identified by SignalP predictions, then the CAZyme and peptidase encoding genes were removed, resulting in 603 S. sclerotiorum and 879 B. cinerea genes encoding for candidate secreted effector proteins [S. sclerotiorum bioinformatics study, aimed at identifying effector-like proteins, combined evolutionary genetics and transcriptomics to predicted 486 in planta expressed secreted proteins and 70 S. sclerotiorum-specific candidate effectors [in planta was not explored, potentially excluding the involvement of the secretome in apothecia and/or sclerotia development. Subsequently, a complete, comparative, analysis of the entire S. sclerotiorum and B. cinerea secretome is required to characterise the repertoire, physical organisation, conservation and regulation of the secretomes from these two necrotrphic fungi.A comparative analysis of the sequenced abolites . In the proteins . An addiffectors . HoweverS. sclerotiorum and B. cinerea secretomes. This comprehensive approach revealed the genomic organisation and functional profile of the secretomes, plus identified candidate effectors. A wide-ranging interspecies comparison of the S. sclerotiorum and B. cinerea secretomes with 115 fungi and oomycetes revealed the species specific and non-specific secreted proteins. Finally, in vitro and in planta expression patterns, plus proteomics analyses, provided support for the secretion of identified proteins and insights into possible function in infection and apothecia/ sclerotia development. By investigating the arsenal of proteins secreted by S. sclerotiorum and B. cinerea, a greater understanding of the necrotrophic infection process may be gained, while additional secreted proteins with biological relevance to fungal development and infection may be identified.Previously studies –29 combiS. sclerotiorum genome of the ‘1980’ strain (ATCC18683) and the B cineria B05.10 genome sequences were downloaded from the Broad Institute [S. sclerotiorum and B. cinerea genomes. Within this gene set, proteins predicted to contain no transmembrane domain (TM) or a single TM in, or just beyond, the signal peptides, plus GPI anchored proteins, were retained. Finally, a ProtComp analysis excluded proteins not predicted to be located in the extracellular space. This resulted in a predicted secretome size of 1,060 and 1,262 proteins for S. sclerotiorum and B. cinerea respectively   which, while SS1G_00891 and SS1G_00892 in gene cluster C012 were both annotated as endoglucanases. Overall, this cluster analysis revealed that 16% of genes coding for the refined S. sclerotiorum secretome (i.e. 69 genes) resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes.The refined nome map ; Fig 2.  . The Blast2GO analysis revealed that 310 S. sclerotiorum and 328 B. cinerea sequences had some form of annotation, i.e. an InterPro (IPO), Gene3D, SUPERFAMILY or PFAM entry, while 122 S. sclerotiorum and 171 B. cinerea sequences had no functional annotation. Proportionally, the S. sclerotiorum secretome possessed a slightly greater amount of functionally annotated proteins than B. cinerea  and the pentose/ glucornate pathways, reflecting the abundance of enzymes required to breakdown plant polysaccharides. Evaluation of the biological processes assigned to the proteins within either secretome confirmed the high representation of polysaccharide catabolic and proteolytic processes in both secretomes, while also showing the greater diversification of function in the S. sclerotiorum secretome  and biological processes (GO annotations) of the refined ecretome .S. sclerotiorum and B. cinerea were identified. Four S. sclerotiorum proteins required for infection were identified in the refined secretome, including the SsCUTA cutinase, the Sspg1 polygalacturonase, a fungal integrin-like protein SsITL, and a novel hypothetical protein, Ssv263   was increased in the B. cinerea secretome compared to S. sclerotiorum. The refined S. sclerotiorum and B. cinerea secretomes also respectively contained 24 and 33 genes involved in lipid degradation and 37 and 32 genes involved in protein degradation  domains were identified within the S. sclerotiorum and B. cinerea secretomes. These enzymes use FAD as a co-factor and are mainly oxygen-dependent oxidoreductases (IPR006094). In fungi, these enzymes are involved in many process including the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation, mainly melanin production [S. sclerotiorum and B. cinerea secretomes. This protein is a heme-containing glycoprotein that is secreted by various fungi (IPR000028) and performs a range of diverse functions including facilitating the decomposition of hydrogen peroxide to oxygen and water and catalysing chloroperoxidase P450-like oxygen insertion reactions (IPR000028). These enzymes have also been described to have lignin degradation activity as they are potential chlorinators of lignin [Beyond the domains potentially involved in plant cell degradation, the Blast2GO analysis describes numerous proteins involved oxidation-reduction processes within the oduction ,36. Seveoduction . Severalf lignin ,39.S. sclerotiorum and B. cinerea secretomes, which are crucial for the degradation of oxalic acid, which is secreted at very high levels and could become potentially toxic if the pathogen does not locally hydrolyse this pathogenicity determinant [S. sclerotiorum the degradation of oxalic acid is important for the regulation of pH-dependent expression of genes during a later stage of the infection process [S. sclerotiorum and B. cinerea secretomes included the necrosis-inducing protein (PF05630) plus the cerato-ulmin hydrophobin (PF06766) and the cerato-platanin (PF07249). Interestingly, the S. sclerotiorum secretome possessed more chitin-binding domains (PF00187) than B. cinerea, which also lacked any LysM (PF01476), CFEM (PF05730), PAN-1 (PF00024) or metallophosphoesterase (PF00149) domains. Hence, the S. sclerotiorum secretome has an increased representation of several effector-like protein domains.Additional pathogenicity-related PFAM domains were identified in the two refined secretomes. Two cupin domains (PF00190) were present in the oxalate decarboxylase enzymes in the erminant ,41. In a process ,42. BothBlumeria graminis f. sp. hordei and wheat infecting basidiomycete rust species, Puccinia graminis f. sp. tritici [Effectors of plant infecting oomycetes and animal infecting malaria parasites, but not fungi, possess a conserved RXLR-dEER motif that facilitates secretion and uptake into the host cell, resulting in the modification of host transcription –46. A st tritici . Formal  tritici .S. sclerotiorum and B. cinerea secretomes were inspected for protein sequences containing RXLR, dEER and Y/F/WxC motifs and their proximity to the N-terminal signal peptide. In total, 21 S. sclerotiorum and 18 B. cinerea proteins from the refined secretomes contain a RXLR motif , dioxygenases  and unannotated proteins . These RXLR containing sequences did not possess a dEER motif downstream of the RXLR domain. None of these S. sclerotiorum genes were found in the physical gene clusters.The refined S. sclerotiorum and 11 B. cinerea protein sequences contained a Y/F/WxC motif within 16 and 20 base pairs of the signal peptide, respectively , an aspartate protease and a histidine acid phosphatase. Of these, SS1G_04662, an alpha-galactosidase was found in gene cluster C005 , a carboxylesterase, an aminopeptidase and a TOS1-like glycosyl hydrolase, plus a carbohydrate binding module. In both species, a homologue of candidate effector 5, from the apple scab fungus Venturia inaequalis [Eutypa lata [Eight ter C005 ; Table 3aequalis  and the ypa lata , with a S. sclerotiorum and B. cinerea secretomes. Both sets of unannotated sequences were inspected for the presence of small (less than 200 amino acids), cysteine-rich proteins (5% or greater), a sequence profile that has previously been used to predict candidate effector genes [C. fulvum plant infection and prevents PAMP triggered recognition by the plant chitin receptors [Z. tritici [S. sclerotiorum refined secretome, 38 proteins had >5% cysteine residues of which 22 were less than 200 amino acids in length and had 6 or more cysteine residues in the mature protein . Most genes had single copies within the genomes except for SS1G_00263/BC1G_00896, SS1G_01086/BC1G_12867, and SS1G_04312/BC1G_12229 where 2 copies are found in both species. SS1G_09841, SS1G_01086 and SS1G_00768 were found in the identified gene clusters C001, C003 and C011, respectively.The entire multispecies dataset was explored for shared S. sclerotiorum and B. cinerea secretomes contain at least one gene homologue (e-100) in 110 and 111 of the 115 species, respectively  (Table 1 in B. cinerea shared the highest number of common sequences (92%). This result was anticipated because these two species share 84% of their total proteomes [S. sclerotiorum secretome was Botryosphaeria dothidea, an ascomycete plant pathogen which infects mainly woody species and shrubs. This proteome shares approximately 77% homology (331 proteins) with the S. sclerotiorum secretome  with S. sclerotiorum. The citrus infecting pathogen, Rhytidhysteron rufulum, which is also a dothideomycete shared 308 proteins (71%) with S. sclerotiorum. Monocotyledonous infecting ascomycete pathogens such as Fusarium verticillioides, Pyrenophora tritici-repentis and Gaeumannomyces graminis shared 596 (56%), 551 (52%) and 496 (47%) proteins respectively.The refined roteomes . The nexS. sclerotiorum and B. cinerea secretomes shared approximately 67% of sequences with Aspergilli and 75% with Fusarium oxysporum, all of which have a saprophytic plus either an animal pathogenic or plant pathogenic lifestyles that is very different to S. sclerotiorum [F. oxysporum can produce oxalic acid in large quantities in vitro [S. sclerotiorum and B. cinerea, the role of this secreted metabolite in animal and plant pathogenicity is unclear [S. sclerotiorum and B. cinerea secretomes. Other phyla of fungi including basidiomycetes share much less homology with the S. sclerotiorum (less than 60%). The chromalveolata phyla which contain oomycete plant pathogens such as Phytophthora spp share no greater than 36% proteome homology with the S. sclerotiorum secretome.The refined rotiorum ,52. Althin vitro , similar unclear . There wS. sclerotiorum and the proteome’s of Alternaria brassicicola and Leptosphaeria maculans was made to explore putative secreted proteins uniquely shared by these three fungal species which are all economically important pathogens of oilseed rape. No proteins unique to only these three species were found. A second specific investigation was made to explore plant pathogens that infect multi dicotyledonous and monocotyledonous species. Again, no genes were specific to this entire group. Although three genes found in the S. sclerotiorum and B. cinerea inter-comparison were subsequently detected only in a limited number of other fungi , 21 genes in the infection cushion library (G866), 146 genes in the infected tomato library (G2118) and 118 genes in the oxidative stress library (G2128). Collectively, this EST analysis revealed that 251 genes present in the refined secretome (58%) had EST support, and of these 101 where found only to be expressed in vitro, including during apothecia and/or sclerotia development, and 49 were found to be only expressed in planta. Similar to the EST analysis, 252 (59%) genes in the secretome were detected in the microarray analysis. Only 72 (17%) secretome-encoding genes showed a significant modulation in transcription post sunflower infection, including 44 up-regulated genes which predominantly encoded hydrolytic enzymes, and 28 down-regulated genes which mainly encoded hypothetical protein.In order to provide transcriptional support for the refined S. sclerotiorum secretome was compared to two proteomic studies which used ESI-q-TOF MS/MS and LC–MS/MS to identify secreted proteins in liquid cultures [S. sclerotiorum secretome so far contains 42 proteins that have been experimentally confirmed to be secreted.Additionally, the refined cultures  or exudacultures . Out of S. sclerotiorum sequences with hits above 40 in any of the EST libraries were investigated further to determine which genes were the most expressed under the seven different conditions explored. Twenty seven genes were identified with 40 or more hits in at least one EST library. Fifteen of the sequences with this high level of EST support have PFAM annotation and 12 sequences had no PFAM domains or IPS entries (Table 12 in S. sclerotiorum only had EST support in the apothecia development library (G787), while SS1G_13599 had an extremely high EST count of 675 in the developing sclerotia library G781, suggesting that these proteins have roles in these developmental processes.Twenty GHs encoding genes had good EST support in both the infected Brassica library (G865) and the tomato infection library (G2118), compared to 11 genes which had only low EST support in the neutral pH mycelia library (G786). This result highlights the possible specific role of these 20 proteins in plant infection rather than just fungal growth. Interestingly, SS1G_03611 only had EST support in the plant infection libraries  and the infection cushion library (G866). SS1G_03611 was predicted to encode a mature protein of 101 amino acids in length with a cysteine residue content of 7.93%. This gene was found in the small gene cluster C030  and was S. sclerotiorum secretome that are predicted to reside within small gene clusters, 8 have high EST support > 40 counts and 7 genes are supported by between 10 and 39 counts and a further 24 have at least one supporting EST  and Z. tritici  genomes [S. sclerotiorum secretome-encoding genes were evenly distributed across the 16 chromosomes whilst several small gene clusters were found that contained no more than 3 genes. These S. sclerotiorum small secretome gene clusters were similar to the F. graminearum small secretome gene clusters and dissimilar to the larger secretome gene clusters (ranging from 3 to 26 genes) in the U. maydis genome, which are known to be required for pathogenicity [S. sclerotiorum clusters did not contain any duplicated genes and only two clusters contained genes coding for similar protein families (C012 and C017). In accordance, the functionally related MCL clusters [C. fulvum CfECP6, M. oryzae Slp1 and Z. tritici Mg3LysM [A refined secretome size of 432  genomes ,29. The genicity ,58. The S. sclerotiorum  and B. cinerea  was similar to Z. tritic , while F. graminearum  possessed a higher proportion of unannotated proteins. Unsurprisingly, both the S. sclerotiorum and B. cinerea secretomes contained a large battery of hydrolysing enzymes which degrade different plant host substrates. The repertoire hydrolytic enzymes in the respective secretomes were comparable in S. sclerotiorum, B. cinerea and F. graminearum, and smaller in Z. tritici [F. graminearum exhibits a much smaller host range, predominantly infecting cereals. As suggested in previous studies [S. sclerotiorum have optimum activities at an acidic pH, fitting with the secretion of oxalic acid ahead of fungal hyphal invasion [S. sclerotiorum secretome, whereas, in B. cinerea the carboxylesterases are the most expanded gene family. This again highlights some potential differences that may underlie the subtle differences in the infection strategies deployed by these two fungal necrotrophic species.Comparatively, the proportion of the functionally annotated secretome in  tritici . Despite studies , many ofinvasion . The polS. sclerotiorum and B. cinerea secretomes revealed a large number of proteins predicted to be involved in oxidation-reduction interactions, representing oxidases involved in multiple biological process including melanin production (tyrosinase) [F. graminearum and Z. tritici secretomes. The redox state of the host substrate has been shown to be extremely important in determining the ability of S. sclerotiorum and B. cinerea to cause disease as demonstrated by the role of oxalic acid and reactive oxygen species, which are produced during infection to suppress the host plant oxidative burst and later to induce plant cell death [S. sclerotiorum and B. cinerea secretomes and would also be anticipated to influence the host or fungal redox balance. This enzyme has already been implicated in the biocontrol of other fungal species such as F. oxysporum [Z. tritici and were found to be prevalent in many other plant pathogens belonging to the Dothidiomycetes family [S. sclerotiorum and B. cinerea. These enzymes may prevent the direct toxicity of oxalic acid to the fungus by hydrolysing excess oxalic acid. This would then allow a local rise in pH, which when sensed by hyphae, could then signal the downstream regulation of other genes involved in later infection processes, for example sclerotia formation.The functional analysis of the refined osinase) , lignin osinase)  and isoaosinase) . This isll death ,13,18,19xysporum . Nine cos family . Two oxaS. sclerotiorum and B. cinerea secretomes were explored for small, cysteine-rich proteins and proteins containing RXLR-dEER or Y/F/WxC motifs, which are characteristics of known effectors [S. sclerotiorum  and B. cinerea  secretomes was significantly lower than that of F. graminearum  and Z. tritici  potentially reflecting the diversification of this protein class in hemibiotrophic ascomycetes. Nonetheless, in S. sclerotiorum several of the unannotated cysteine-rich proteins had good in planta EST support. This included a small CFEM domain-containing protein that had strong EST support in the infected plant libraries and was highly induced during the microarray analysis of sunflower infection. CFEM domains have previously been implicated in plant pathogenesis [S. sclerotiorum motif-containing candidate effectors were cysteine-rich proteins. This is in contrast to B. cinerea, which possessed four, and Z. tritici, which possessed 10, cysteine-rich proteins < 250 amino acids in length that contained a Y/F/WxC motif. Interestingly, the SS1G_02025 and BC1G_14481 proteins, which contained the Y/F/WxC motif, had good homology to a candidate effector 5 protein that has been found in two other fungal plant pathogens, namely the apple scab fungus Venturia inaequalis [Eutypa lata [The refined ffectors . The repogenesis . The seaaequalis  and the B. cinerea proteome shared the closest homology to the S. sclerotiorum secretome across all 432 proteins. This was an expected result because the similarity between the two species and their lifestyles is well documented [S. sclerotiorum primarily secretes oxalic acid in very high levels during infection whereas B. cinerea secretes oxalic acid [B. cinerea was shown to have a reduced number of, or was lacking, chitin-binding, LysM, CFEM and metallophophoesterase domain containing proteins, while the S. sclerotiorum secretome possessed multiple copies. In a recent study, B. cinerea was shown to secrete small RNAs that subsequently enter the plant cells and inhibit the expression of specific target genes [S. sclerotiorum infection of plant tissue a role for small RNAs has not been reported. Therefore, the two closely related necrotrophs may adopt different mechanisms to influences the host plant cell.The multispecies comparison confirmed that the cumented . The ~8%lic acid  as well lic acid  and botrlic acid . In thiset genes . During S. sclerotiorum and B. cinerea, whereas no secreted proteins were found to be specific to a small group of fungal pathogens that could infect oilseed rape or amongst a larger group of ascomycete plant pathogens of dicotyledonous plants. This finding is unlike the result reported for the analysis of the refined Z. tritici secretome in which nine proteins were identified as being specific to fungal pathogens infecting wheat (Triticum spp) or other cereals [http://tolweb.org/angiosperm), monocotyledonous plants are grouped as a single taxon of Angiosperms whereas the plants we now know as Dicotyledonous are classified across a range of different taxa. It is tempting to speculate that fungal pathogens with a wider host range would require a larger and more diverse arsenal of secreted proteins, some of which would be conserved or overlap with pathogens with more specific host ranges. This may explain why in this study the proteome of B. dothidea, a plant pathogen infecting woody dicotyledonous species, was found to share such a large set of proteins with the secretome of S. sclerotiorum.Additionally, the multispecies analysis identified 30 proteins specific to  cereals . One triS. sclerotiorum EST libraries was extremely valuable when trying to assign some form of function to the unannotated putative secreted proteins. EST support was available for 57% of the putative proteins within the S. sclerotiorum secretome in a range of infection conditions and developmental stages. Similarly, a microarray analysis of S. sclerotiorum grown in vitro compared with sunflower cotyledon infection also provided transcriptional support for 57% of the secretome. Two secreted proteome studies, interrogating in vitro and sclerotia development extracellular fluids, confirmed the physical secretion of 42 proteins identified in the refined S. sclerotiorum secretome, which is of significant importance when validating the bioinformatics pipeline. The other secreted proteins which were not identified in these studies may be expressed during different conditions compared to those investigated via proteomics. For example, some protein secretion will only be induced during specific in planta infection conditions or at an earlier stage of spore germination. In addition, in both proteome studies did not use the sequenced S. sclerotiorum strain which may have contributed to protein differences in the computational search.The analysis of the seven in silico characterisation of the refined S. sclerotiorum and B. cinerea secretomes has provided new insight into how secreted proteins, alongside the secreted metabolites, may contribute to the necrotrophic infection. Although no direct associations can be made, many candidate proteins have now been identified. This fundamental area of research is anticipated to expand with the identification and functional characterisation of novel types of effector proteins, which will ultimately lead to the discovery of new targets for disease intervention, while providing new options for the control of economically destructive fungal pathogens such as S. sclerotiorum and B. cinerea.In summary, this S. sclerotiorum (WT1980) and B. cinerea (B05.10) genomes used in this investigation were downloaded from the Broad Institute. Sclerotinia sclerotiorum and Botrytis cinerea Sequencing Project, Broad Institute of Harvard and MIT, (http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiHome.html and http://www.broadinstitute.org/annotation/genome/botrytis_cinerea/MultiHome.html). The S. sclerotiorum genome of the ‘1980’ strain (ATCC18683) and the B cineria B05.10 genome sequence were downloaded in 2012. The S. sclerotiorum and B. cinerea genomes were analysed using a previously described and published software pipeline [Both the pipeline –29. BrieThe first stage of the analysis followed the previously described method ,29, whichttp://www.cbs.dtu.dk/cgi-bin/nph-sw_request?targetp) or a Signal P D-score = Y  were combined [http://www.cbs.dtu.dk/cgi-bin/nph-sw_request?tmhmm) and all proteins with 0 TMs or 1 TM, if located in the predicted N-terminal signal peptide, were kept. GPI-anchor proteins were predicted by big-PI (http://mendel.imp.ac.at/gpi/cgi-bin/gpi_pred_fungi.cgi) [http://www.softberry.com). WoLF PSORT analysis was done using ‘‘runWolfPsortSummaryfungi” in the WoLF PSORT v0.2 package, which estimates where proteins are located after secretion with a sensitivity and specificity of approximately 70% [Initially all proteins from downloaded genomes with a Target P Loc = S  . ProtComtely 70% .ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd/) and the rpsblast program in the NCBI blast+ software package (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/). http://pfam.sanger.ac.uk/ was also accessed for direct inspection of protein domains. The number of cysteine residues within the mature peptide and the search for degenerative YFWxC and RXLR motifs were computed using custom python scripts. The number of internal sequence repeats was found using RADAR (http://www.ebi.ac.uk/Tools/Radar/) [S. sclerotiorum genes of interest was explored by BLASTN analysis (e-100) of the 7 designated EST libraries available from the Broad website: (http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiDownloads.html). All nucleotide and amino acid sequences were aligned in Geneious Pro5.5.6 created by Biomatters. Available from http://www.geneious.com/. ClustalW and MUSCLE alignments were used for the analysis.Additionally, PFAM analysis was done using the PFAM database (/Radar/) . The detF. graminearum [Z. tritici [http://pfam.sanger.ac.uk.The second stage of the analysis generated the refined secretome for both species from which secreted proteins for both genomes could be identified. A comparison between the refined secretomes was made to explore the relatedness and evolution of the two pathogens. Initially only sequences starting with a methionine were selected. Then sequences predicted with an extracellular WolF-PSORT score of 18 and above were kept in the final secretome dataset. This selection ‘cut-off’ point has been tested using a range of experimentally verified secreted fungal proteins from other pathogens including minearum  and Z. t tritici . Any mathttp://www.cazy.org/) [http://www.kegg.jp/) and Brenda (http://www.brenda-enzymes.info/).Genes identified in the refined secretome with a PFAM domain were inspected for gene function and grouped into the four CAZy classes; glycoside hydrolases, glycosyl transferases, polysaccharide lyases and carbohydrate esterases listed on the Carbohydrate-Active EnZymes database (CAZy) (zy.org/) . Enzyme S. sclerotiorum was generated using the free software, OmniMapFree (http://www.omnimapfree.org) [A genome map for ree.org) . This alhttp://www.blast2go.com/b2glaunch) [http://www.ebi.ac.uk/interpro/ [The protein set within the predicted refined secretome was analysed using Blast2GO to explore whether any additional gene annotation existed (glaunch) . From thnterpro/ ,72.S. sclerotiorum EST libraries were downloaded from the Broad Institute online database to determine whether there was any expression data to support the gene models of interest. The EST libraries were generated from different biological materials and culture conditions Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


Clostridium difficile infection (CDI) is a common cause of antibiotic-associated diarrhea and can be difficult to cure. Most patients with CDI present only colonopathy and in fewer cases extra intestinal features were described such as reactive arthritis (ReA). We present a series of cases with CDI-associated reactive arthritis (CDI-AReA), admitted between 2011-2014 in the National Institute for Infectious Diseases “Prof. Dr. Matei Balş”.Four patients, one male (22 years) and 3 female (median age 63 years) were diagnosed with CDI-AReA. They had monoarthritis, mild leukocytosis, elevated CRP and were treated with anti clostridial antibiotics and anti-inflammatory drugs with favorable outcome.Clostridium difficile toxin EIA was positive she was admitted in our Institute with CDI and ATLAS score 2. She received vancomycin p.o.250 mg QID for 14 days. After six days she developed left knee pain and swelling. She denied any local trauma, conjunctivitis, rash, mucous membrane lesions or dysuria. The lung, cardiovascular and genito-urinary exams were normal. Musculoskeletal exam revealed left knee swelling, tenderness and painful with movement. Laboratory studies showed CRP 18.6 mg/L but normal WBC, ESR, acid uric, CPK and rheumatoid factor. Blood culture, urine culture and serology for Chlamydia trachomatis and Borrelia were negative. Stool culture was negative for Salmonella, Shigella, Yersinia, and Campylobacter. Arthrocentesis of the left knee reveled a cloudy synovial fluid with Rivalta 3+, WBC count over 50,000/cmm with 85% neutrophil, 15% mononuclear cells, fibrin 3+, high protein level, LDH 2212 IU/L and glucose 107 mg/dL. Cristal exam, gram stain and culture were negative. X-rays of the knee was negative for abnormalities and ultrasound show important joint effusion. Diagnosis of CDI-AReA was made. The patient received nonsteroidal anti-inflammatory meloxicam 15 mg/day. The diarrhea resolved quickly and no recurrent CDI occurs but the effusion persisted and slowly resolved several weeks after discharge.We include the case of a 63-year-old female admitted in May 2014 to the Adults 3 Department of INBI for watery diarrhea. One week previous to her admission, she underwent cholecystectomy and had received ceftriaxone for five days. Two days after discharge she developed 6-7 watery stools, without fever and any other symptoms. Because Clostridium difficile should be recognized as a rare cause of reactive arthritis. We emphasize the importance of a proper diagnosis and treatment of arthritis. It is important that clinicians avoid unnecessary antibiotic therapy for CDI-AReA.Written informed consent was obtained from the patients for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N2-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. Plant available nitrogen is a precious commodity in many agricultural soils and the most commonly limiting nutrient in plant growth. The supply of plant available nitrogen to nitrogen (N)-deficient farming systems is thus vital to productivity . The app2) into bioavailable N that can be used by the plant for growth. Pasture legumes, including the clovers that comprise the Trifolium genus, are major contributors of biologically fixed N2 to mixed farming systems throughout the world [Trifolium spp. have developed large and symbiotically diverse populations of Rhizobium leguminosarum bv. trifolii (R. l. trifolii) that are able to infect and form nodules on a range of clover species. The N2-fixation capacity of the symbioses established by different combinations of clover hosts (Trifolium spp.) and strains of R. l. trifolii can vary from 10 to 130% when compared to an effective host-strain combination [In this symbiotic association, rhizobia reduce atmospheric dinitrogen  and formation of white (Fix-) pseudo-nodules on T. subterraneum cv. Clare [R. leguminosarum bv. trifolii strain SRDI565 together with its genome sequence and annotation.yn. N8-J ) was isov. Clare ,11. HereR. l. trifolii strain SRDI565 is a motile, Gram-negative rod  [ar (½LA)  at 28°C.R. l. trifolii strain SRDI565 in a 16S rRNA sequence based tree. This strain clusters closest to R. l. trifolii T24 and Rhizobium leguminosarum bv. phaseoli RRE6 with 99.8% and 99.6% sequence identity, respectively.Minimum Information about the Genome Sequence (MIGS) is provided in R. l. trifolii SRDI565 forms nodules on (Nod+), and fixes N2 (Fix+) with, a range of annual and perennial clover species of Mediterranean origin (-) nodules with annual clovers T. glanduliferum and T. subterraneum cv. Clare, and with the perennial clovers T. pratense and T. polymorphum. SRDI565 does not form nodules on T. vesiculosum.n origin . SRDI565This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database  and an iRhizobium leguminosarum bv. trifolii strain SRDI565 was cultured to mid logarithmic phase in 60 ml of TY rich media [ch media  on a gyrch media .Rhizobium leguminosarum bv. trifolii strain SRDI565 was sequenced at the Joint Genome Institute (JGI) using Illumina [+ 58 bp was used to generate 18,700,764 reads and an Illumina long-insert paired-end library with an average insert size of 8,446 + 2,550 bp was used to generate 21,538,802 reads totalling 6,036 Mbp of Illumina data .The genome of Illumina  data. AnAll general aspects of library construction and sequencing performed at the JGI can be found at the JGI user homepage . The iniGenes were identified using Prodigal  as part The genome is 6,905,599 nucleotides with 60.67% GC content  and comp