The hicAB locus consists of genes hicA and hicB encoding a toxin and its cognate antitoxin, respectively. BLAST analysis revealed that hicAB is prevalent in approximately 36% of P. aeruginosa strains and locates in the same genomic region. RT-PCR demonstrated that hicAB forms a bicistronic operon that is cotranscribed under normal growth conditions. Overproduction of HicA inhibited the growth of Escherichia coli, and this effect could be counteracted by co-expression of HicB. The Escherichia coli kill/rescue assay showed that the effect of HicA is bacteriostatic, rather than bactericidal. Deletion of hicAB had no effect on the biofilm formation and virulence of P. aeruginosa in a mice infection model. Collectively, this study presents the first characterization of the HicAB system in the opportunistic pathogen P. aeruginosa.Toxin-antitoxin (TA) systems are small genetic modules that are widely distributed in the genomes of bacteria and archaea and have been proposed to fulfill numerous functions. Here, we describe the identification and characterization of a type II TA system, comprising the  Toxin-antitoxin (TA) systems are abundant, diverse and small genetic modules that are widely distributed in the genomes of bacteria and archaea ,2. TA syEscherichia coli and Mycobacterium tuberculosis possesses 36 and 79 TA loci, respectively [In the increasingly high-throughput genome sequencing era, a large collection of sequence data become available in several databases. Analyses of genome sequences have revealed that TA systems are prevalent in nearly all bacterial species, including pathogens and non-pathogens ,8. Diffeectively . Since tectively ,9,10. HiPseudomonas aeruginosa inhabits diverse ecological niches, causes significant morbidity and mortality among immune-compromised individuals and resists treatment with antibiotics due mainly to its notable biofilm formation and multi-drug resistance [P. aeruginosa will be beneficial to gain further insight into the biological characteristics and pathogenesis of this versatile opportunistic pathogen.sistance . To datesistance . IdentifhicAB locus belongs to one of the well-characterized type II TA systems. This locus was first described as an insertion into the major pilus gene cluster in several strains of Haemophilus influenza and subsequently was predicted to be a novel TA system using a comparative-genomic approach [E. coli K-12, the hicAB locus was first identified as an active TA system, in which ectopic production of toxin HicA induced cleavage in three model mRNAs  and tmRNA by a ribosome-independent manner, concomitantly reducing the global rate of translation, while HicB functions as an antitoxin and neutralizes HicA [Yersinia pestis, which belongs to the HicAB family, has been determined, showing that HicB3 forms a tetramer that can bind two HicA3 toxin molecules and occludes the HicA3 active site through its N-terminal domain [hicAB locus has been found in numerous bacterial and archaeal genomes and characterized in several bacterial species [P. aeruginosa still remain unknown.The approach ,14. In El domain . Severall domain ,17. Hith species ,19,20,21hicAB locus was identified in P. aeruginosa by homology search, and its prevalence was investigated. The results showed that hicAB forms a bicistronic operon that is cotranscribed under normal growth conditions and constitutes an active TA system. The HicAB system appears to be not involved in the biofilm formation and virulence of P. aeruginosa, and its biological role needs to be further elucidated. To our knowledge, this is the first characterization of the HicAB system in P. aeruginosa.In this study, the P. aeruginosa PA1, the gene PA1S_06925 encodes a 60-aa protein annotated as a putative mRNA interferase [E. coli HicA toxin, thus termed hicA. The other gene, PA1S_06920, encoding a 140-aa protein that shares 30% identity with the E. coli HicB antitoxin, was named hicB. Protein homology modeling using CPHmodels predicted the structures of P. aeruginosa HicA and HicB based on the available solved three-dimensional structure of proteins TTHA1913 (PDB: 1WHZ) and TTHA1756 (PDB: 2YZT), respectively [E. coli HicA, B. pseudomallei HicA and Y. pestis HicA1 toxins and has been experimentally verified in some species [N-terminal domain, adopting a partially-degraded RNase H fold , glycoside hydrolase (PA1S_06935), terminase  and portal protein (PA1S_06960). These results indicate the horizontal gene transfer (HGT) of hicAB, which is consistent with previous analysis [P. aeruginosa that possess the hicAB locus, hicAB is linked to sequences encoding phage-related proteins and has the same genomic location as in PA1. In addition, HicA and HicB of P. aeruginosa PA1 share 100% and 78% amino acid identity with other homologues among these strains, respectively  and −10 (TTGTATAAT) sites  sites A. FindTehicA and hicB, a reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. cDNA was synthesized using cellular total RNA extracted from log-phase cultures and amplified using a set of primer pairs /pLysS. In this system, the hicB and hicA genes were cloned successively into the pJS298 expression vector [BADP and IPTG-inducible promoter T7P control the expression of hicA and hicB on the same vector backbone in trans, respectively /pLysS cells harboring the pJSHicAB plasmid showed no difference in growth in the presence of IPTG that induced hicB gene expression alone, as indicated in l-arabinose (Ara), the bacteria growth was inhibited /pLysS cells showed that the normal-sized cells became aggregated after induction of HicA, and this phenotype can be reverted by subsequent induced HicB .To investigate the possible biological role of the hicAB of P. aeruginosa is involved in biofilm formation, a microtiter dish assay was performed. The results showed that there were no significant difference of biofilm formation between PA1 and PA1∆HicAB, either in the case of LB or M63 broth (a standard biofilm assay medium for P. aeruginosa)  . To asseP. aeruginosa was rarely documented. This study identified and characterized the first HicAB system (a type II TA system) in P. aeruginosa. Based on the predicted identity of the amino acid sequence and their secondary structure, transcriptional analysis and the cell growth assay, we demonstrated that the hicAB locus has the properties required for a typical TA system belonging to the HicAB family [E. coli K-12 suggested that HicA functions as a ribosome-independent mRNA interferase that blocks translation by cleaving mRNAs and causes cell growth inhibition [E. coli HicA, we therefore reasoned that the P. aeruginosa HicA induces cell growth arrest via a similar mechanism.Due to its ubiquitous distribution in bacterial genomes and various functions, TA systems have attracted increasing concern in recent years. To date, TA systems have been identified in numerous bacterial genomes, with many being well characterized. Unfortunately, the related knowledge of the notorious opportunistic pathogen B family ,15. Analhibition . As the hicAB locus in numerous bacterial genomes showed that hicAB is transferred horizontally [hicAB cassettes were found to be encoded in prophages and plasmids that could serve as the vehicles for HGT. Moreover, no virtual collinearity in the localization of hicAB was found in the genomes of closely-related species. In this study, we found that the flanking regions of hicAB encode several proteins homologous to phage-related proteins, including holin and terminase. Thus, we deduced that hicAB of P. aeruginosa appears to be a mobile element acquired through HGT. Furthermore, analysis of the hicAB locus in different P. aeruginosa strains revealed that hicAB locates in a conserved genomic region, implying that hicAB may be involved in a particular biological process that remains unknown and needs to be further characterized.Previous studies of the distribution of the zontally . ConsistE. coli has been usually used as the host for verification of heterogenic TA components due mainly to its well-established conditional expression systems [E. coli BL21(DE3)/pLysS cells, HicA functions as a toxin and leads to cell stasis; meanwhile, HicB could rescue the bacteria cells from growth inhibition by co- or subsequent induction, indicating that the toxicity of HicA is bacteriostatic and can be alleviated by HicB. These results are consistent with previous reports of HicAB in several bacterial species [E. coli cells became aggregated after induction of HicA, and subsequently induced HicB could revert this phenotype. The underlying mechanism is still mysterious. Cell aggregation has been proposed to be an important adaptive strategy ensuring the survival and growth of bacteria in adverse environments [E. coli cells to cope with the stress triggered by toxin overproduction. systems ,32,33. I species ,15,19. Aronments ,35. ThushicAB in P. aeruginosa, a hicAB knockout mutant was constructed. No significant difference between the WT and the hicAB deletion mutant was observed in terms of their biofilm formation and virulence in a mice infection model. Indeed, most TA deletion mutants show no discernible phenotype [E. coli induced a cumulative effect on persister cell formation [M. tuberculosis [To investigate the possible biological role of henotype , which ihenotype ,37. For ormation , and crorculosis ,39. Anotrculosis .P. aeruginosa is a notorious opportunistic pathogen and resists lots of antibiotics due mainly to its notable biofilm formation and intrinsic drug resistance; thus, P. aeruginosa-associated infections are always intractable challenges in the clinical setting. Based on the wide spread of TA systems in bacterial genomes and the ability of toxins to regulate bacterial cell growth, the exploitation of TA systems as a novel antibacterial strategy via artificial activation of toxin has been proposed and has considerable potential [P. aeruginosa TA systems (including HicAB) might be greatly beneficial to develop novel and effective strategies to control this versatile opportunistic pathogen.otential ,42,43. TP. aeruginosa and E. coli strains were cultured in LB broth at 37 °C with shaking at 200 rpm or plated on LB agar, unless otherwise specified. When necessary, antibiotics were added at the following concentrations: for E. coli, gentamicin, 15 μg/mL; kanamycin, 50 μg/mL; for P. aeruginosa, gentamicin, 100 μg/mL.The bacterial strains and plasmids used in this study are listed in http://www.ncbi.nlm.nih.gov/BLAST) and on the Pseudomonas Genome Database (http://www.pseudomonas.com/) [http://www.ebi.ac.uk/Tools/msa/clustalw2/) [http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) [P. aeruginosa HicA and HicB were generated using the CPHmodels 3.2 Server (http://www.cbs.dtu.dk/services/CPHmodels) [http://linux1.softberry.com/berry.phtml) and FindTerm (http://www.softberry.com/berry.phtml?topic=findterm&group=programs&subgroup=gfindb), respectively.Sequence homology searches and conserved domain analyses were carried out using the Basic Local Alignment Search Tool (BLAST) on the NCBI website (as.com/) . Multiplstalw2/) , and theipt.cgi) . The modHmodels) . The putP. aeruginosa using the TIANamp Bacteria DNA Kit  according to the manufacturer’s instructions. Total RNA was isolated from log-phase cultures using the Tripure Isolation Reagent  following the manufacturer’s protocol. cDNA was synthesized from total RNA using random primers with RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s recommendations. gDNA and cDNA were used as templates for PCR amplification and coupling transcriptional analysis of the hicAB locus and the flanking sequences.Genomic DNA (gDNA) was extracted from overnight cultures of hicAB locus encodes an active TA system, the conditional expression plasmid pJSHicAB was constructed to expression of hicA and hicB under the l-arabinose-inducible BADP promoter and the IPTG-inducible T7P promoter, respectively. Briefly, the hicB gene was amplified from PA1 genomic DNA using primers HicB-T7F and HicB-T7R  and cloned under the T7P promoter into pJS298, resulting in pJSHicB. The hicA gene was amplified using primers HicA-BADF and HicA-BADR and cloned behind the BADP promoter of pJSHicB using Sac I and Nco I restriction sites, generating the recombinant plasmid pJSHicAB.To determine if the HicB-T7R , digesteE. coli BL21(DE3)/pLysS cells harboring the plasmid pJSHicAB were cultured in 100 mL of LB broth supplemented with 50 μg/mL kanamycin and 0.2% glucose to an OD600 of ~0.3. For selective expression of hicA and hicB genes, the culture was divided into four equal parts, of which three were provided with 1 mM of IPTG, 0.2% l-arabinose (Ara) and both , respectively. The cultures were continued at 37 °C for 6 h, and aliquots were removed every 30 min for measurement of OD600 and enumeration of CFU. In the kill/rescue assay, E. coli culture was supplemented with 0.2% l-arabinose at an OD600 of ~0.3 and continued for 2 h. Then, the culture was divided into two equal parts, of which one was supplemented with 1 mM of IPTG. The cultures were continued for 4 h, and the OD600 and CFU were assessed every 1 h.In growth assays, E. coli cells was performed as described previously [E. coli BL21(DE3)/pLysS cells harboring the plasmid pJSHicAB were grown to an OD600 of 0.3, then induced with and without HicA expression and continued for 4 h. Gram staining of the cultures was performed; at the same time, the HicA overproduction culture was induced with HicB expression and continued for 2 h. Gram staining was done again to test whether HicB could revert the aggregated phenotype. Microscopic images were captured digitally (×400).Microscopic examination of the eviously . BrieflyhicAB deletion mutant of PA1 was constructed through homologous recombination. Briefly, the left flanking region (~1000 bp) and right flanking region (~1000 bp) of the hicAB locus were amplified using primer pairs of LA-F/LA-R and RA-F/RA-R, respectively. The gentamicin resistance cassette was amplified from plasmid pUCP24 using primers Gm-OF and Gm-OR. The fragment of LA + Gm + RA was obtained using overlap PCR and cloned into plasmid pEX18Tc using BamH I and Hind III restriction sites, resulting in plasmid pEX∆HicAB. Then, pEX∆HicAB was electroporated into PA1 as described previously [hicAB deletion mutant was screened on LB agar supplemented with 100 μg/mL gentamicin and confirmed by PCR detection and sequencing.The eviously . The hic550 was quantified to represent biofilm formation.The biofilm formation assay was performed as described previously . Briefly7 CFU. Each strain (1 mL) was injected intraperitoneally into 6- to 8-week-old BALB/c female mice, and animals were monitored for 24 h. All the experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Mililary Medical University .The mouse infection experiment was performed as described previously . Bacterit-test. Differences were considered statistically significant at p < 0.05.All statistical analyses were carried out using GraphPad Prism . Where appropriate, the data were analyzed using Student’s


Staphylococcus aureus is one of the most frequent pathogens that causes severe morbidity and mortality throughout the world. S. aureus can infect skin and soft tissues or become invasive leading to diseases such as pneumonia, endocarditis, sepsis or toxic shock syndrome. In contrast, S. aureus is also a common commensal microbe and is often part of the human nasal microbiome without causing any apparent disease. In this review, we explore the immunomodulation and disease tolerance mechanisms that promote commensalism to S. aureus.The Gram-positive bacterium  Staphylococcus aureus is a Gram-positive bacterium of the Micrococcae family [S. aureus is commonly found as commensal bacteria located superficially on the human anterior nares, its primary reservoir [e family . It has e family . S. aureeservoir ,2. From S. aureus is well equipped with a variety of virulence factors such as microcapsules, toxins, and drug resistance genes that contribute to pathology [S. aureus occur with a seasonal trend, being highest in the summer and lowest in the winter [S. aureus is the primary cause of local skin and soft tissue infections (SSTI) such as impetigo, dermatitis, and cellulitis [S. aureus include bacteremia, pneumonia, osteomyelitis, endocarditis, sepsis, and toxic shock syndrome (TSS) [S. aureus have the highest annual death toll for any single infectious agent in the US, with close to 20% mortality [S. aureus (MRSA). MRSA caused an estimated 80, 641 infections in US during 2011. Of these infections, 78% occurred in outpatients or within the community and carried a mortality rate of over 10% [Although it is a commensal, athology . Infectie winter . S. aurellulitis ,4. Systeortality ,4. One rover 10% .S. aureus and a fewer amount of those (around 20%) are persistently colonized [S. aureus since serum immunoglobulin G (IgG) antibodies against staphylococcal antigens can be found in the entire population despite the lack of history of S. aureus infection [S. aureus carriage rates differ based on ethnicity, gender, and age [S. aureus carriage but are gradually increasing [S. aureus include cattle, sheep, and goat, yet the strain lineages appear to have evolved distinctively in each host [S. aureus is reported to have a large economic impact in the dairy industry since it is the primary cause of mastitis [S. aureus and display a relative resistance to both the bacterium and its toxins, something to keep in mind when considering mouse models of disease [At any moment in time, 30%–50% of healthy adults are colonized with olonized ,2. It canfection . S. aure and age . Currentcreasing . In addiach host . S. auremastitis . Mice ar disease .S. aureus a pathobiont [S. aureus colonization in their nares or skin [S. aureus bacteremia cases in carriers were caused by their endogenous strain [S. aureus tend to have better outcomes to S. aureus bacteremia than non-carriers [S. aureus colonization [We have termed thobiont . This clthobiont . Over 90 or skin , and 80%s strain . In factcarriers ,11,12. IS.aureus. In addition, chemical “elements” of defense such as anti-microbial peptides  are secreted to the outer layer to control bacterial growth and breaching [S. aureus. Phagocytic cells and keratinocytes recognize MAMPs through their pathogen recognition receptors (PRRs) triggering a cutaneous immune response [S. aureus recognition, NOD1/2 recognize muramyl dipeptides, which are PGN-derived fragments [The uppermost layer of the skin, the corneal layer, acts as a physical barrier to the microbiota, including reaching . Below tresponse . PRRs arresponse , and traresponse . In the ragments . CLRs biragments . TLR2 reragments .S. aureus and other Gram-positive bacteria [Since TLR2 has been reported as the dominant receptor for bacteria ,18,19,20bacteria . Recruitbacteria . Ultimatbacteria .H1/TH17 pro-inflammatory response through the production of IL-12 and IL-23 [Plasticity in TLR2 signaling has been reported depending on the cell type expressing the receptor and the structure of the TLR2 ligands. Recently, our group has reported the existence of two sets of MAMPs that can trigger either predominantly pro-inflammatory or predominantly anti-inflammatory responses to TLR2 signaling . TLR2 sind IL-23 .S. aureus [S. aureus pathogenicity is enhanced by a neutrophil-rich environment [At the cellular level, neutrophils are the first cells to attempt the clearance of . aureus . They ph. aureus . Howeverironment ,24. SomeS. aureus requires a mixture of TH1, TH17, and humoral antibody responses. For example in SSTI both cellular and humoral responses (primary or secondary) are observed [H1/TH17 response is associated with the production of IL-1 and IL-17A to promote abscess formation at the site of infection [S. aureus infections and is required for the clearance of bacteria via phagocytosis and oxidative burst [When innate immune mechanisms are not sufficient to clear the bacterial infection, adaptive immune mechanisms are activated. An adaptive immune response to observed . A TH1/Tnfection . Abscessve burst .H17 responses in controlling S. aureus infections. In this syndrome, a mutation in the stat3 gene results in lower levels of retinoid-related orphan receptor (ROR) γt [H17 lineage differentiation. Thus, these patients show defective IL-17A production and increased susceptibility to S. aureus infections [H2 phenotype concomitant with the absence of TH17 cells [Candidiasis but not to S. aureus [Candida and Mycobacterium infections but not to increased susceptibility to S. aureus [The Hyper-IgE Syndrome (HIES) highlights the importance of the T(ROR) γt , the masfections . HIES is17 cells  but it i. aureus . In addi. aureus .H1 response during S. aureus infections is commonly associated with TSS where staphylococcal superantigens (SAgs) activate roughly 20% of T cells leading to their massive proliferation and production of cytokines [H1 response, and can be found in pathogenic as well as commensal isolates. However, this is not unique to SEA because all SAgs have been found to share similar superantigenicity when levels of pro-inflammatory/TH1 cytokines were measured in vitro [A Tytokines . This acytokines . SAg expytokines ,30. Anotin vitro .S. aureus infections. In the serum of patients with S. aureus bacteremia, IgG antibodies targeting eight conserved extracellular proteins can be found. Of these targets, seven are coded by the bacterial core genome [In addition to T cell responses, humoral responses have been found to play a role in the outcome of e genome . The proe genome . These ae genome .S. aureus in humans is still unclear [S. aureus. This observation is in line with challenges we have in developing a vaccine against this microbe. In mice, recent studies suggest that IL-17A may be important for the development of a memory state because a high IL-17A-to-IFNγ ratio in mice has been linked to protective memory to SSTI [S. aureus may reflect a balance between nominal antigen-induced TH17 responses and SAg-induced TH1 responses. If so, the former response would conduce to protection upon re-exposure, whereas the later would not. However, this may not be applicable to the same extent in humans because patients with a defect in RORγt or IL-17 receptor signaling, which lead to impaired TH17 responses, show no increased susceptibility to repeated S. aureus infections [The role of immunological memory to  unclear ,31,32. T to SSTI . This IL to SSTI . This evfections ,28.S. aureus has a plethora of sophisticated mechanisms that allow it to induce immunomodulation that promote disease tolerance and establish commensalism. The precise molecular and cellular basis of immunomodulation is starting to emerge.The capacity of a microbe to induce anti-inflammatory responses and to restrain host pro-inflammatory responses to that microbe is referred to as immunomodulation. In addition to triggering host innate and adaptive immunity, S. aureus relies on MAMPs binding to PRRs on host cells. These cells include epithelial cells, macrophages, DCs and neutrophils [Like other microbes, recognition of trophils . PGN frotrophils . The quatrophils ,34,35. Rtrophils .S. aureus isolates express at least one SAg [S. aureus carriers is low [S. aureus PGN-embedded TLR2 ligands induce IL-10 production and apoptosis of APCs, down-regulating SAg-induced T cell activation and preventing TSS [Although most  one SAg ,38,39, ts is low ,41. Thisting TSS . Consistting TSS .S. aureus strain resulted in enhanced innate and adaptive immune responses, compared with infection with wild-type strain, as illustrated by increased neutrophil infiltration, influx of innate IL-17+ γδ T cells, recruitment of TH1 and TH17 cells, and enhanced pro-inflammatory cytokine and chemokine production. These effects were correlated with reduced lesion size. The enhancement of the response to S. aureus was also observed with the administration of an α-toxin neutralizing antibody prior to the staphylococcal infection [In addition to cell wall components, secreted staphylococcal toxins can mediate immunomodulation in SSTI. In a murine model of dermonecrosis, infection with an α-toxin-deficient nfection . AltogetS. aureus. IL-10 inhibits TH1 and TH17 responses to S. aureus [H2 responses [Emerging evidence suggests that IL-10 is an important mediator of immunomodulation by . aureus . In humaesponses . In addiesponses , resultiesponses  and co-sesponses ,49, leadS. aureus. The production of IL-10 by monocytes and macrophages is between 4 and 20 times higher than that by DCs [S. aureus triggers a robust TH1/TH17 response [S. aureus is dependent on the type of APC. Consequently, one would expect different outcomes of S. aureus detection in different tissues based on different cellular composition. For example, in the nose, macrophages are the primary APCs, whereas in the skin Langerhans cells and other DCs are more abundant. This might explain why S. aureus usually acts as a commensal in the nose  and a pathogen in the skin .Quantitatively, monocytes and macrophages are the main source of IL-10 in response to t by DCs . In contresponse . This diS. aureus can also induce immunomodulation through the regulation of monocyte/macrophage function and differentiation. In a mouse model of catheter-associated biofilm infection, S. aureus attenuated phagocytosis by macrophages, and polarized these cells towards an alternatively activated M2 phenotype, altogether down-regulating host inflammatory responses [S. aureus phagocytosis and are polarized to a M2 phenotype compared to macrophages from chronic rhinosinusitis patients without nasal polyps. This correlates with more staphylococcal colonization in the former patients. These findings suggest that alternative activation of macrophages contributes to S. aureus chronic infection and colonization [S. aureus skews macrophage differentiation has been linked to Akt1 signaling and subsequent regulation of the miR-155/SOCS1 axis [esponses . Targetiesponses . In linenization . The mecCS1 axis .S. aureus infection. It is unlikely that S. aureus directly interacts with T cells to induce IL-10 production, since human resting T cells do not express TLR2 [S. aureus can imprint T cells to produce IL-10. For example, it has been reported that S. aureus primes naïve T cells to differentiate into IL-10-producing TH17 cells [in vitro polarization study, S. aureus imprinted neonatal cord blood CD4+ T cells to differentiate into FOXP3+CD25+CD127l°w Treg cells through a PD-1/PD-L1 dependent mechanism [In addition to monocytes and macrophages, T cells can produce IL-10 upon ess TLR2 . However17 cells . In anotechanism .S. aureus can also facilitate immune evasion, which under certain circumstances is a form of immunomodulation. IL-10 inhibits pro-inflammatory immune responses and establishes an anti-inflammatory environment [Induction of IL-10 production by ironment ,57. UndeS. aureus-induced immunomodulation. S. aureus or its PGN are able to down-regulate staphylococcal SAg-induced expression of chemokines such as CXCL10, and prevent T cell recruitment. Such a regulation is not due to IL-10 production, but rather to S. aureus-triggered activation of MAPKs p38 and ERK, and inhibition of STAT1 signaling .Recent evidence has suggested the involvement of IL-10-independent mechanisms in S. aureus has demonstrated a role of Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) in immunomodulation [S. aureus infection model has corroborated the important role of MDSC in immunosuppression [Recently, a study on cutaneous infection by dulation . Signalipression . In thisS. aureus can trigger an anti-inflammatory response. Emerging evidence indicates that TLR2 ligands embedded in staphylococcal cell wall are involved in S. aureus-induced immunomodulation. LTA is one of these ligands that down-regulates host innate and adaptive immune responses [S. aureus than commercially prepared staphylococcal PGN, which implies that ligands other than PGN-derived molecules are responsible for the immunomodulation. The exact immune regulatory molecules still need to be identified. In addition to cell wall components, the staphylococcal secreted protein Map  can act as an immunomodulator that interferes with T cell-mediated responses [In addition to the pro-inflammatory properties, the cell wall of esponses ,59. Howeesponses . Furtheresponses , althougS. aureus can be uncoupled. Nasal S. aureus isolates have a different IL-10-inducing capacity, which does not correlate with the TNFα-inducing capacity [S. aureus cell wall may induce the formation of different TLR2 signaling complexes. The balance between these receptor complexes may determine the difference in anti-inflammatory capacity. For example, TLR2/6 dimers, rather than TLR2/1 dimers, have been often linked to immunomodulation [S. aureus are required for the pro- but not the anti-inflammatory response [S. aureus. The PI3K/Akt/mTOR and extracellular signal-regulated kinase (ERK) pathways are responsible for the immunomodulation, while the MAPK p38 pathway is essential for pro-inflammatory response [Recent studies have demonstrated that the pro-inflammatory properties and immunomodulatory properties of capacity . The comdulation . Accessodulation . Phagocyresponse . In addiresponse . The MyDresponse .Signaling from TLRs trigger activation and nuclear translocation of several transcription factors that drive IL-10 expression. NFκB , C/EBPβ S. aureus might be determined by several factors, such as the site of infection, strain of S. aureus, host genetics, host diet and environment. The nostril is a common commensal colonization site of S. aureus, while skin colonization by this microbe is usually associated with disease, such as AD , or through hyper-activation of the host immune system . The mechanisms of S. aureus-induced damage are balanced by “disease tolerance” mechanisms that prevent unnecessary tissue destruction caused by the microbe or the host [S. aureus-induced tissue damage and pathology during infection but also for S. aureus to establish commensalism.During infection, the host . Diseasethe host . These mthe host ,77,78. WS. aureus pathogenesis was recently highlighted by the observation that autophagy prevents tissue damage caused by α-toxin [i.e., Atg16L1HM mice) were susceptible to S. aureus pneumonia and sepsis independently of bacterial burden. They established that susceptibility was conferred by production of the S. aureus α-toxin, and that autophagy down-regulated the expression of the α-toxin receptor ADAM10 in epithelial cells, thereby limiting damage during S. aureus infection. Interestingly, anthracyclines, a family of DNA damaging chemotherapeutic agents, also induced autophagy in the lung and protected mice from cecal-ligation and puncture sepsis [S. aureus infections. Whether a similar effect is seen in the upper respiratory tract during S. aureus commensalism is currently unknown. However, given the importance of the integrity of the nasal epithelial layer during commensalism, it is reasonable to speculate that autophagy in the nose would confer disease tolerance to S. aureus as well. Indeed, autophagy-deficiencies have been identified in nasal polyps [S. aureus to limit pathology. It is, therefore, likely that autophagy mediates disease tolerance in the nose to promote S. aureus commensalism.The importance of disease tolerance in limiting  α-toxin . In thise sepsis , suggestl polyps , and stal polyps ,82. Morel polyps ,84, perhThe hyper-active immune response during staphylococcal systemic complications causes severe tissue damage through immunopathology. In particular, the cytokine storm resulting from massive T cell activation by SAgs leads to multi-organ failure, a hallmark of staphylococcal TSS ,86. Any The breakdown of disease tolerance during staphylococcal TSS may explain the shortage of current treatments. The limited success of many of these treatments may be due to the focus on eliminating the microbe, and ignoring the extensive tissue damage that is occurring. Implementing treatment strategies that combine tissue damage control and antibiotics that inhibit toxin production  may proS. aureus commensalism. IL-10 is an essential anti-inflammatory molecule that mediates tolerance to the gut microbiota, and mutations and/or deficiencies in IL-10 cause severe enterocolitis in mice [S. aureus commensalism by limiting the inflammatory response to S. aureus and tissue damage caused by its toxins. For example, IL-10 would protect against nasal epithelium dysregulation caused by staphylococcal SAgs [S. aureus isolates do carry SAgs as frequently as clinical isolates [Streptococcus pyogenes [S. aureus colonization in an HLA-DR4 transgenic mouse [S. aureus commensalism is an intriguing possibility that must be explored further. Additional disease tolerance mechanisms by particular receptors  reporteS. aureus pathobiosis is the ability of this microbe to evade the host immune response. Immune evasion can be broadly classified into two main types: evasion by avoiding host detection, or evasion by modulating the host immune response. Here, we will only briefly outline the mechanisms of S. aureus immune evasion as they have been recently reviewed extensively elsewhere [Another important aspect of lsewhere ,98.S. aureus produces a plethora of virulence factors to avert the essential components of the host immune response involved in the elimination of S. aureus. These include the complement system, leukocyte recruitment mechanisms, and phagocyte function. For example, S. aureus has developed multiple, parallel strategies to subvert opsonization and chemotaxis by the complement system. Some examples are the staphylococcal C3 convertase assembly inhibitor [S. aureus also inhibits the anaphylatoxins C3a and C5a through proteolysis [S. aureus (CHIPS) [To avoid detection, nhibitor , fibrinonhibitor  and C3 cnhibitor , and pronhibitor . In additeolysis ,103, or  (CHIPS) . The redS. aureus. As such, S. aureus has evolved multiple mechanisms to avoid them by regulating their recruitment and function. For example, capsule-producing S. aureus are more resistant to phagocytosis and killing [S. aureus can resist the oxidative burst by producing superoxide dismutase and catalase to reduce superoxide and hydrogen peroxide into non-lethal compounds [S. aureus can also mask the MAMPs in its PGN layer with wall teichoic acids [S. aureus may also contribute to this immune evasion strategy by limiting the pro-inflammatory response while maintaining the IL-10 induction [Mycobacterium tuberculosis infection [S. aureus by professional phagocytes.Professional phagocytes are the most effective cells in battling  killing ,106, ind killing , and are killing . Moreove killing . If enguompounds ,111. S. ic acids  or by acic acids , avoidinic acids . Moreovenduction . In factnfection ,115. AltS. aureus can evade its host by modulating the immune response it triggers. The staphylococcal adenosine synthase A (AdsA) is a PGN-anchored protein that converts adenosine phosphates  to adenosine, a potent anti-inflammatory molecule. S. aureus strains deficient in AdsA are more susceptible to neutrophil killing and cause less severe disease in mouse models of staphylococcal infections [H1/TH17 clearance response and towards an immunomodulatory phenotype that promotes microbial survival and colonization. Therefore, AdsA is an important staphylococcal virulence factors that promotes evasion by modulating the host immune response.More pertinent to this review is the fact that fections . Adenosifections , decreasfections ,119. It fections . CollectS. aureus can also alter the response of neutrophils to promote its survival upon phagocytosis. Although neutrophils are essential for the clearance of S. aureus, their recruitment to the site of infection is associated with enhanced bacterial burden and pathogenicity [S. aureus has developed mechanisms to evade neutrophil function. One mechanism involves S. aureus upregulating the “don’t eat me” signal CD47 on neutrophils to prevent their uptake by responding macrophages [S. aureus to trigger a specific type of neutrophil cell death program that releases viable S. aureus into the extracellular environment [H17 response that recruits many neutrophils may end up benefiting the microbe more than the host.In addition to the mechanisms mentioned above, genicity . This imrophages . This prironment . Thus, iS. aureus induces can also promote immune evasion through modulation of the host response in a site-dependent manner [H1 responses [H1/TH17 responses, allows S. aureus to spread and cause severe invasive infections. A similar observation has been described for Bordetella [The robust IL-10 response that t manner  and co-st manner , and cant manner  and TH1 esponses . The immesponses . This rordetella  and may S. aureus may facilitate the development of new methods to prevent and treat staphylococcal infections.Current treatments for staphylococcal infections rely heavily on antibiotics, which will select for antibiotic-resistance strains  . DespiteS. aureus in a given individual suggests that the initial infection does not generate host protective immunity to subsequent infections [S. aureus can be partially explained by immunomodulation. For example, SpA binds to IgG antibodies and acts as a B cell SAg, limiting host responses to other staphylococcal virulence factors [S. aureus vaccination by limiting T cell activation and help. Therefore, purification of a low IL-10-inducing staphylococcal antigen can be considered as a strategy for vaccine development.The observation of recurrent infections by fections . The rea factors . In addiS. aureus-induced immunomodulation, down-regulating SAg-induced T cell activation, and preventing TSS. Thus, IL-10 is a potential therapeutic agent in inflammatory diseases. Indeed, administration of recombinant IL-10 is protective in a mouse model of TSS [As discussed above, IL-10 plays an important role in l of TSS ,131. Howl of TSS .S. aureus can be uncoupled and are mediated by different signaling pathways [i.e., PI3K/Akt/mTOR and MAPKs, are usually involved in many aspects of cellular function. Therefore, direct manipulation of these signaling cascades is likely to cause numerous side effects. For example, pan-class I PI3K inhibitors, used to treat rheumatoid arthritis (RA), have been reported to cause hyperglycemia, gastrointestinal symptoms, and psychiatric side effects [The pro-inflammatory and immunomodulatory properties of pathways . One wou effects . Similar effects ,135. MAP effects , the res effects ,139,140.S. aureus-induced immunomodulation. Compared with heat-killed S. aureus, clindamycin-treated S. aureus better preserves the anti-inflammatory properties, in terms of IL-10-inducing capacity [S. aureus. In line with this observation, clinical data show that treating TSS with bacteriostatic antibiotics  is more effective and has better clinical outcomes than bactericidal antibiotics [Staphylococcal cell wall-embedded TLR2 ligands are predominantly responsible for capacity . This ilibiotics ,141.S. aureus opens up the search for anti-inflammatory ligands in the cell wall. The study on the uncoupling of responses to S. aureus suggests that the staphylococcal cell wall contains qualitatively different TLR2 ligands with distinct IL-10-inducing capacities. Although the anti-inflammatory TLR2 ligands are yet to be identified, it is very unlikely that a single ligand will solely induce IL-10 without inducing pro-inflammatory cytokines. However, identification of a predominant IL-10-inducing TLR2 ligand may provide a template for novel immunomodulatory drug development.The uncoupling of the immunomodulatory properties from the pro-inflammatory properties of S. aureus is very effective at immunomodulating innate and adaptive immune responses, to promote either disease tolerance or immune evasion. The type of immune response to S. aureus that will develop is determined by various factors, such as site of infection, bacterial virulence and host genetics, all together translating into distinct clinical outcomes. A better understanding of the mechanisms of immunomodulation leading to disease tolerance and commensalism will provide further insights into staphylococcal diseases and the development of novel clinical treatments for these conditions.


Helicobacter pylori infection, a common infection in many countries, is related to the clinical course of upper gastrointestinal diseases. Gastroesophageal reflux disease (GERD) is a common esophageal disease in Western countries and its prevalence is increasing in Asian countries. The pathophysiology of GERD is multifactorial. Although no single factor has been isolated as the cause of GERD, a negative association between the prevalence of H. pylori and the severity of GERD, including Barrett's esophagus, has been demonstrated in epidemiological studies. The high prevalence of H. pylori infection affects the incidence of GERD in Asian countries. In the subjects with East Asian CagA-positive strains, acid injury may be minimized by hypochlorhydria from pangastritis and gastric atrophy. Additionally, host genetic factors may affect the development of GERD. The interactions between genetic factors and the virulence of H. pylori infection may be the reason for the low prevalence of GERD in Asian countries. H. pylori eradication is not considered pivotal in GERD exacerbation based on evidence from Western studies. A recent meta-analysis demonstrated that eradication therapy of H. pylori was related to a higher risk of developing de novo GERD in Asian studies. H. pylori infection remains an inconclusive and important issue in GERD in Asian countries. H. pylori and GERD have been found to be negatively associated and strongly related to cytotoxin-associated gene product- (CagA-) positive strains of H. pylori [ H. pylori have been reported in Asian countries [ H. pylori infection is an important issue in GERD, especially in Asian populations. In addition, H. pylori eradication has been presumed to exacerbate GERD due to improvement of gastritis and the recovery of hypochlorhydria; several studies have been conducted to clarify this controversy.The prevalence of gastroesophageal reflux disease (GERD) in the general population has been estimated to be 10–20% . Convers. pylori . Howeverountries , which iountries . GERD maountries –11. Ther H. pylori infection. Gastric secretion can increase, decrease, or remain steady depending on the pattern of H. pylori-related inflammation [ H. pylori infection include the density of H. pylori colonization, its distribution, and the severity of the mucosal inflammatory response to the infection. Patients with a duodenal ulcer and H. pylori infection have antrum-predominant gastritis, which leads to hypergastrinemia and acid hypersecretion. In contrast, patients with gastric ulcer or gastric cancer present mainly with corpus-predominant gastritis or pangastritis, which is characterized by intense destruction or atrophy of acid-secreting glands. Patients who have corpus-predominant gastritis or pangastritis also show gastric acid hyposecretion [ H. pylori induces primarily corpus-predominant gastritis or pangastritis with hypochlorhydria. And, East Asian CagA-positive strain is strongly associated with gastric cancer. A Japanese study revealed different sequences of CagA between the regions where gastric cancer is prevalent or not. The authors defined the East Asian CagA-positive strains which showed the specific repeat sequences located in the 3′ region of cagA gene. In the study, most CagA-positive strains in Asian countries were East Asian CagA-positive strains and most CagA-positive strains in Western countries were Western CagA-positive strains [ H. pylori-infected subjects because their specific genotypes are linked to corpus atrophy, gastric cancer, and hypochlorhydria [ H. pylori infection due to high gastric mucosal IL-1β levels [β. Additionally, several genetic risk factors for GERD, including polymorphisms in G-protein beta 3 subunit gene (GNB3) [Gastric acid plays a key role in the etiology of GERD and is an element of the disease that may be modified byammation . The majecretion , 14. Bac strains . In Asiaorhydria –20. Thusβ levels . Howeverβ levels . These oe (GNB3) , IL-10 [e (GNB3) , CYP2C19e (GNB3) , glutathe (GNB3) , 27, cyce (GNB3) , and DNAe (GNB3) , may be  H. pylori infection may be the reason for the low prevalence of GERD in Asian countries [The interactions between genetic factors and the virulence ofountries , 27, 29. H. pylori infection and reflux esophagitis or Barrett's esophagus in the western countries and East Asian countries [ H. pylori infection with virulent CagA-positive strains in Western countries [ H. pylori infection is inversely correlated with the risk and severity of reflux esophagitis; [ H. pylori infection suggests a protective role in both Barrett's esophagus and esophageal adenocarcinoma [ountries –35. Thisountries , 37. Thehagitis; , 37, 38 arcinoma . H. pylori infection [ H. pylori infection, recommend H. pylori eradication in GERD patients requiring long-term PPIs [ H. pylori eradication reduces the risk of gastric adenocarcinoma in patients with this condition.Long-term maintenance therapy of proton pump inhibitors (PPIs) for GERD induces gastritis and progression of gastric atrophy and intestinal metaplasia to gastric adenocarcinoma in patients withnfection , 47. Thenfection . Currenterm PPIs . However H. pylori and GERD in cross-sectional studies, the results are less consistent in prospective studies of H. pylori eradication in patients with GERD. Early studies revealed that H. pylori eradication was positively associated with reflux esophagitis or GERD symptoms in patients with gastric and duodenal ulcer diseases [ H. pylori strains have been reported to be risk factors for newly developed reflux esophagitis after H. pylori eradication [ H. pylori eradication in patients with peptic ulcer disease and nonulcer dyspepsia [ H. pylori eradication does not exacerbate preexisting GERD or affect treatment efficacy [ H. pylori was related to a significantly higher risk of developing de novo GERD in Asian studies [ Helicobacter pylori infection states that H. pylori eradication does not affect the development or clinical course of GERD [Despite the inverse relationship betweendiseases , 51. Hiadication . Howeveryspepsia , 54. Theefficacy . A recen studies . In cont studies –45.  Tab studies , 57. Thu of GERD .H. pylori infection and GERD are highly prevalent conditions globally. The prevalence of H. pylori varies geographically and among ethnicities. Many epidemiological studies have shown a negative correlation between H. pylori infection and GERD. A specific virulence factor, such as CagA, and specific host genotypes may affect the diverse prevalence and other aspects of GERD owing to individual differences in acid secretion. A high prevalence of CagA-positive strains has been reported in Asian countries. The diversity of H. pylori infection between Western and Asian countries should be considered when analyzing the results of studies of H. pylori eradication in GERD patients. To date, cohort studies and randomized controlled trials of the effects of H. pylori eradication on GERD are inconclusive. The decreasing prevalence of H. pylori and the recovery of acid secretion capacity after eradication in patients with CagA-positive H. pylori and corpus gastritis are possible causes of the higher prevalence of GERD in Asian countries. These issues necessitate a more detailed study.


Bursaphelenchus sycophilus n. sp. is described. The species was found in syconia of a fig species, Ficus variegata during a field survey of fig-associated nematodes in Japan. Because it has a well-developed stylet and pharyngeal glands, the species is considered an obligate plant parasite, and is easily distinguished from all other fungal-feeding species in the genus based upon these characters. Although B. sycophilus n. sp. shares an important typological character, male spicule possessing a strongly recurved condylus, with the “B. eremus group” and the “B. leoni group” of the genus, it was inferred to be monophyletic with the “B. fungivorus group”. The uniquely shaped stylet and well-developed pharyngeal glands is reminiscent of the fig-floret parasitic but paraphyletic assemblage of “Schistonchus”. Thus, these morphological characters appear to be an extreme example of convergent evolution in the nematode family, Aphelenchoididae, inside figs. Other characters shared by the new species and its close relatives, i.e., lack of ventral P1 male genital papilla, female vulval flap, and papilla-shaped P4 genital papillae in males, corroborate the molecular phylogenetic inference. The unique biological character of obligate plant parasitism and highly derived appearance of the ingestive organs of Bursaphelenchus sycophilus n. sp. expands our knowledge of the potential morphological, physiological and developmental plasticity of the genus Bursaphelenchus.A new nematode species,  Ficus L. are pollinated by highly specialized fig wasps (Agaonidae). This fascinating relationship has become a model system for studying cospeciation and host switching The fig syconium provides a unique and interesting habitat for microbes and microscopic invertebrates. Trees of the genus Parasitodiplogaster Poinar, which parasitizes the fig wasps, has been examined as a model system of species radiation and the evolution of pathogenicity Ficus species that occur worldwide (>700 species), the diversity of fig-associated nematodes (and mites) is far from being fully understood, and further intense surveys of diversity are needed.However, regardless of this apparently closed system, many different groups of phoretic and parasitic invertebrates, e.g., nematodes Bursaphelenchus was isolated from F. variegata Blume. Although two lethal plant pathogens are known Bursaphelenchus Fuchs is generally regarded as beetle (Coleoptera) or bee (Hymenoptera)-phoretic fungal feeders, and even the plant-parasitic species retain many of the morphological  characters of fungal feeding in their ingestive organs B. sycophilus n. sp., and its molecular phylogenetic status and morphological and biological characters are described and discussed.During a field survey of fig and fig wasp-associated nematodes in Japan, a species of No specific permissions were required for these locations/activities. Field studies did not involve endangered or protected species. The detailed location information is provided in F. variegata, F. septica Burm. F. and F. bengtensis Merrill, and dissected on site using a portable dissecting microscope. The figs were pealed to remove the outer layers containing latex, and cut into small pieces in sterilized distilled water using a sterilized knife. Emerging nematodes were hand-picked with sterilized stainless needles for further analyses. They were heat-killed and fixed in TAF fixative  for morphological specimens, or directly fixed in DESS A field survey of fig-associated nematodes was conducted during May to June, 2013 at the Ishigaki and Iriomote Islands, Okinawa, Japan. Various stages of fig syconia, i.e., unpollinated (young) and pollinated and developing (mature) ones, were collected from The TAF-fixed materials were examined under a dissecting microscope and separated into morphotypes. Each morphotype was processed using a glycerin–ethanol series with the modified Seinhorst's method B. sycophilus n. sp. were then transferred individually to 30 µl of nematode digestion buffer B. sycophilus n. sp. following the methods of Kanzaki and Futai For molecular analysis, DESS-fixed materials were washed and rehydrated in the sterilized distilled water, and observed using high magnification light microscopy to determine morphotypes. These observed specimens of B. sycophilus n. sp. was determined based upon SSU and D2/D3 LSU ribosomal RNA gene sequences using Bayesian, Maximum Likelihood (ML) and Maximum parsimony (MP) analyses. The SSU was compared with a wide-range aphelenchids and other infraorder species, and D2/D3 LSU, which is more suitable for lower level phylogenetic comparisons, was compared with those of closely related species. The species  compared with B. sycophilus n. sp. were determined according to the results of a BLAST homology search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the OTUs used in the previous studies on aphelenchid phylogeny 6 generations. Markov chains were sampled at intervals of 100 generations 6 generations as ‘burn in’, the remaining topologies were used to generate a 50% majority-rule consensus tree. The PhyML 3.0 online version The molecular phylogenetic status of Botrytis cinerea Pers., a standard feeding resource fungus for mycophagous nematodes, and alfalfa callus (Medicago sativa L.), a standard feeding resource for plant-parasitic nematodes. The nematodes were extracted from the additionally-collected samples of F. variegata syconia, washed several times with sterilized distilled water, and transferred to fungal lawns on 2.0% malt extract agar  or alfalfa callus donated by Dr. T. Mizukubo .Attempts were made to culture the nematode using the grey mold B. cinerea and alfalfa callus.The transferred nematodes were kept at 23°C for a month, and were examined under a dissecting microscope every 5–10 days. Culturing attempts were replicated five times for both http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:325625B8-D150-4836-B1A8-FCE3C8AC311C. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Bursaphelenchus sycophilus Kanzaki, Tanaka, Giblin-Davis & Davies n. sp. urn:lsid:zoobank.org:act:6109FB02-E6FA-4570-8959-F918341319A9The holotype male, nine paratype males and 10 paratype females were deposited in the United States Department of Agriculture Nematode Collection (USDANC), Beltsville, Maryland, USA, and 10 paratype males and 10 paratype females were deposited in the Forest Pathology Laboratory Collection, FFPRI, Tsukuba, Japan.Morphometric values are summarized in Intermediate in body size, 738–964 µm and 666–933 µm in length for males and females, respectively. Body cylindrical, slender, weakly ventrally arcuate when killed by heat treatment. Male tail strongly recurved ventrally. Cuticle thin, finely annulated with a lateral field with four incisures. Lip region distinctly offset from body, separated from other body parts by a clear constriction, sub-rectangular or rounded, ca twice as broad as high in lateral view. A cuticular plate present at the anterior end. The edge of plate a little off-set, and appears like two cuticular projections in lateral view. Stylet very well-developed, separated into two parts: a conus occupying approximately 50% of the total stylet length and a shaft with a conspicuous and large basal swelling, but not forming a clear basal knob. Procorpus cylindrical, ca 1.5 metacorpal lengths ( = ca 1 stylet length) long, connected to a well-developed muscular oval-shaped metacorpus (median bulb) occupying ca 90% of the body diameter. Dorsal pharyngeal gland orifice opening into the lumen of the metacorpus midway between the anterior end of the metacorpal valve and the anterior end of the metacorpus. Metacorpal valve conspicuous, ca 1/5 of the metacorpal length, located at the centre or a little posterior to the centre of the metacorpus. Pharyngo-intestinal junction immediately posterior to the posterior end of the metacorpus. Pharyngeal glands well-developed. Dorsal pharyngeal gland ca 5–6 metacorpal lengths long, overlapping the intestine dorsally, ca 50% of the corresponding body diameter at the broadest part. Nerve ring surrounding pharyngeal glands and intestine ca 1 metacorpal length posterior to pharyngo-intestinal junction. Excretory pore visible, located near level of the nerve ring, i.e., varying between the posterior end of the metacorpus to ca 2 metacorpal lengths posterior to the metacorpus. Hemizonid ca 2 metacorpal lengths posterior to the metacorpus.vas deferens, containing several well-developed sperm. Posterior end of vas deferens and intestine fused to form a narrow cloacal tube around the spicule. Sperm amoeboid, spermatocytes arranged in multiple (3–5) rows for anterior 1/5 of testis length, two rows for next 1/5, single row for middle part, with the well-developed sperm packed as 2–5 rows in the posterior part of testis. Tail region strongly arcuate ventrally, terminus claw-like in lateral view. Spicules paired, separate, mitten-shaped, stout, i.e., the length (chord from anterior end of condylus to cucullus) is ca 3 times the length of the widest part of the calomus–lamina complex: condylus short, rounded with anterior tip strongly recurved dorsally. Rostrum triangular, with pointed tip. Capitulum with clear depression immediately anterior to the anterior base of the rostrum. Lamina with two clear lines connecting the dorsal root of the condylus to the blunt tip, smoothly ventrally arcuate. Calomus–lamina complex widest at the posterior end of rostrum and calomus smoothly tapered along with lamina to distal tip. A cuticular limb present between calomus and lamina, extending from the middle of calomus to near the root of condylus. Connection between rostrum and calomus indistinctive, i.e., rostrum and calomus are connected with smooth curvature. Cucullus absent. Six (three pairs) genital papillae present: all are papilla-shaped, i.e., not gland-like (glandular papillae). First pair (P2) subventrally located, adcloacal, i.e., at level of cloacal opening (CO). Second pair (P3) subventral ca 1/2 tail length posterior to CO. Third pair (P4) located ventrally ca 1/2 cloacal body diameter posterior to P3. Bursal flap present, covers the distal part from the level of P3, having oval shape with three projections at the posterior end. The middle projection longer and narrower than the others, appears as hair-like extension in the lateral view.Gonad single, outstretched in most individuals. Posterior 1/5 of gonad forms receptaculum seminis) constructed of rounded cells, present as a branched overlapping of oviduct, i.e., branching out from anterior end of crustaformeria, slightly irregular oval shape, sometimes filled with well-developed sperm. Crustaformeria not conspicuous, formed of rather large, rounded cells. Uterus short with thick wall, sometimes containing a developing egg and several sperm. A sac-like expansion present on both sides of the uterus, which could be a part of the uterus. Dorsal uterine wall thickened at the uterus/vagina/post-uterine sac junction and a three-celled structure, where each cell has a cuticular  pronged structure, present at both right and left sides of the wall, but the structure is rather vague in fixed and mounted materials. Vagina slightly inclined anteriorly. Vulval opening lacking flap apparatus, both anterior and posterior vulval lips slightly expanded, and forming a dome-shaped slit in ventral view. Post-uterine sac conspicuous, filled with well-developed sperm in many individuals. Rectum present, seemingly functional, ca 1 anal body diameter (ABD) long, intestine–rectum junction constricted by sphincter muscle. Anus a small dome-shaped slit in ventral view; posterior anal lip slightly expanded in lateral view. Tail smoothly tapering to distal part, ca 3–6 ABD long. Tail tip region short, conical, with a hair-like projection.Reproductive tract composed of ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac (branch). Ovary single, anteriorly outstretched, anterior end reflexed once in some individuals. Ovary constructed of flat, plate-like cells. Oocytes present in multiple (2–5) rows in anterior 1/2–4/5 of ovary with a couple of well-developed oocytes in a single row at the posterior end. Oviduct tube-like, constructed of large oval-shaped cells, connecting ovary and crustaformeria, sometimes occupied by well-developed oocytes. Spermatheca , B. scolyti Massey, B. yongensis Gu, Braasch, Burgermeister, Brandstetter & Zhang, B. clavicauda Kanzaki, Maehara & Masuya, B. uncispicularis Zhuo, Li, Li, Yu & Liao) and the “B. leoni group” (B. eidmanni (Rühm), B. leoni Baujard, B. silvestris (Lieutier & Laumond) and B. borealis Korentchenko) sensu Braasch et al. B. maxbassiensis (Massey). The new species and these species share the strongly dorsally arcuate condylus of the male spicule B. maxbassiensis is most similar to the new species, i.e., these two species share the extremely well-developed stylet B. maxbassiensis by its lip morphology, with cuticular plate vs umbrella-like horizontal expansion, pharyngeal glands well-developed and extended vs. relatively short, position of excretory pore, posterior vs. anterior to median bulb, male bursal flap possessing three projections vs. rounded distal end, male spicule, the dorsal curvature in condylus is stronger in B. sycophilus n. sp., female post-uterine-branch occupying ca half of vulva-anus distance vs. occupying 2/3 or more of vulva-anus distance, female vulval structure, without any flap apparatus vs. slightly elongated anterior lip forming short flap and female tail, smoothly tapered with short conical distal end vs. smoothly tapered to distal end Based on the male spicule morphology, i.e., the initial typological character of the genus B. sycophilus n. sp. were consistent among analyses, only Bayesian trees are shown .The type materials were obtained on June 6, 2013 from a syconium from a B. cinerea and alfalfa callus were not successful. Bursaphelenchus sycophilus n. sp. showed no feeding behaviour on B. cinerea hyphae or on alfalfa callus parenchymal cells, and died out within one month of inoculation. Thus, the feeding preference (host range) of the nematode appears to be narrow, possibly specific to fig syconium tissue. Because a host-specific fig wasp species, Ceratosolen appendiculatus (Mayr), was the only insect found with the syconium from which type materials were obtained, the wasp is hypothesized to be the carrier (or host) insect of the nematode.The culturing attempts using The species was named after its characteristic habitat, the fig syconia.B. sycophilus n. sp. are contradictory to each other, i.e., the new species shares a characteristic recurved condylus with members of the “B. eremus group”, “B. leoni group” and B. maxbassiensis, but is molecular phylogenetically close to members of the “B. fungivorus group” , lack of a cucullus on the spicule and possession of a ventral limb of the spicule , infesting green ash, Fraxinus pennsylvanica Marsh. from North Dakota Hylesinus californicus sometimes bores into living trees B. maxbassiensis may also have the ability to feed on live tree tissue. Re-isolation of B. maxbassiensis and detailed molecular and morphological comparison are necessary to examine the origin of plant parasitism of B. sycophilus n. sp.The lip and stylet morphology of the new species is very unusual within the genus, and is similar to Bursaphelenchus, Parasitaphelenchus Fuchs, Sheraphelenchus Nickle and Ruehmaphelenchus Goodey Bursaphelenchus, the other three genera are included in the genus as subclades B. maxbassiensis shared a common ancestor, then the resurrection of the genus Omemeea Massey may be justified, if the genus (subfamily) is split into many small genera.The inferred molecular phylogenetic relationships and morphological characters are not congruent to each other in the subfamily Parasitaphelenchinae, which contains B. cinerea and alfalfa callus as food were not successful. The feeding resource preferences of several different nematodes, including Bursaphelenchus spp., have been previously studied, and in many cases, several different species of plant callus and ascomycete fungi, including B. cinerea were considered to be suitable food for aphelenchid nematodes B. sycophylus n. sp. does not appear to feed on fungus, and the species is hypothesized to be an obligate plant parasite.In the present study, culturing attempts using B. cocophilus can be cultured using fresh palm tissue Ficus variegata may be available for the culture of B. sycophilus n. sp.The obligate plant parasite, Bursaphelenchus, and they are phylogenetically distant from each other (B. xylophilus (Steiner & Buhrer), the pathogen of pine wilt disease B. cocophilus Cobb, the pathogen of red ring disease B. sexdentati Rühm, which has moderate to strong pathogenicity to pine trees Bursaphelenchus nematodes in their feeding abilities, e.g., digestive enzymes. However, the morphology of the ingestive/digestive organs in these plant-parasites, including the obligate plant-parasite, B. cocophilusBursaphelenchus species B. sycophilus n. sp. may represent the potential for morphological and developmental plasticity in the genus Bursaphelenchus.Several obligate and facultative plant parasites (pathogens) are known in the genus ch other , e.g., BB. sycophilus n. sp. clearly belongs to the genus Bursaphelenchus, its biological and morphological characters are similar to those of Schistonchus spp. The genus Schistonchus Cobb is known as to parasitize fig syconia, and are phoretically/parasitically associated with fig wasps Schistonchus spp. is very similar to that of B. sycophilus, i.e., it has a long conus and distinct basal swellings. Interestingly, the genus is clearly paraphyletic B. sycophilus n. sp. , detailed host range and distribution range. However, because the genotypes of B. sycophilus n. sp. isolated from several different locations on the Ishigaki and Iriomote Islands were identical, it is considered to be commonly distributed on these two islands. The distribution of F. variegata is widespread, from Ishigaki Island, Japan through South Eastern Asia to Northern Australia. However, regardless of multiple surveys of F. variegata in Northern Australia, B. sycophilus n. sp. has not been isolated from the area B. sycophilus, more surveys in South Eastern Asia and Northern Australia are needed.Some biological characters of F. variegata, and was not found from the other two species, F. septica and F. bengtensis. Thus, F. variegata is considered as the specific fig host of B. sycophilus n. sp.In the present study, three species of figs were examined for their nematode association. The new species was isolated only from Schistonchus species has potential tissue specificity, which could lead to partitioning of the microhabitat inside the syconium. In the present study, detailed histological analysis was not conducted. Because B. sycophilus n. sp. often shares the same syconium with a currently undescribed Schistonchus species  B. tadamiensis) B. sycophilus n. sp. and its related Bursaphelenchus spp., the new species seems to have adapted to this closed environment relatively rapidly. More detailed analyses, e.g., comparative genomic analyses using the new species and its close relatives, may yield interesting information concerning the genes involved in switching feeding habitats and the development of plant parasitism.Because fig syconia represent an exclusive habitat relative to nematode immigration, i.e., only fig wasps and parasitic wasps enter the young figs, the history of multiple aphelenchoidid lineage introductions into different Table S1GPS coordinates of sampling sites and nematode species (morphotypes and/or genotypes) isolated from the materials.(XLS)Click here for additional data file.Table S2Accession numbers and species names of nematodes used in the phylogenetic analysis.(XLS)Click here for additional data file.


Acinetobacter baumannii complex-associated infections, especially bacteremia, in health care settings poses a great threat to public health. We proceeded to investigate the risk and prognostic factors for MDR A. baumannii complex bacteremia in mainland China.The increasing prevalence and mortality of multidrug-resistant (MDR) A. baumannii complex bacteremia were included as the case group, while those infected with non-MDR A. baumannii complex were selected as the control group. The clinical data were collected and analyzed.This retrospective study was conducted at West China Hospital from January 2009 to December 2013. Using a computer-assisted microbiology laboratory database, patients with MDR A. baumannii complex blood isolates identified in our research, with the overall rate of multidrug resistance reaching 75.52% over the past five years. Using multivariate logistic analysis, being in the intensive care unit (ICU) , increased Pittsburgh bacteremia score  and use of carbapenem  were independent risk factors for MDR acquisition among patients with A. baumannii complex bacteremia. Older age , being post-transplantation , having a higher Pittsburgh bacteremia score  and having a lower level of albumin  were identified as independent risk factors for 30-day mortality in patients with MDR A. baumannii complex bacteremia.There were 241 non-duplicated A. baumannii complex bacteremia, which may be used to prioritize infection control practices and prognostic evaluations.In conclusion, our research revealed the risk factors associated with acquisition of and mortality from MDR  Acinetobacter baumannii, a non-fermentative Gram-negative coccobacillus, has become an increasingly notorious pathogen in health care settings due to its enhanced environmental resilience [A. baumannii comprises 4% to 7% of ventilator-associated pneumonia and 1% to 2% of nosocomial bloodstream infections [A. baumannii have reached a range from 10% to 43% in intensive care units (ICUs) and from 7.8% to 23% outside ICUs, which significantly increased the outlay of the infirmary [silience . It is efections . Based onfirmary .A. baumannii usually leads to a high rate of treatment failure, and the clinically-isolated multidrug-resistant (MDR) A. baumannii also tend to be increasing over time [A. baumannii can invade different parts of the body, such as the respiratory tract, bloodstream, or skin and soft tissue, MDR A. baumannii bacteremia usually leads to especially high attributable mortality [A. baumannii bacteremia has become extremely urgent.It is generally recognized that the propensity for antimicrobial resistance in ver time –9. Althoortality . AccordiA. baumannii bacteremia and its outcomes [A. baumannii as controls to identify risk factors only for A. baumannii bacteremia [A. baumannii complex bacteremia in mainland China. This study was performed in West China Hospital, a 4,000-bed tertiary hospital in Chengdu, southwest China, using our computer-assisted microbiology laboratory database.However, until recently, few retrospective studies have evaluated the factors influencing drug-resistance acquisition in outcomes –13. SeveA. baumannii complex were selected, and their clinical data were obtained from the electronic medical records for review. For patients with multiple episodes of A. baumannii complex bacteremia, only the first episode was included for analysis. Those included patients were further divided into cases or controls according to antibiotics susceptibility results. The case group was comprised of patients with MDR A. baumannii complex bacteremia, and the control group included patients with any other A. baumannii complex bacteremia.This retrospective study was conducted at West China Hospital from January 2009 to December 2013. Using the microbiology laboratory records, patients aged > = 18 years, with at least one blood culture positive for This study was approved by the ethics review committee of West China Hospital, Sichuan University, Chengdu, China, which waived informed consent.A. baumannii complex described here included the following 4 species: A. baumannii, A. nosocomialis, A. pittii, and A. calcoaceticus, which cannot been distinguished by conventional biochemical methods [During the study period, blood specimens were obtained under sterile conditions and cultured using the BacT/Alert 3D blood culture system . Identification of microorganisms was conducted using the VITEK 2 compact , and  methods ,15. Susc methods . Multidr methods .A. baumannii complex bacteremia, the sample date and report date of the first positive blood culture for A. baumannii complex, co-infection with other pathogens in the bloodstream, antibiotic susceptibility results, and the level of serum albumin.A standardized case report form was designed to collect the relevant demographic, clinical, and laboratory data following medical record review. The following details were gathered: age, gender, body mass index (BMI), underlying diseases, primary admission diagnosis, sources of bacteremia, Pittsburgh bacteremia score , duratioThe criteria for infection diagnosis were based upon National Healthcare Safety Network criteria . Bacteret test or the Mann-Whitney U test as appropriate. Categorical variables were calculated using a chi-square test or Fisher’s exact test as appropriate. The trend of proportion of isolates that were MDR was assessed using a linear by linear association test. Univariate analysis was used to identify significant factors for MDR acquisition and mortality, with results presented as odds ratios (OR) and 95% confidence intervals (95% CI). Independent covariates with a p value <0.10 were included in a multivariate logistic regression analysis. The survival curve was generated using the Kaplan-Meier method, and the log-rank test was used to compare survival between groups. All tests of significance were 2-sided, and significance was set at p value <0.05. Statistical analyses were performed using SPSS software .Continuous variables were compared using the student A. baumannii complex blood isolates between January 2009 and December 2013, including 182 MDR A. baumannii complex blood isolates. As shown in p = 0.032) from 2010 to 2013. The susceptibilities of A. baumannii complex isolates to 15 types of routinely reported antibiotics in the MDR group and the non-MDR group were showed in A. baumannii complex was highly resistant to all antibiotics (>90%) except amikacin (52.0%), tobramycin (87.4%) and trimethoprim-sulfamethoxazole (69.3%). However, non-MDR A. baumannii complex exhibited low resistance to all antibiotics (<50%), with the exceptions of cefotaxime (61.9%), ceftriaxone (71.2%) and aztreonam (93.1%).We identified 241 non-duplicated A. baumannii complex isolates from blood sample with their MDR constituent ratios across different hospital department are shown in A. baumannii complex (48.1%) and MDR A. baumannii complex (59.9%), followed by two surgical departments  and the hematological department.The distribution of A. baumannii complex bacteremia and non-MDR A. baumannii complex bacteremia. By univariate logistic analysis .There were no differences in gender, BMI, prior invasive therapy or prior surgical procedures between the patients with MDR analysis , patientanalysis , stayingEnterococcus faecium bacteremia and those with lower levels of albumin were associated with poorer prognosis. Older age , being post-transplantation , high Pittsburgh bacteremia score  and lower level of albumin  remained independent risk factors for 30-day mortality in patients with MDR A. baumannii complex bacteremia in the multivariate analysis  [A. baumannii complex bacteremia [In this study, patients with MDR  studies –37. Lee cteremia . They recteremia ,40.A. baumannii complex bacteremia had a 30-day mortality rate of 29.1%, which was similar to that in previous studies [A. baumannii complex bacteremia were shown to be related to patients’ co-morbidity, sources of bacteremia, bacteremia severity, and inappropriateness of empirical therapy [A. baumannii complex bacteremia, and the subsequent survival analysis also confirmed this conclusion. As one of the negative acute phase proteins, the level of albumin in the plasma of critical patients likely reflects the change in vascular permeability rather than changes in the synthesis or catabolism of the protein [A. baumannii complex support the theory that albumin could be a good indicator of prognosis in critically ill patients, which is consistent with previous reports [A. baumannii complex bacteremia did not differ from the prognosis of patients with monomicrobial MDR A. baumannii complex bacteremia. One possible reason is that MDR A. baumannii complex may be the predominant determinant of prognosis compared with other organisms in polymicrobial bacteremia.Our patients with MDR  studies . Such hi therapy . Our pre therapy ,44. In o protein . Thus, a protein . However reports . In addi reports . HoweverA. baumannii complex bacteremia. However, the decision for empiric therapy against MDR A. baumannii infection needs to identify the risk factors of both MDR acquisition and A. baumannii complex bacteremia. In that situation, a case-case control study might be more effective in guiding clinical practice [Our study should be interpreted with caution given its several limitations. First, the retrospective design may preclude the validity and reliability of comparisons. To diminish potential biases, multivariate logistic analysis was employed to discover independent risk factors. Additionally, all the data included in our analysis were retrieved from computerized records in order to reduce non-systematic information bias. Second, our data from a single center also limit the applicability of our results to other geographical areas or institutions. Third, as molecular studies cannot be performed because most clinical isolates in our center are not routinely preserved, resistance mechanisms of specific bacteria were not identified, and molecular epidemiology was not investigated. Lastly, our research focused on investigating the factors associated with the acquisition of MDR among practice .A. baumannii complex bacteremia. Patients with MDR A. baumannii complex bacteremia had worse outcomes, and their mortality risk factors were confirmed to be associated with older age, severity of illness and their immune status. The risk factors associated with acquisition of and mortality from MDR A. baumannii complex bacteremia may be used to prioritize infection control practice and prognosis evaluation.In conclusion, our research revealed that exposure to carbapenem and previous ICU admission were independent risk factors for acquiring MDR among S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


Didymella pinodes is the principal causal agent of ascochyta blight, one of the most important fungal diseases of pea (Pisum sativum) worldwide. Understanding its host specificity has crucial implications in epidemiology and management; however, this has not been clearly delineated yet. In this study we attempt to clarify the host range of D. pinodes and to compare it with that of other close Didymella spp. D. pinodes was very virulent on pea accessions, although differences in virulence were identified among isolates. On the contrary, studied isolates of D. fabae, D. rabiei, and D. lentil showed a reduced ability to infect pea not causing macroscopically visible symptoms on any of the pea accessions tested. D. pinodes isolates were also infective to some extend on almost all species tested including species such as Hedysarum coronarium, Lathyrus sativus, Lupinus albus, Medicago spp., Trifolium spp., Trigonella foenum-graecum, and Vicia articulata which were not mentioned before as hosts of D. pinodes. On the contrary, D. lentil and D. rabiei were more specific, infecting only lentil and chickpea, respectively. D. fabae was intermediate, infecting mainly faba bean, but also slightly other species such as Glycine max, Phaseolus vulgaris, Trifolium spp., Vicia sativa, and V. articulata. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to confirm identity of the isolates studies and to determine phylogenetic relationship among the Didymella species, revealing the presence of two clearly distinct clades. Clade one was represented by two supported subclusters including D. fabae isolates as well as D. rabiei with D. lentil isolates. Clade two was the largest and included all the D. pinodes isolates as well as Phoma medicaginis var. pinodella. Genetic distance between D. pinodes and the other Didymella spp. isolates was not correlated with overall differences in pathogenicity. Based on evidences presented here, D. pinodes is not specialized on pea and its host range is larger than that of D. fabae, D. lentil, and D. rabiei. This has relevant implications in epidemiology and control as these species might act as alternative hosts for D. pinodes. Arx (anamorph A. rabiei (Pass) Labr.) are the causal agents of ascochyta blights on faba bean (Vicia faba L.), lentil (Lens culinaris Medik.), and chickpea (Cicer arietinum L.), respectively  Petrak, Phoma medicaginis var. pinodella (L.K. Jones) Morgan-Jones and K.B. Burch and Phoma koolunga Davidson, Hartley, Priest, Krysinska-Kaczmarek, Herdina, McKay, and Scott  Vestergr., anamorph Ascochyta pinodes L.K. Jones) is the most predominant and damaging pathogen and under some conditions can cause yield losses up to 70%  to assess the susceptibility/resistance of different accessions within each of these legume species to nine isolates of D. pinodes from different geographical origin; (iii) to compare the host range of D. pinodes with that of other Didymella species; and (iv) to relate fungal isolates by ITS molecular markers.The aims of this study were therefore (i) to further refine the host range of D. pinodes, two isolates of D. fabae, one of D. lentil, and one of D. rabiei, all from IAS-CSIC fungal collection, were used in the experiments , barrel medick (M. truncatula Gaertn.), button medick (M. orbicularis (L.) Bartal.), chickpea (Cicer arietinum L.), common bean (Phaseolus vulgaris L.), common vetch (Vicia sativa L.), faba bean (Vicia faba L.), fenugreek (Trigonella foenum-graecum L.), grass pea (Lathyrus sativus L.), lentil (Lens culinaris Medik.), oneflower vetch (Vicia articulata Hornem.), pea (Pisum sativum ssp. sativum L.), prinkly scorpion's tail (Scorpiurus muricatus L.), red clover (Trifolium pratense L.), soybean (Glycine max (L.), subterranean clover (T. subterraneum L.), sulla (Hedysarum coronarium L.), tawny pea (P. fulvum Sibth. & Sm.), white clover (T. repens L.), and white lupin . From 1 to 6 accessions per species were tested  filled with a 1:1 mixture of sand and peat in a rust-free growth chamber. Plants were pre-germinated and sown at 3 days intervals in order to be able to select seedlings at the same growing stage at the time of inoculation. There were three independent replicates per fungal isolate, arranged in a complete randomized design. Each replicate consisted of 3 pots with 5 plants each per accession. Experiments were repeated three times. Pea cv. Messire was included in each replication as a common susceptible check. Plants were grown in a growth chamber at 20°C, under a photoperiod of 14/10 h day/night regime, with 148 μmol/m6 spores/ml. Tween 20 (VWR) was added as wetting agent (two drops per 500 ml pycnidiospore suspension). The pycnidiospore suspensions were sprayed at the 4–5-leaf stage using a handheld sprayer at a rate of 1 ml per plant. After inoculation, plants were covered with a polyethylene sheet during the first 24 h in darkness, and high humidity was ensured by ultrasonic humidifiers operating for 15 min every 2 h. Later on, the polyethylene cover was removed and plants were maintained 9 more days in a growth chamber (under conditions described above). Every 2 days, water was added to the trays to maintain high relative humidity (95–100%).Plants with 4-5 leaves were inoculated as described by Fondevilla et al.  with somPlant response to infection was visually assessed 10 days after inoculation using two separate assessments. Disease severity (DS) was assessed by a visual estimation of the percent of diseased tissue per plant , gently scraping the colony with a glass rod and filtering the suspension through two layers of sterile cheesecloth. Three Petri dishes per isolate were used, in order to ensure sufficient amount of fungal material. Suspension was centrifuged at maximum speed  and pellet was collected. DNA was extracted from ground mycelium using the DNeasy plant minikit . DNA concentration was determined using an ND-1000 spectrophotometer (NanoDrop Technologies) and adjusted to 20 ng μl/1 for PCR. Primers ITS1 and ITS2 were used to amplify the nuclear ribosomal internal transcribed spacer (ITS) region ITS1-5.8S-ITS2 following the protocol described by White et al. . PCR proAscochyta pisi, Didymella pinodes, Phoma koolunga, and P. medicaginis var. pinodella .All isolate x species combinations  were arranged in a completely randomized design in a controlled condition growth chamber. For the whole data set, only final disease severity values were included in the statistical analysis. Disease severity was first analyzed by taking into account differences in pathogenicity between the 13 Didymella spp. isolate between accessions within each species. The whole experiment was repeated three times. Before performing analyses of variance, the normality and equality of variances were checked using Shapiro–Wilk's .Disease severity was assessed for every Disease rating (DR) was visually estimated as the mean disease score over the first, second and third leaves of each accession within each specie.http://nhm2.uio.no/norlex/past/download.html, with the following settings: covariance matrix type, four principal components, 1-fold change threshold for clusters, and 0.3 correlation thresholds for clusters. PCA results were represented as a biplot, with accessions more susceptible to a specific Didymella spp. isolate (according to both DS and DR) located in the same area of the graph.The entire data set was analyzed by Principal Component Analysis (PCA) using the web-based software PAST  were defined as weak support, scores between 75 and 89% BS as moderate support and scores > 90% BS as strong support. A likelihood ratchet employs multiple sequential truncated searches on different starting trees created by fast algorithmic searches on reweighed data, in the hope of exploring a larger pro- portion of tree space, analogous to the parsimony ratchet   confirming expectations , Trifolium spp., Medicago spp., V. articulata, C. arietinum, and L. culinaris . Some infection was also observed on G. max, V. sativa, L. sativus, H. coronarium, P. fulvum, S. muricatus, V. faba, and T. foenum-graecum although at the level of resistance . P. vulgaris did not showed any symptoms of fungal infection  Table  as well Didymella spp. showed that the legume species under study displayed differential resistance/susceptibility to each isolate as indicated by significant specie x isolate interactions in ANOVA , fungal isolates , and their interaction , indicating that not all D. pinodes isolates displayed the same infection pattern toward the legume species involved in this study.Results from cross inoculations performed with different P. sativum accessions showed DR values = 4 against all D. pinodes tested . As for P. sativum, accession IFPI3260 was immune to other Didymella spp. isolates tested , showing level of infection that varied depending on the isolate tested . Isolates Dp-Esc-13 and Dp-ANN-13 were the most virulent , being differences significant among accessions and isolates (P < 0.01) .1) Table . V. artiMedicago spp. accessions showed from resistance to susceptibility to D. pinodes infections. Nevertheless, differences between plant species were not consistent  . By contrary, D. rabiei did not cause any symptoms on lentils and D. fabae was only slightly infective  Table . As for %) Table . By contPI08100 from G. max showed from moderate to high resistance against D. pinodes infections  and susceptible to D. fabae  and susceptible to Dp-KHM-13 , with no significantly difference among accessions (Table V. sativa showed a fully compatible interaction with both D. fabae isolates in spite of a reduced severity (DS < 10%). Nevertheless, both D. rabiei and D. lentil caused foliar symptoms at reduced rates  although significant differences between accessions were found  with exception of D. fabae that caused compatible interactions (DR ≥ 3.3) although with reduced DS values  was only virulent on L. culinaris accessions, while isolate Dr-Pt04 (D. rabiei) showed symptoms on C. arietinum and, although limited, on G. max. Finally, G. max, P. vulgaris, T. pratense, V. sativa, and V. faba were susceptible to isolates from D. fabae  showed that two principal axes gave eigenvalues greater than 1, while the other axis all had eigenvalues lesser than 1 Table . Hence, d Figure . On the d Figure .ITS analysis by MEGA6 originates an optimal tree with the sum of branch length = 0.06595538. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches Figure . The treD. pinodes from the others . The isolate of P. medicaginis var. pinodella was also included in this clade although it was divergent and on a branch apart from the rest of the isolates included.From the dendrogram generated, using UPGMA with the genetic distance coefficient, the 17 isolates could be classified into two main clusters that clearly separate all isolates belonging to s Figure . ClusterD. fabae, one isolate from D. lentil, one isolate from D. rabiei and one isolate from A. pisi. D. fabae isolates showed to be monophyletic since they were included in a unique well-supported branch ([BS] = 99). By contrary, isolates from D. lentil and D. rabiei clustered together in other strongly supported branch ([BS] = 97) where A. pisi was apart ([BS] = 80). Finally, isolate from P. koolunga did not fit with any other isolates.Clade II ([BS] = 71) comprised two isolates of Didymella pinodes was reported as the principal agent causing aschochyta blight on peas  and common vetch. Results for D. lentil and D. rabiei agreed with previous studies which demonstrated that artificial inoculations with Ascochyta fungi in the greenhouse and/or growth chambers are host-specific  displayed a certain degree of partial resistance. These results confirms that only incomplete resistance is available for cultivated pea, while the highest levels of resistance are available in related Pisum species. In fact, sources of resistance to D. pinodes were recently found in accessions belonging to P. fulvum, P. sativum ssp. syriacum, and P. sativum ssp. elatius  and pea rust (Uromyces pisi (Pers.) Wint) , lentil (L. culinaris), fenugreek (T. foenum-graecum) and oneflower vetch (V. articulata) is described here for the first time, expanding the current knowledge of D. pinodes's host range.y Weimer  who studP. vulgaris) against several D. pinodes isolates may indicate that this species is a non-host species or that the fungus had invaded the host tissues internally although no visible symptoms were observed. This has been previously found for D. rabiei, which was recovered consistently from inoculated tissue of pea without causing any visible symptoms , faba bean (V. faba), and soybean (G. max) may be defined as a host plant under conditions of high inoculum pressure, but all genotypes studied displayed a very high level of partial resistance against the set of fungal isolates tested. As the conditions used in this study were very favorable for disease development on plants, the results would require confirmation by testing under different infection conditions such as in the field since growth habit, canopy morphology, lodging and precocity can affect D. pinodes development  suggest that the reaction may therefore be described as cultivar specific since the fungal ability to infect these other species depends on the susceptibility of the cultivar chosen  were significantly more aggressive, hence dangerous if introduced in other fields. Migration of invasive organisms might lead to selective emergence of adapted isolates in novel geographic regions and on specific host genotypes (Leo et al., D. pinodes is a teleomorph of A. pinodes that reproduces asexually by pycnidia containing splash-dispersed pycnospores (Roger and Tivoli, D. pinodes isolates is still a matter of concern since there are numerous reports analyzing differential reaction of fungal isolate collection on various hosts leading to ambiguous conclusions (Ali et al., D. pinodes isolates used for the study. Both results from D. pinodes host range as well as molecular ITS analysis indicate a lack of pathotypes within the fungal collection used here.Knowledge of the host range is important to determine whether other crops could be affected. Understanding of population diversity and identification of pathogenic variation within plant species will assist in the management of ascochyta blight diseases.D. pinodes as our results suggest, the use of this specie may have positive effect on soil infestation and subsequent disease development. Conversely, the use of grass pea, clover, lentil, oneflower vetch, white lupin might considerably increase the inoculum potential of the soil, having a deleterious effect on the subsequent pea crop. Ascospores produced in pseudothecia on overwintered debris of alternative hosts may serve as important sources of primary inoculum and/or inoculum necessary for secondary infections later in the growing season, as other aschochyta species did (Trapero-Casas et al., If common bean is a non-host to D. pinodes isolates from one legume with those from another, as demonstrated with Ascochyta spp. by Hernandez-Bello et al. (A. pisi and A. fabae isolates. This is especially important in light of the plasticity of D. pinodes which is highly adaptable under the influence of biotic and abiotic factors (Le May et al., The use of chickpea, medick, sulla or fenugreek cultivars with qualitative resistance could be considered, but studies on the risk of resistance breakdown are required. As well, it would be important to determine if and which species could act as bridging hosts allowing for the crossing of o et al.  for A. pEB designed experiments. Together with MC carried out the experimental work. EB carried out most of the data analysis and contributed to the writing of the manuscript. DR contributed to the interpretation of results and writing of the manuscript. MC also contributed to critical reading.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


T is a nitrogen-fixing symbiont of Centrosema pubescens, and comprises the recently described species Bradyrhizobium viridifuturi. Its draft genome indicates that it belongs to the Bradyrhizobium elkanii superclade. SEMIA 690T carries two copies of the regulatory nodD gene, and the nod and nif operons resemble those of Bradyrhizobium diazoefficiens.SEMIA 690 Bradyrhizobium is probably the main symbiotic bacteria (–Glycine max (L.) Merrill] , isolated from Centrosema pubescens (Benth.) Kuntze.A major process for global nitrogen balance results from the symbiotic association of nitrogen-fixing rhizobia and legume plants , 2. In tbacteria , and thebacteria –6. The gacteria –, to rootMerrill] , 9. Recedifuturi . Here, wde novo assembled by Newbler version 2.9 (Roche). Shotgun sequencing allowed a 28-fold coverage, and the genome analysis revealed that the strain has one circular chromosome. Sequences were submitted to RAST  and processed at the Ion PGM platform (Life Technologies) at the LNCC, Petrópolis, Brazil. The FASTQ files were  to RAST , and the to RAST . The majT indicates highest similarity (~90%) with B. elkanii and B. pachyrhizi (nod) and nitrogen fixation (nif) genes of SEMIA 690T resemble those of B. diazoefficiens USDA 110T, but with different levels of similarity. As in USDA 110T, there are two copies of the regulatory nodD gene , BioProject (PRJNA290320), BioSample (SAMN03890369), and Accession (LGTB00000000). The version described in this paper is the first version.