Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged  Drosophila imaginal discs, the epithelial structures in the larva that will form the adult animal during metamorphosis, have been an important model system for tissue repair and regeneration for over 60 years. Here we show that damage-induced JNK signaling leads to the upregulation of a gene called moladietz, which encodes a co-factor for an enzyme, NADPH dual oxidase (Duox), that generates reactive oxygen species (ROS), a key tissue-damage signal. High expression of moladietz induces continuous production of ROS in the regenerating tissue. The sustained production of ROS then continues to activate JNK signaling throughout the course of regeneration, ensuring maximal tissue regrowth.Regenerating tissue must initiate the signaling that drives regenerative growth, and then sustain that signaling long enough for regeneration to complete.  The capacity to regenerate damaged or lost organs or limbs is significantly greater in some animals than others. The use of model organisms with varying degrees of regenerative capacity, from whole-body regeneration in planaria and hydra, to limb regeneration in amphibians, organ and fin regeneration in zebrafish, and the limited tissue regeneration that occurs in mammalian models, has advanced our understanding of this process , antz (mol)  , AdoR (F0039747) , and kay0001297)   , Nlaz (FE FBgn00117 activi0003900) , and zfh0004606)  showed u (corto) . Some offeration ,73, and % were dipuckered, can be observed . Vari. VariDroresponse , during response ,85 and aresponse . After acontrols . A seconneration . Additio (Alp-4) , the 4E-4) , an89) /TM6B (gift from M. Uhlirova)[nlaZ:GFP[R2] (gift from M. Ganfornina)[w1118; P{10xStat92E-GFP}1 (BL26197)[cn1P{PZ}dve01738/CyO; ry506 (BL11073)[cn1P{PZ}sm05338/CyO; ry506 (BL11403)[y1w*; P{PTT-GB}LamCCB04957ttvCB04957/SM6a (BL51528)[y1w*; Mi{PT-GFSTF.1}AdoRMI01202-GFSTF.1/TM6C, Sb1Tb1 (BL60165)[y1w*; P{lacW}Thork13517 (BL9558)[y1w*; Mi{PT-GFSTF.0}kayMI05333-GFSTF.0 (BL63175)[w1118; PBac{corto-GFP.FPTB}VK00037 (BL42268), w1118; PBac{Hr78-GFP.FLAG}VK00037 (BL38653), w1118;PBac{NC2β-GFP.FPTB}VK00033 (BL56157), y1 w*; Mi{PT-GFSTF.0}CatMIO4522-GFSTF.0 (BL60212)[HmlΔRFP (gift from K. Bruckner)[TRE-red and gstD-GFP (gifts from D. Bohmann)[Ets21Cf0369 (BL18678)[y1w*; Mi{MIC}CG9336MI03849 (BL36397)[y1w67c23; P{lacW}Col4a1K00405/CyO (BL10479)[y1w67c23; P{lacW}vkgk00236 (BL10473)[P{PZ}Thor06270cn1/CyO; ry506 (BL11481)[w1; P{UAS-Sod1.A}B36 (BL24754), w1; P{UAS-CatA}2 (BL24621), w1; P{UAS-Sod2.M}UM83 (BL24494), w1118; PBac{RB}mole02670/CyO (BL18073)[y1sc*v1; P{TRiP.HMS02560}attP40 (UAS-molRNAi) (BL42867), y1v1; P{TRiP.GL00678}attP40 (UAS-DuoxRNAi) (BL38907), y1v1; P{TRIP.GL00678}attP40 (UAS-NoxRNAi) (BL32902), y1w*; Mi{MIC}NoxMI15634/SM6a (BL61114)[pucE69 [UAS-JNKDN [w* hepr75/FM7C (BL6761)[w*; P{neoFRT}82B p38a1 (BL8822)[Flies were reared on standard molasses medium at 25° C except during regeneration experiments. The following (BL37920), y1,w67c(BL10440), y1, w* (BL56274), P{PZ}Al(BL12285), w1118; BL11515), y1w67c(BL12173), ry506PS (BL684), w;;pBACUhlirova), nlaZ:GFnfornina), w1118; (BL26197), cn1P{P(BL11073), cn1P{PBL11403), y1w*; (BL51528), y1w*; (BL60165), y1w*; (BL63175), w1118; (BL60212), HmlΔRFP15, y1w603, y1w*(BL36397), y1w67c(BL11481), w1; P{U(BL61114), pucE69 AS-JNKDN , w* hepr (BL6761), w*; P{n (BL8822).Immunostaining was carried out as previously described .Anitbodies and dilutions used were Anti-Nubbin (1:500) (gift of S. Cohen) , mouse aAlexa Fluor (AF) secondary antibodies from Molecular Probes were AF488, AF555 and AF633 (used at 1:500). Nuclei were labeled with DAPI (Sigma)(1:5000).EdU incorporation was carried out using the click-it EdU Alexa Fluor 594 Imaging kit (Molecular Probes) as previously described . SamplesImmunostained samples were imaged on a Zeiss LSM 700 confocal microscope and images were processed using ZenLite, Adobe Photoshop, and Image J software. Bright-field imaging of adult wings was done on an Olympus SZX10 microscope using the CellSens Dimension software, and images were processed using Image J.ROS were detected in imaginal discs using Dihydroethidium (DHE)  using the protocol described in Owusu-Ansah et al. , with slgstD-GFP, whose expression was not uniform and thus was quantified by measuring GFP intensity in the entire pouch area. Average intensities of multiple wing discs were combined to calculate the final average intensity plotted in the graphs. For measuring the pouch area, a maximum projection of all the confocal slices was taken and the Nubbin-expressing area measured in Image J. Graphs were plotted in Excel, R, and GraphPad Prism 7.0.Fluorescence intensity analysis was performed using single confocal slices. Average intensity was calculated by measuring intensity values in three equal-sized boxes in the pouch region of the wing disc in Image J, except for the For imaginal disc measurements and immunofluorescence quantifications, the Welch’s t-test was performed using R and GraphPad Prism 7.0. For the adult wing size assay, chi-squared tests were performed using GraphPad online tools. Statistical analyses for adult wing measurements were performed using Welch’s t-test.nub-GFP/+; rn-Gal4, GAL80ts, UAS-rpr /+, while the mock-ablated controls had the genotype nub-GFP/+; rn-Gal4, GAL80ts/+. Cells were isolated for the transcriptional profile as previously described [The ablated regenerating discs had the genotype escribed . Brieflyescribed . RNA quaDrosophila melanogaster genome  with a maximum of 2 mismatches permitted. Intron length was set between 20 and 150,000, and a gene model was provided as GTF . FPKM estimation was done using Cufflinks (v.0.0.7) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101797).Fastq reads were trimmed using FASTQ Quality Trimmer (v.1.0.0) and adaptor sequences were removed using Clip (v.1.0.1) in Galaxy . Reads wv.0.0.7) , and botv.0.0.7) , geometrv.0.0.7) . All biov.0.0.7) . The datv.0.0.7)  and are S1 Fig(A) Variation in the size of the wing pouch as marked by anti-Nub immunostaining in undamaged and ablated wing discs. Note the minimal variation in pouch size at R0, indicating consistency of ablation. Undamaged n = 8, regenerating n = 28. Error bars are SEM. (B) Quantification of percentage of regenerating animals that had pupariated by each day, showing the asynchronous progression to pupariation that occurred after tissue damage. Undamaged animals were w1118;; rnGAL4, Gal80TS/+, regenerating animals were w1118;; rnGAL4, UAS-reaper, Gal80TS/+, and both experienced the 24-hour temperature shift. Three independent experiments, total undamaged n = 144 pupae, regenerating n = 176 pupae. (C) Percentage of wings of different sizes on w1118;; rnGAL4, UAS-reaper, Gal80TS/+ animals that eclosed on different days after egg laying due the asynchronous development induced by tissue damage. Note that the animals that eclosed first (day 18) had smaller wings than those that eclosed on day 19, and those that eclosed on day 20 had the largest wings. Thus, variation in wing size after disc regeneration is partly determined by length of time for regeneration. Three independent experiments, total n = 371 wings.(TIF)Click here for additional data file.S2 Fig(A) Venn diagrams showing genes at least 1.3-fold upregulated or downregulated in three transcription profiles  of r(TIF)Click here for additional data file.S3 FigAdoR-GFP MiMIC enhancer trap. (B-B’) Kayak-GFP protein trap. (C-C’) Thor-lacZ enhancer trap. (D-D’) Corto-GFP protein trap. (E-E’) NLaz-GFP MiMIC enhancer trap. (F-F’) anti-Twist. (G-G’) zfh1-lacZ enhancer trap. Blue dashed line outlines the wing primordium. Scale bars are 100 μm. (H) Quantification of upregulation of mol, Nox, and Ets21C expression using qPCR. Four biological replicates each. Error bars are SEM, *p<0.05.Undamaged (A-G) and regenerating (R24) (A’-G’) wing discs. (A-A’) (TIF)Click here for additional data file.S4 Figfru3/+ males had larger wings after regeneration than controls. Three independent experiments, w1118 n = 112 wings, fru3/+ n = 95 wings, p<0.001 by a chi-squared test. (B) Adult htlAB42/+ animals had larger wings after regeneration than controls. Three independent experiments, w1118 n = 316 wings, htlAB42/+ n = 223 wings, p<0.001 by a chi-squared test. Error bars are SEM.(A) Adult (TIF)Click here for additional data file.S5 Fig(A-D) Hemolectin-RFP (Hml-RFP) (green) showing hemocytes near undamaged  and R24  wing discs.  Anti-Nimrod (green) also showing hemocytes near an R24 wing disc, confirming the Hml-RFP results. (B), (D), and (F) are orthogonal slices with the columnar epithelium or the disc proper (DP) toward the bottom and the peripodial epithelium (PE) toward the top of the images. (G) Schematic of an undamaged and a regenerating epithelium showing the location of a hemocyte outside the PE.(TIF)Click here for additional data file.S6 FigmolRNAi caused smaller adult wings after regeneration of the imaginal discs than controls. Three independent experiments, w1118 n = 402 wings, UAS-molRNAi n = 221 wings, p<0.0001 by a chi-squared test. (B-C) Cellular debris is visually distinct from the regenerating epithelium. Regenerating w1118 (B) and mole02670/+ (C) discs at R24, expressing the nub-GFP enhancer trap and stained with DHE. The speckled, grainy GFP tissue is cellular debris, outlined with yellow. The smooth GFP tissue is the intact epithelium, outlined with blue. The side panels are zoomed-in views of debris and epithelium, to show that they are easily distinguished.  Undamaged and R24 w1118 discs stained with DAPI and the ROS detector H2DCFDA. (F-J) gstD-GFP expression in undamaged w1118 (F), and regenerating w1118  and mole02670/+  wing discs at R24  and R48 . (K-O) Anti-Myc immunostaining in w1118  and mole02670/+  regenerating wing discs at R24  and R48 . (O) Quantification of immunofluorescence in Myc staining. R24 w1118 n = 14 discs, mole02670/+ n = 12 discs. R48 w1118 n = 11 discs, mole02670/+ n = 14 discs. Scale bars are 100 μm. Error bars are SEM except where noted. *p<0.02.(A) Expression of (TIF)Click here for additional data file.S7 Figw1118 and UAS-DuoxRNAi animals. Three independent experiments. w1118 n = 390 wings, UAS-DuoxRNAi n = 200 wings, p = 0.0005 using a chi-squared test. (B) Sizes of adult wings after disc regeneration in w1118 and UAS-NoxRNAi animals. Two independent experiments, thus error bars are SD. w1118 n = 299 wings, UAS-NoxRNAi n = 257 wings, p<0.0001 using a chi-squared test. (C) Sizes of adult wings after disc regeneration in w1118 and NoxMI15634/+ animals. Three independent experiments. w1118 n = 349 wings, NoxMI15634/+ n = 180 wings, p<0.0001 using a chi-squared test.  qPCR showing effectiveness of Duox (D) and Nox (E) RNAi. The RNAi was expressed under rn-GAL4 control in the pouch of normally developing wing discs for 24 hours before collecting for qPCR. Three biological replicates each, *p<0.05. (F) Total number of mitotic cells as identified by anti-phospho-Histone H3 staining in the wing pouch as identified by anti-Nub staining in the indicated genotypes. R0 w1118 n = 14 discs, NoxMI15634/+ n = 15 discs, UAS-NoxRNAi n = 15 discs, R24 w1118 n = 12 discs, NoxMI15634/+ n = 17 discs, UAS-NoxRNAi n = 15 discs, R48 w1118 n = 11 discs, NoxMI15634/+ n = 18 discs, UAS-NoxRNAi n = 19 discs. (G) Area of the wing pouch as marked by anti-Nub staining was measured at R0, R24 and R48 for the indicated genotypes. R0 w1118 n = 14 discs, NoxMI15634/+ n = 15 discs, UAS-NoxRNAi n = 15 discs, R24 w1118 n = 12 discs, NoxMI15634/+ n = 17 discs, UAS-NoxRNAi n = 15 discs, R48 w1118 n = 11 discs, NoxMI15634/+ n = 18 discs, UAS-NoxRNAi n = 19 discs. (H) Pupariation timing for regenerating animals of the indicated gentoypes. Three independent experiments. w1118 n = 204, NoxMI15634/+ n = 113, UAS-NoxRNAi n = 206 (I) Pupariation timing for normally developing animals that did not experience the thermal shift and so did not ablate and regenerate the wing primordia. Note that because these animals did not experience the thermal shift, the pupariation timing cannot be compared directly to the pupariation timing in panel H, in which the animals did experience the thermal shift. w1118 n = 97, NoxMI15634/+ n = 49, UAS-NoxRNAi n = 122. Error bars are SEM.Regeneration assays using adult wing size to assess extent of regenerative growth in the imaginal discs. (A) Sizes of adult wings after disc regeneration in (TIF)Click here for additional data file.S8 Figmol-lacZ reporter (green) in w1118 undamaged discs (A), and in regenerating wing discs at R0 (B), R24 (C) and R48 (D). (E) Quantification of mol-lacZ expression changes. Undamaged n = 3, R0 n = 7, R24 n = 10, R48 n = 10.  Anti-β-galacosidase immunostaining showing expression of the mol-lacZ reporter (green) in w1118 (F) and p38a1/+ (G) R24 discs. (H) Quantification of the fluorescence from the immunostaining. Two independent experiments, for a total w1118 n = 10 discs, p38a1/+ n = 12 discs. Scale bars are 100 μm. Error bars are SEM.(A-E) Anti-β-galacosidase immunostaining showing expression of the (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Staphylococcus aureus , including methicillin-resistant S. aureus (MRSA); streptococci , including Streptococcus pneumoniae, viridans group streptococci, and beta-hemolytic streptococci; Enterobacteriaceae, including Escherichia coli ; Haemophilus influenzae ; and Moraxella catarrhalis . Omadacycline merits further study in serious infections where resistant pathogens may be encountered.Omadacycline was tested against 21,000 bacterial isolates collected prospectively from medical centers in Europe and the United States during 2016. Omadacycline was active against  Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterobacteriaceae that produce extended-spectrum β-lactamases (ESBLs) and carbapenemases, and multidrug-resistant (resistant to ≥3 classes of agents) strains of Acinetobacter spp. and Stenotrophomonas maltophilia  were collected prospectively from hospitalized patients in 68 medical centers in the United States and Europe for the 2016 SENTRY Antimicrobial Surveillance Program. Isolate identifications were established by the participating medical centers and confirmed at JMI Laboratories , when necessary. Omadacycline MIC values were determined using the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (Enterobacteriaceae isolates were classified as susceptible (S) to ceftazidime , nonsusceptible (NS) to ceftazidime , R to imipenem , R to tetracycline , and NS to tigecycline . Other resistant phenotypes included MRSA (oxacillin MIC ≥ 4 mg/liter or cefoxitin MIC > 8 mg/liter), vancomycin-NS enterococci (MIC ≥ 8 mg/liter), tetracycline-R A. baumannii, staphylococci, enterococci , and Streptococcus pneumoniae (MIC ≥ 4 mg/liter), macrolide-R S. pneumoniae (erythromycin MIC ≥ 1 mg/liter and azithromycin MIC ≥ 2 mg/liter), and macrolide-R β-hemolytic streptococci (BHS) (erythromycin MIC ≥ 1 mg/liter). Haemophilus influenzae isolates were divided into β-lactamase-positive and -negative groups. QC strains were tested concurrently and included Escherichia coli ATCC 25922 and ATCC 35218, S. aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and S. pneumoniae ATCC 49619. All QC results, including all omadacycline MIC values, were within published ranges.Bacterial isolates  isolates were methicillin R, 40.0% of Enterococcus faecium isolates were vancomycin NS, 11.6% of S. pneumoniae isolates were penicillin R, 31.4% of S. pneumoniae isolates were macrolide R, 14.4% of E. coli isolates were ceftazidime NS, 28.6% of Klebsiella pneumoniae isolates were ceftazidime NS, 27.9% of Enterobacter cloacae species complex (SC) isolates were ceftazidime NS, and 8.1% of K. pneumoniae isolates were imipenem R , 43.6% of BHS, 32.8% of Enterobacteriaceae, 66.4% of Acinetobacter baumannii, 0.2% (data not shown) of H. influenzae, and 11.1% (data not shown) of Haemophilus parainfluenzae  , omadacycline inhibited 99.9% of isolates at ≤2 mg/liter, including 100.0% of methicillin-S S. aureus (MSSA) and 99.7% of MRSA isolates . Tetracycline resistance had little effect on omadacycline MIC values against S. aureus  or CoNS  isolates  than against E. faecalis  and was not adversely affected by vancomycin or tetracycline resistance , VGS , and BHS  isolates, regardless of species and susceptibility to penicillin, macrolides, or tetracycline  , indole-positive Proteus spp. , and tigecycline-NS K. pneumoniae    and K. pneumoniae  (50/MIC90 2/4 mg/liter) against ceftazidime-NS  E. cloacae isolates as it was against ceftazidime-S  E. cloacae isolates , K. pneumoniae , Klebsiella oxytoca , E. cloacae SC , and Citrobacter spp.   inhibited 71.2% of isolates at ≤4 mg/liter  at ≤4 mg/liter (data not shown). Omadacycline  demonstrated good in vitro activity against S. maltophilia (Table 2). Omadacycline was equally active against β-lactamase-negative and -positive isolates of H. influenzae  and was also very active against Moraxella catarrhalis isolates  (Omadacycline MIC distributions are shown in isolates . All CoNsistance  and S1. acycline  and S1. g/liter)  except Pg/liter)  and S2. g/liter)  and S2. ctively)  and S2. isolates  and S2. olates])  and S2. mg/liter . Omadacyg/liter)  and S2.Compared to older tetracyclines, omadacycline has advantages that include a low propensity for selection of resistance, enhanced binding to the 30S ribosomal subunit, the ability to overcome common tetracycline resistance mechanisms, a lack of effect of other resistance mechanisms, availability as intravenous or oral formulations, a prolonged half-life, and once-daily administration . Omadacyin vitro activity of omadacycline against bacterial isolates (United States and Europe) from the 2016 SENTRY survey. Overall, omadacycline provided broad coverage against Gram-positive and fastidious Gram-negative bacteria (S. pneumoniae isolates (E. coli and somewhat less active against ESBL-producing K. pneumoniae and ceftazidime-NS E. cloacae strains. Tetracycline-R Enterobacteriaceae were slightly less susceptible to omadacycline than tetracycline-S strains. Imipenem and amikacin were the most active agents against Enterobacteriaceae, including the resistant subsets. Omadacycline was the only agent with useful activity against A. baumannii, and omadacycline and trimethoprim-sulfamethoxazole were the only agents with useful activity against S. maltophilia isolates.This study documents the isolates  and S1. These data build on previous SENTRY surveillance surveys  and indi


However, IDH mutations make classification of gliomas according to the WHO2007 edition controversial. Here, we characterized IDH-1R132H mut status in a cohort of 670 adult patients with different WHO2007 grades of diffuse glioma. Patient characteristics, clinical data and prognoses were obtained from medical records. Patients with IDH-1R132H mut were younger and had better clinical outcomes than those without mutations. Differences in age among patients with astrocytomas of different WHO2007 grades were eliminated after patients were grouped based on IDH-1R132H status. IDH-1R132H mut was present more often in patients with lower Ki-67 and MGMT protein levels and higher mutant p53 levels. Ki-67 was also strongly associated with WHO2007 grade independently of IDH-1R132H mut status. Moreover, patients with Ki-67<30 survived longer than those with Ki-67≥30, regardless of IDH-1R132H mut status. Patients in the IDH-1R132H mut group with lower MGMT protein levels also had better clinical outcomes than those in other groups. Our results indicate that to better treat gliomas, IDH mutation status should be included when determining WHO2007 grade in glioma patients.WHO The median OS of 36.230 months (95% CI 31.103–41.357) in patients with Ki-67<30 was higher than the OS of 15.370 months (95% CI 12.842–17.898) in patients with Ki-67≥30 . We subdivided IDH-1R132H mut or IDH-1R132H wt gliomas based on Ki-67<30, and found differences in mOS among the four groups  vs. 19.000 months (95% CI 13.754–24.246); p<0.001, Breslow test, Figure R132H wt [median 17.400 months (95% CI 13.627–21.173) vs. 13.270 months (95% CI 11.141–15.399); p=0.039, Breslow test, Figure R132H mut/Ki-67≥30 and IDH-1R132H wt /Ki-67<30 patients .IDH-1s Figure . Patientp<0.001, Breslow test). Furthermore, mOS differed among IDH-1R132H-mut-MGMTneg , IDH-1R132H mut-MGMTpos , IDH-1R132H wt-MGMTneg  and IDH-1R132H-WT-MGMTpos  patients .The mOS of 15.670 months (95% CI 13.273–18.067) in MGMT-positive patients was shorter than the mOS of 32.500 months (95% CI 22.439–42.561) observed in MGMT-negative patients  and IDH wt (median OS=355 days) groups. The median OS in our study was closer to that found by Zeng et al. These results indicate that Ki-67 index is a reliable candidate for determining prognosis in glioma patients in addition to IDH-1 status. Although the prognostic value of MGMT protein levels is controversial [Recent data suggests that IDH-1 mutations, Ki-67 index, and MGMT protein levels are prognostic factors for diffuse gliomas , 30–32. oversial , we founR132H mut in a large cohort of glioma patients. IDH-1R132H mut was associated with specific WHO2007 histological grades and younger age. Age differences between different WHO2007 grades of astrocytoma were strongly influenced by IDH-1R132H mutation status. Low Ki-67 index values occurred much more often in patients with lower WHO2007 grades and IDH-1R132H mutation. Finally, our study indicated that Ki-67 index and MGMT protein levels, together with IDH mutation status, were predictive of prognosis in different glioma subtypes.In summary, we characterized the expression of IDH-1Tumor samples were obtained from Sanbo Brain Hospital. Informed consent was obtained from all patients prior to the study. All experiments using human tissues were approved by the Institutional Review Board of Sanbo Brain Hospital. 670 adult patients with diffuse supratentorial gliomas were involved in the study. WHO classification of all specimens was performed by two independent neuropathologists . In the R132H (Dianova 1:100), p53 (1:100 Invitrogen), MGMT (1:150 Invitrogen), and Ki-67 (1:200 Invitrogen) were used. The cutoff values were 10% for IDH-1R132H mut, 10% for mutant p53, 10% for MGMT, and 30% for Ki-67. Representative images of high and low IDH-1R132H mut  was considered statistically significant.SPSS 22.0 was used for all statistical analyses. The χ


Nature Communications8:312 doi:10.1038/s41467-017-00390-1; Article pubilshed online: 22 Aug 2017Nat. Commun.7, 12374 (2016).”We regretfully omitted to give credit to a previous figure upon which the surface-tension scheme in Fig. 1b is based. The caption to Fig. 1 should have included the following: “The surface-tension scheme in Fig. 1b is adapted from Fig. 1a in Noh, J., Jeong, S. & Lee, J.-Y. Ultrafast formation of air-processable and high-quality polymer films on an aqueous substrate.


Xiphinema includes a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We make available the first detailed information on the diversity and distribution of Xiphinema species infesting wild and cultivated olive in a wide-region in southern Spain that included 211 locations from which 453 sampling sites were analyzed. The present study identified thirty-two Xiphinema spp. in the rhizosphere of olive trees, ten species belonging to Xiphinema americanum-group, whereas twenty-two were attributed to Xiphinema non-americanum-group. These results increase our current knowledge on the biodiversity of Xiphinema species identified in olives and include the description of four new species , and two new records for cultivate olives . We also found evidence of remarkable prevalence of Xiphinema spp. in olive trees, viz. 85.0% (385 out of 453 sampling sites), and they were widely distributed in both wild and cultivated olives, with 26 and 17 Xiphinema spp., respectively. Diversity indexes  were significantly affected by olive type. We also developed a comparative morphological and morphometrical study together with molecular data from three nuclear ribosomal RNA genes . Molecular characterization and phylogenetic analyses allowed the delimitation and discrimination of four new species of the genus described herein and three known species. Phylogenetic analyses of Xiphinema spp. resulted in a general consensus of these species groups. This study is the most complete phylogenetic analysis for Xiphinema non-americanum-group species to date.The genus  Soil is most likely one of the more species-rich habitats of terrestrial ecosystems because over one quarter of all living species on Earth are inhabiting the soil , 2. One Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.), likewise the ectoparasitic nematodes belonging to the family Longidoridae Thorne, 1935 [Xiphinema Cobb, 1913 [Nepovirus, family Comoviridae) that cause significant damage to a wide range of crops [Xiphinema was divided into two different species groups [Xiphinema americanum-group comprising a complex of about 60 species [Xiphinema non-americanum-group which comprises a complex of more than 215 species [Xiphinema is based mainly on classical diagnostic features; however, due to a high degree of intraspecific morphometric variability can lead to overlapping among Xiphinema species and increase the risk of species miss-identification [The most important nematodes economically include endoparasitic species such as the root-knot  species , 17, 18.fication .Xiphinema species  have been characterized molecularly by ribosomal genes , constituting a useful tool for molecular-based species identification [Xiphinema species identification becomes difficult when dealing with morphological closely species that co-occur in a sample or region, as often detected in the Iberian Peninsula [Xiphinema spp. detected in the Iberian Peninsula [Xiphinema have been reported in Spain, mainly associated with woody, ornamental and vegetable plant species [Recently, 96 fication –33. Xipheninsula , 28. Seveninsula –36. In p species , 38.Xiphinema spp.) [Xiphinema spp. associated with olive trees, except for the recent contributions of Archidona-Yuste et al. [Xiphinema macrodora Archidona-Yuste et al., 2016, Xiphinema oleae Archidona-Yuste et al., 2016, Xiphinema plesiopachtaicum Archidona-Yuste et al. 2016, and Xiphinema vallense Archidona-Yuste et al. 2016 [Xiphinema spp. infecting wild and cultivated olives in southern Spain, we surveyed a total of 211 localities at the eight provinces of Andalusia where both olive forms were present. This survey raised 385 populations of Xiphinema species, apparently morphologically related to other known Xiphinema spp. This prompted us to carry out an integrative taxonomic study to identify the species within this complex genus.Olive, in wild and cultivated forms, is widely distributed in the Mediterranean Basin, and particularly in southern Spain , 39–41. ma spp.) , 42. Howe et al. , 18, 28 al. 2016 , 18, 28.Xiphinema species and to test the resemblance between morphological and molecular data within Xiphinema species, and the specific objectives were: i) to identify the 385 Spanish populations of Xiphinema spp. detected in wild and cultivate olives; ii) to carry out a molecular characterisation of these Xiphinema populations based on sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene, the ITS1 of rRNA, and partial 18S rRNA sequences; and iii) to study the phylogenetic relationships of Xiphinema spp.The general objectives of this research was to study the occurrence and abundance of No specific permits were required for the described fieldwork studies. Permission for sampling the olive orchards was granted by the landowner. The samples from wild olives were obtained in public areas, forests, and other natural areas studied and do not involve any species endangered or protected in Spain. The sites are not protected in any way.et al. [Nematodes were surveyed from 2012 to 2015 during the spring season in wild and cultivate olives growing in Andalusia, southern Spain , Fig 1. et al. .3 sub-sample of soil by a modification of Cobb´s decanting and sieving method [et al. [Xiphinema.Nematodes were extracted from a 500-cmg method . Since rg method , 44, theg method . The nem [et al. . PPN froXiphinema spp. populations detected infesting soils from olives in Andalusia, conventional ecological and diversity indexes were performed in order to evaluate the distribution and changes in the diversity in wild and cultivated olives. In this regard, abundance and prevalence of each Xiphinema species identified were estimated. For each sampling site, abundance was calculated as the mean number of Xiphinema nematodes per 500 cm3 of soil for all samples. The prevalence was computed by dividing the number of samples in which a Xiphinema species was detected by the total number of samples and expressed as a percentage.Based on the P ≤ 0.05 using the general model procedure of SAS .Several diversity indexes including Hill´s diversity, Hill´s reciprocal of D (Simpson´s dominance index) and Hill´s evenness indexes  were calXiphinema specimens for light microscopy were killed by gentle heat, fixed and examined Xiphinema specimens as described by Archidona-Yuste et al. and Seinhorst [viz. Xiphinema cadavalense Bravo & Roca 1995 [viz. Xiphinema conurum Siddiqi, 1964 [Xiphinema species already described were analysed morphologically and molecularly in this study and proposed as standard and reference populations for each species given until topotype material becomes available and molecularly characterized. Voucher specimens of these described species have been deposited in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.einhorst , 50. Theeinhorst , 27, 51.oca 1995 , and Dr.qi, 1964 . NematodXiphinema. Following morphological confirmation, the specimens were removed from the slides and DNA extracted.For molecular analyses, in order to avoid mistakes in the case of mixed populations, two live nematodes from each sample were temporary mounted in a drop of 1M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed to the unidentified populations of et al. [et al. [Detailed protocols for nematode DNA extraction, PCR and sequencing were applied as described by Castillo et al. . The D2-et al. , 55–58.  [et al. . The newXiphinema spp. from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previous published data [http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using the less stringent option . Percentage similarity between sequences was calculated using a sequence identity matrix in BioEdit. For that, the score for each pair of sequences was compared directly and all gap or place-holding characters were treated as a gap. When position of both sequences has a gap they do not contribute as a difference. Phylogenetic analyses of the sequence data sets were performed based on Bayesian inference (BI) using MrBayes 3.1.2 [6 generations, respectively. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees were visualised using TreeView [D2-D3 expansion segments of 28S rRNA, ITS1, and partial 18S rRNA sequences of different hed data , 17, 18.hed data , strateghed data  and edithed data  in Castres 3.1.2 . The beses 3.1.2  with theTreeView .http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:CE945C7D-7B14-46DD-8A17-A93A05750590. 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 (ICZN), and hence the new names contained herein are available under that Code from the electronic edition. 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 Xiphinema spp. found in this study including specimens of sampling sites used in morphological and/or molecular analyses, are shown in Xiphinema spp. and sampling sites are presented in Xiphinema spp. were detected in the rhizosphere of olive trees, ten species belonging to X. americanum-group, whereas 22 were attributed to X. non-americanum-group  Kirjanova 1951 [X. parachydermum Gutiérrez-Gutiérrez et al., 2012 [X. turcicum Luc, 1963 [X. vallense), while the remaining 21 identified species where present either in wild or cultivated olives only.All um-group . From aled olive ; Fig 1. l., 1992 , X. coxian, 1964 , X. duril., 1993 , X. incel., 1983 , X. italyl, 1953 , X. macrl., 1992 , X. pachova 1951 , 71, X. l., 2012 , X. turcuc, 1963  and X. vXiphinema spp. were present in low to high densities  in both wild and cultivated olives, being observed in cultivated olives in higher densities than in wild olives (X. americanum-group species was significantly higher (P < 0.01) in cultivated than wild olives  in both olive types in the Xiphinema non-americanum-group  (P < 0.001) for X. americanum group than X. non-americanum-group species (Xiphinema species with the highest nematode density was X. pachtaicum (414 nematodes per 500 cm3 of soil), which showed a higher average density in cultivated than wild olives , both belonging to X. non-americanum-group, showing lower average density in cultivated than in wild olives  , which r species . On the d olives . Howeverd olives .Xiphinema spp. in olive was 85.0% (385 out of 453 sample sites) in Andalusia  than cultivated olives    that was detected in both olive types in the most of the Andalusia provinces, at exception of Almería , that was found in both olive types in Almería, Cádiz, Huelva and Málaga provinces, but only in wild olive in Córdoba, Granada, Jaén and Seville provinces  . In addive types . As indie sites) . The sub Almería . Anotherrovinces .Xiphinema spp. in wild and cultivated olive (Xiphinema spp. detected in each sampling site (Richness index) was significantly affected (P < 0.05) by olive type (P < 0.001) in wild than cultivated olives (X. non-americanum-group species were significantly higher (P < 0.05) in wild than in cultivated olive (X. americanum-group species (P < 0.001) in X. americanum-group than in X. non-americanum-group (P < 0.05) for both diversity indexes were observed when Xiphinema species groups were considered separately  than X. americanum-group (Tables P < 0.01) among them when it was considered both olive types (P < 0.01) than that of wild olives (P < 0.05) detected in cultivated than wild olives (X. americanum-group was significantly higher (P < 0.001) than that of X. non-americanum-group species , and Hill´s evenness ), and teed olive . Overallive type , showingd olives . Similared olive , but the species . Overallum-group . Diversium-group . Thus, sparately . On the  P < 0.00 in wild p Tables  resultedve types . Evennesd olives  accordind olives . On the  species .Nematoda Linnaeus, 1758 Dorylaimida Pearse, 1942 Longidoridae Thorne, 1935 [Longidorinae Thorne, 1935 [Xiphinema Cobb, 1913 [bb, 1913 Xiphinema andalusiense Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov.urn:lsid:zoobank.org:act:95E9BE47-B822-4AAF-A11C-50EF7A016137Figs 5Olea europaea subsp. silvestris (Miller) Lehr) , at Belmez, Córdoba province, Spain; collected by G. Leon Ropero, March 14, 2015; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number AR093-2).Adult female, collected from the rhizosphere of wild olive  of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers AR093-5-AR093-7); two females at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (AR093-8); and one female at USDA Nematode Collection, Beltsville, MD, USA (T-6774p); collected by G. Leon Ropero, March 14, 2015.Xiphinema andalusiense sp. nov. is an apparently parthenogenetic species belonging to morphospecies Group 5 from the Xiphinema non-americanum-group species [pars dilatata uteri, and small spiniform structures and crystalloid bodies in low number; female tail short, convex-conoid to conical shape with distinctly digitate terminus, and bearing three pairs of caudal pores; c´ ratio (1.0–1.3); and specific D2-D3, ITS1 rRNA and partial 18S rRNA sequences . According to the polytomous key of Loof & Luc [ species . It is cof & Luc , the newThe species epithet refers to the autonomous community from Spain, Andalusia, where the species was detected and moderately distributed.ca 76–88% of lip region diam. and located at ca two-thirds of lip region height. Odontostyle long, 1.6–1.9 times longer than odontophore, and the latter with moderate-developed flanges 9.5–12.5 μm wide. Guiding ring with average guiding sheath length of 16.0 μm. Pharynx occupying about 8–15% of body length, consisting of an anterior slender narrow part 346–541 μm long and extending to terminal pharyngeal bulb occupying ca 19–27% of total pharyngeal length, 112–139 μm long and 22.5–29.5 μm wide. Glandularium 99.5–119.0 μm long. Nucleus of dorsal pharyngeal gland (DN) located at beginning of basal bulb (10.4–14.3%), ventrosublateral nuclei (SVN) situated ca halfway along bulb (46.9–59.4%)  similar to that of female, including almost conoid tail shape ending in a digitate terminus  were identified using morphological characters such as body length, length of replacement and functional odontostyle , Fig 5 , 76. Speterminus , becominXiphinema andalusiense sp. nov. was collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) of two localities belonging to Córdoba and Jaén provinces, being one of the new species described in this work which has a broader distribution in Andalusia, concretely on North of Andalusia , slightly lower c´ ratio (1.0–1.3 vs 1.1–1.8), the presence of spiniform structures or crystalloid bodies along tubular portion of uterus vs absent, and the absence vs presence of males [X. andalusiense sp. nov. mainly differs from X. cadavalense in having a shorter odontostyle and odontophore length  resulting in a shorter stylet length (215.5–239.5 vs 244.5–278.5 μm), a narrower lip region (12.0–15.5 vs 14.0–19.5 μm), and higher a and c´ ratios  [X. andalusiense sp. nov. differs from X. turdetanense in having a slightly longer odontostyle length (137.0–151.0 vs 121.0–142.0 μm), a slightly narrower lip region (11.5–15.5 vs 14.0–16.0 μm), higher number of globular bodies present in the Z-differentiation (11–16 vs 6–8), size and number of spiniform structures presents along tubular part of uterus , presence of crystalloid bodies along uterus vs absence, and the absence vs presence of males [According to the polytomous key by Loof & Luc  and sortl., 2013 , X. cadal., 2013 . Xiphineof males . On the ctively) . Finallyof males .X. andalusiense sp. nov. is molecularly related to X. macrodora, but it can be clearly differentiated in having a smaller nematode body and odontostyle length  [In addition, ctively) .X. andalusiense sp. nov. (KX244884-KX244888) was 97% similar to X. baetica , X. macrodora  and X. cadavalense (KX244900); sequence variation among these species was from 24 to 34 nucleotides and from 3 to 8 indels  in relation to the ITS1 region were also X. baetica , X. cadavalense , and X. macrodora . Intraspecific variation for this marker was 44 nucleotides and 23 gaps amongst the five studied populations , with several Xiphinema spp. such as X. baetica (KC567148-KC567149), X. cadavalense (KX244932), X. macrodora (KU171050) and X. coxi europaeum (KC567153).D2-D3 region of 8 indels . Xiphineulations . FinallyXiphinema celtiense Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov.urn:lsid:zoobank.org:act:17E565E4-18E8-4D60-AA57-55253F3E257EFigs 7Olea europaea subsp. silvestris (Miller) Lehr) , at Peñaflor, Seville province, Spain; collected by A. Archidona-Yuste, April 22, 2014; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number AR083-01).Adult female, collected from the rhizosphere of wild olive  of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers AR083-03-AR083-06); two females and one juvenile at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (AR083-22); two females and two juveniles at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT 852); and two females and two juveniles at USDA Nematode Collection, Beltsville, MD, USA (T-6775p); collected by A. Archidona-Yuste, April 22, 2014.Xiphinema celtiense sp. nov. is a Xiphinema non-americanum-group species belonging to morphospecies Group 5 sensu Loof & Luc [of & Luc . It is aof & Luc , the newCelti’, where the type specimens were collected.The species name is derived from originating Roman city of Peñaflor, ‘ca 58–78% of lip region diam. Guiding ring with average guiding sheath length of 15.5 μm. Odontostyle long, 1.4–1.8 times longer than odontophore, and the latter with well-developed flanges 13.0–16.5 μm wide. Pharynx very long occupying about 10–14% of body length, consisting of an anterior slender narrow part 379–510 μm long and extending to pharyngeal bulb, 126.0–168.0 μm long and 22.5–36.0 μm wide. Glandularium 110–155 μm long. Nucleus of dorsal pharyngeal gland (DN) located at beginning of basal bulb (11.5–16.1%), ventrosublateral nuclei (SVN) situated ca halfway along bulb (50.5–62.3%)  μm wide and 7.2 ± 1.4 (4.5–9.5) μm high. Amphidial fovea aperture extending for  Coomans . Vestigiize Figs . Small cExtremely rare, only one male specimen was found in type locality. Male genital tract diorchic with testes containing multiple rows of different stages of spermatogonia. Tail short, convex-conoid with a broadly rounded terminus and thickened outer cuticular layer. Spicules moderately long and slightly curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. One pair of adanal and 4 mid-ventral supplements.ca 75 μm. Tail morphology of second-stage juvenile similar to J1 expect to absence of knob-like expansion, and tail conoid and subdigitate with rounded terminus for third-stage juvenile. In J4 tail conoid with a short bulge rounded terminus  were identified using morphological characters such as body length, length of replacement and functional odontostyle , Fig 5 , 76. Speterminus . All juvterminus , Fig 7.Xiphinema celtiense sp. nov. was found in the rhizosphere soil of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) in one additional locality belonging to Córdoba province. , posterior vulva position (50.0–55.0 vs 46.0–51.0%), a longer odontostyle and odontophore  resulting in a longer stylet length (241.0–263.05 vs 213.0–234.0 μm), a narrower lip region (13.5–16.0 vs 15.5–17.0 μm), frequency of males (extremely rare vs frequent), and the female and J1 tail shape , posterior vulva position (50.0–55.0 vs 47.1–51.8%), and presence vs absence of crystalloid bodies along uterus [X. turcicum by slightly higher a and c ratios , presence vs absence of crystalloid bodies in the tubular portion of uterus, and different shape of J1 tail  although in both species the tail ends in a knob-like expansion more or less separated from the anterior part of tail , and female tail shape  [X. cohni it mainly differs by the presence vs absence of Z-differentiation containing numerous granular bodies, and female tail shape  , 65. AndX. celtiense sp. nov. (KX244889-KX244890) differed with the closest related species, X. hispanum (GU725074) by 24 nucleotides and 3 gaps (97% similarity) and from X. cohni  from 27 nucleotides and 1 indel (97% similarity). Intraspecific variation of D2-D3 segments detected between the two studied population of X. celtiense sp. nov. consisted of 7 nucleotides (99% similarity), and no indels (X. hispanum (GU725061) and 86% (141 nucleotides and 34 indels) with X. cohni (KX244933). Intraspecific variation of the ITS1 for these sequences (KX244926-KX244927) was 44 nucleotides and 18 gaps, 95% similarity (X. celtiense sp. nov. (KX244943) showed a high level of similarity (99%) with several sequences deposited in GenBank such as X. hispanum (GU725083), X. adenohystherum (GY725084), and X. nuragicum (GU725080).D2-D3 sequences from o indels . ITS1  , at Iznájar, Córdoba province, Spain; collected by J.E. Palomares-Rius, December 3, 2014; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number JAO-25-1).Adult female, collected from the rhizosphere of cultivated olive  of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers JAO-25-2-JAO-25-7); one female and one male at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (JAO-25-12); two females and one juvenile at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT 853); and two females, one male, and one juvenile at USDA Nematode Collection, Beltsville, MD, USA (T-6777p); collected by J.E. Palomares-Rius, December 3, 2014.Xiphinema iznajarense sp. nov. is an amphimictic species belonging to morphospecies Group 5 from X. non-americanum-group species sensu Loof & Luc [ca 71 μm), and one pair of adanal supplement plus 4–5 pairs of ventromedian supplements; and specific D2-D3, ITS1 rRNA and partial 18S rRNA sequences . According to the polytomous key of Loof & Luc [of & Luc . It is cof & Luc , the newThe species epithet refers to the type locality, Iznájar, where the species was detected.ca 63–74% of lip region diam. and located at ca two-thirds of lip region height. Guiding ring with average guiding sheath length of 12.0 μm. Odontostyle moderately long, 1.5–1.8 times longer than odontophore, and the latter with well-developed flanges in the most of specimens studied, 11.5–22.0 μm wide. Pharynx consisting of an anterior slender narrow part 265–414 μm long, extending to a cylindrical, terminal pharyngeal bulb occupying ca 23–36% of total pharyngeal length, cylindrical, 117–153 μm long and 20–29 μm wide. Glandularium 101–135 μm long. Nucleus of dorsal pharyngeal gland (DN) located at beginning of basal bulb (11.6–12.6%), ventrosublateral nuclei (SVN) situated ca halfway along bulb (50.5–57.8%) , a reflexed oviduct with well-developed pars dilatata oviductus separated from uterus by a well-developed sphincter. Uteri tripartite, comprising a developed pars dilatata uteri continuing into a narrower, muscular tube-like portion, and a well-developed Z-differentiation with weakly muscularised wall and containing numerous small granular bodies. Uterine wall wrinkles present along uterus, being more numerous in the proximal part of pars dilatata uteri and ovejector . Ovejector well-developed 35.5–56.0 μm wide, vagina perpendicular to body axis, 18.0–24.0 μm long in lateral view. Vulva slit-like, pre-equatorial. Tail conoid and short, dorso-ventrally convex, ending in a rounded and broadly terminus, bearing in four to five pairs of caudal pores on each side.Habitus in specimens killed by gentle heat usually almost straight anterior to the vulva, more curved behind the vulva, occasionally open C-shaped. Cuticle 2.0–4.0 μm thick at mid-body, more thickened in the lip region (4.0–6.0 0 μm wide) and tail tip region (5.5–10.0 μm wide). Lateral hypodermical chords occupying about 26–46% of the corresponding maximum body diameter. Lip region hemispherical, broadly rounded frontally, usually low and offset from body contour by a shallow constriction; 15.5–17.0 μm wide and 5.5–7.5 μm high. Amphidial fovea aperture extending for vejector . Small svejector . In someFrequent but less abundant than female (ratio = 1: 2). Morphologically similar to female except for genital system and more curved posterior part of body. Male genital tract diorchic with testes containing multiple rows of different stages of spermatogonia. Tail short, convex-conoid with short bulge rounded terminus and thickened outer cuticular layer Figs . Spiculeca 63 μm. Tail morphology in second-stage juvenile similar to J1 expect for the presence a slightly depression at the level of the hyaline region in both sides. On the other hand, the tail was conoid and subdigitate with a rounded terminus for J3, while for fourth-stage juvenile was conoid with rounded terminus and short bulge  were identified using morphological characters such as body length, length of replacement and functional odontostyle , Fig 5 , 76. In rt bulge . All juvrt bulge .Xiphinema iznajarense sp. nov. was only found in type locality, Iznájar (Córdoba province), being extracted from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (Morphometric variability is described in paea L.) , Fig 1.X. iznajarense sp. nov. closely resembles with X. celtiense sp. nov., X. coronatum and X. turcicum. Xiphinema iznajarense sp. nov. can be differentiated from X. celtiense sp. nov. by the characters discussed above. From X. coronatum it differs in having a longer body (4.5–5.8 vs 3.8–4.6 mm), higher a ratio (75.2–106.3 vs 65.5–75.5), a shorter odontophore and lower oral aperture-guiding ring distance , frequency of males (frequent vs extremely rare), presence vs absence of crystalloid bodies in the tubular portion of uterus, female tail shape , and shape of J1 tail (conoid elongate with rounded terminus vs a long clavate peg) , a shorter odontostyle length (132.0–151.0 vs 152.0–182.0 μm), the presence vs absence of crystalloid bodies along uterus, the frequency of males (frequent vs rare), the female tail shape , and shape of J1 tail  , lower c´ ratio (0.7–1.1 vs 1.4–2.2), a longer odontostyle (132.0–151.0 vs 107.0–131.0 μm), and the presence vs absence of crystalloid bodies along uterus  was 97% similar (26 nucleotides and 1 indel) to X. adenohystherum (GU725075), X. hispidum (KC567181) and 95% similar (36 nucleotides and 2 indels) to X. hispanum (GU725074). No intraspecific variation of D2-D3 segments was detected amongst the studied individuals (100% similarity) (X. hispanum (GU725061), X. adenohystherum (GU725063) and X. hispidum (HM921367) with similarity values of 88% (131 nucleotides and 31 indels), 87% (145 nucleotides and 29 indels) and 84% (175 nucleotides and 52 indels), respectively (X. iznajarense sp. nov. (KX244944) closely matched with several species of Xiphinema, some of them were X. adenohystherum (GU725084), X. hispanum (GU725083), X. gersoni Roca & Bravo, 1993 [X. sphaerocephalum Lamberti, Castillo, Gómez Barcina & Agostinelli, 1992 [D2-D3 region of ilarity) . Similarectively . ITS1 alvo, 1993   , at Mengíbar, Jaén province, Spain; collected by J. Martín-Barbarroja, March 25, 2012; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number O3C4-01).Adult female, collected from the rhizosphere of cultivated olive  of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers O3C4-02-O3C4-08); one female, one male and one juvenile at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (O3C4-19); one female and one male at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT 854); and one female and one male at USDA Nematode Collection, Beltsville, MD, USA (T-6776p); collected by J. Martín-Barbarroja, March 25, 2012.Xiphinema mengibarense sp. nov. belongs to the X. non-americanum Group 5 in Loof and Luc [Xiphinema non-americanum Group 5 and has the following specific alphanumeric codes (codes in parentheses are exceptions): A4-B2+3-C5a-D6(5)-E6(5)-F45-G32-H2-I2-J6-K2-I2. and Luc ; and it  and Luc , the speThe species epithet refers to the type locality, Mengíbar, where the species was detected.ca 64–78% of lip region diam. and located at ca two-thirds of lip region height. Guiding ring and guiding sheath variable in length depending on degree of protraction/retraction of stylet. Odontostyle moderately long, 1.5–1.7 times longer than odontophore, in the most specimens the latter with moderate-developed flanges, but in some specimens it was observed weaker, 8.5–14.0 μm wide. Pharynx composed by an anterior slender narrow flexible part 317–417 μm long, and a posterior muscular expanded part with three nuclei. Terminal pharyngeal bulb variable in length, 120–173 μm long and 19.5–29.5 μm wide. Glandularium 104–148 μm long. Nucleus of dorsal pharyngeal gland (DN) located at beginning of basal bulb (9.2–15.0%), ventrosublateral nuclei (SVN) situated ca halfway along bulb (45.7–58.0%) , with slightly bulging rounded terminus with a distinct terminal blind canal. Three to four caudal pores present on each side.Body cylindrical in an open C-shape when heat relaxed. Cuticle 3.1 ± 0.3 (2.0–4.5) μm thick at post-lip region, 2.8 ± 0.5 (2.0–4.0) μm wide at mid-body, but thicker just posterior to anus, 6.4 ± 1.8 (5.0–10.0) μm thick. Lateral chord 13.0 ± 4.8 (8.0–20.0) μm wide, occupying 17–42% of corresponding body diam. Lip region flatly rounded, slightly offset from body contour by a depression, 13.9 ± 0.7 (12.5–15.5) μm wide and 6.9 ± 0.8 (5.5–8.5) μm high. Amphidial fovea aperture extending for  Coomans ). CardiaFunctional, less abundant than females (ratio = 1: 2). Reproductive system diorchic with testes occupying 45–57% of body length, and spindle-shaped sperm. Spicules dorylaimoid, massive, well sclerotised, 57.5–66.0 μm long, ventrally curved with tubular lateral guiding pieces 13.5–18.0 μm long. One pair of adanal supplements located at 16.6 ± 1.2 (15.5–19.0) μm from cloacal opening and a series of four to five ventromedian supplements. Tail similar to that of female, dorsally more convex than female, and ending in a rounded terminus with short bulge.ca 53 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. Tail morphology in second and third juvenile stages similar to J1, becoming progressively shorter and stouter in each progressively moult. However, tail shape in fourth-stage similar into that of female, broadly dorsally convex-conoid with slightly bulging rounded terminus , being extracted from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (Morphometric variability is described in paea L.) , Fig 1.X. mengibarense sp. nov. groups with X. herakliense Tzortzakakis et al., 2015 [X. hispanum, and X. lanceolatum Roca & Bravo, 1993 [Xiphinema mengibarense sp. nov. can be differentiated from X. herakliense by higher a and c ratios , a shorter odontostyle (120.0–131.5 vs 127.0–157.0 μm), shorter spicules (57.5–66.0 vs 70.0–81.0 μm) [X. mengibarense sp. nov. mainly differs from X. hispanum in having higher a ratio (80.0–98.2 vs 73.1–83.9), a shorter odontostyle (120.0–131.5 vs 131.2–142.3 μm), the number of spiniform structures present in the Z-differentiation (lower vs abundant), the presence vs absence of crystalloid bodies in the tubular portion of uterus, and the frequency of males (frequent vs rare) , a shorter odontostyle and odontophore  resulting in a shorter stylet (194.5–215.0 vs 255.5–283.0 μm), a slightly narrower lip region (12.5–15.5 vs 14.5–18.0 μm), posterior vulva position (48.5–57.0 vs 43.5–50.0%), the presence vs absence of males, and the number of spiniform structures and crystalloid bodies (lower vs very abundant)  was 94% similar to several Xiphinema species such as X. italiae , X. pyrenaicum Dalmasso, 1969 [X. sphaerocephalum . Xiphinema mengibarense sp. nov. showed a high homogeneity for the D2-D3 region  in the three sampled populations (X. hispanum (GU725061) and X. cohni (KC567159), with a similarity of 84%  (X. mengibarense sp. nov. (KX244945) closely matched (99% similarity) those for X. italiae (FJ713154), X. pyrenaicum (GU725085) and X. gersoni (KC567154).D2-D3 region of so, 1969  (GU72507ulations . The cloctively) . Low intX. adenohystherum, X. baetica, X. cohni, X. coxi europaeum, X. duriense, X. hispanum, X. hispidum, X. incertum, X. index Thorne & Allen, 1950 [X. italiae, X. lupini Roca & Pereira, 1993 [X. macrodora, X. madeirense Brown, Faria, Lamberti, Halbrendt, Agostinelli & Jones, 1993 [X. nuragicum, X. oleae, X. opisthohysterum Siddiqi, 1961 [X. pachtaicum, X. parapachydermum, X. plesiopachtaicum, X. rivesi Dalmasso, 1969 [X. santos Lamberti, Lemos, Agostinelli & D’Addabbo, 1993 [X. sphaerocephalum, X. turcicum, X. turdetanense, and X. vallense have been previously recorded within studies of dagger and needle nematodes infesting olives and vineyards in southern Spain [viz. X. cadavalense, X. conurum and X. pseudocoxi Sturhan, 1985 [Morphological and morphometrical data, and molecular delineation (rDNA) of en, 1950 , X. italra, 1993 , X. macres, 1993 , X. nuraqi, 1961 , X. pachso, 1969 , X. santbo, 1993 , X. spharn Spain , 18, 28.an, 1985 ), a brieXiphinema collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) at Espiel (Córdoba province) corresponds fairly well with studied paratypes and original description of X. cadavalense. This population was characterised by a long body; lip region hemispherical, rounded both anteriorly and laterally and set off from body contour by slightly depression; long odontostyle and odontophore; reproductive system didelphic-amphidelphic with both branches about equally developed with a well-developed Z-differentiation with weakly muscularised wall and comprising 9–15 sclerotized bodies of variable size and petal shape, each one consisting of a large portion, irregularly spherical surrounded by a variable number of refractive pieces; spiniform structures and crystalloid bodies in very small size and low number present along the narrower and muscular tube-like of uterus; tail dorsally convex-conoid  ending in a terminal peg with blind canal , posterior guiding ring position from oral aperture (149.5–167.0 vs 126.5–148.5 μm) [X. cadavalense has the following specific alphanumeric codes (codes in parentheses are exceptions): A4-B2+3-C5a-D65-E56-F5-G4-H2-I3-J-K-L1.The amphimictic population of nd canal . The obs and Luc , which aavalense . In addi48.5 μm) . These d48.5 μm) , this SpX. cadavalense (KX244900) was 98% similar (14 nucleotides and no indels) to X. baetica (KC567168), 97% similar (24 nucleotides and 3 indels) to X. andalusiense sp. nov. (KX244884-KX244888) and 96% similar (30 nucleotides and 10 indels) to X. macrodora . ITS1 sequence (KX244932) region also agrees with results obtained from D2-D3, this sequence was 90% similar (105 nucleotides and 28 indels) to X. baetica (KC567157), 89% (121 nucleotides and 35 indels) to X. andalusiense sp. nov. (KX244921-KX244925) and 86% (157 nucleotides and 70 indels) to X. macrodora (KU171048). The partial 18S region of X. cadavalense (KX244946), was very similar to several sequences of Xiphinema spp., including X. diversicaudatum  Thorne, 1939 [X. baetica (KC567149) and X. bakeri Williams, 1961 [D2-D3 segments of nse KX2440 was 98%et al. [The Spanish population of this species from the rhizosphere of olive was characterised by a lip region rounded offset from the rest of the body by a conspicuous depression, two equally developed female genital branches, vulva slightly anterior to mid-body, uterus with uterine differentiation, presence of Z-differentiation with small granular bodies plus small spines (in low number), female tail conical, ventral profile nearly straight, dorsal profile regularly curved with rounded terminus . The moret al. . Up to oX. conurum (KX244902) matched well, 99% similar with former sequences from Tunisia deposited in GenBank (KX062671-KX062673); and ITS1 (KX244934) was 95–96% similar with former sequences from Tunisia deposited in GenBank (KX062696-KX062697). And partial 18S (KX244947) was provided for the first time in this research, being 99% similar to several Xiphinema spp. such as X. nuragicum (GU725081) or X. israeliae Luc, Brown & Cohn, 1982 [D2-D3 sequence for TS1 KX2444 was 95–Xiphinema collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) at Alcaracejos (Córdoba province) agrees fairly well with original description of X. pseudocoxi. This population was characterised by a moderately long body in an open C-shaped after fixation; lip region distinct from the body contour by a depression, frontally rounded; female reproductive system didelphic-amphidelphic having both branches about equally developed; Z-differentiation with weakly muscularised wall formed by 6–10 globular bodies similar in size, and irregularly spherical surrounded by a variable number of refractive pieces; no spiniform structures and, crystalloid bodies nor sperm cells observed along uterus; female tail convex-conoid, varying slightly in shape, and ending in a terminal peg with a blind canal : A4-B2-C5a-D45-E4(5)-F4(5)-G2-H2-I3-J-K-L1.The amphimictic population of nd canal , Table 9 and Luc , which ieudocoxi . Additioeudocoxi , 94, 95.eudocoxi , the newX. pseudocoxi (KX244915-KX244916) were obtained for the first time in this study. The closet species regarding D2-D3 segments of X. pseudocoxi (KX244915-KX244916) were X. globosum Sturhan, 1978 [X. diversicaudatum  and X. coxi europaeum . Similarly, ITS1 region (KX244939-KX244940) also showed some similarity with X. globosum , X. diversicaudatum  and X. coxi europaeum . Finally, the partial 18S of X. pseudocoxi (KX244948) matched closely (99%) with several Xiphinema spp., such as X. globosum (GU549476), X. diversicaudatum (EF538761), X. bakeri (AY283173), X. vuittenezi Luc, Lima, Weischer & Flegg, 1964 [X. index (AY687997).Sequences for an, 1978   were used for further phylogenetic studies. Sequences for X. andalusiense sp. nov., X. cadavalense, X. celtiense sp. nov., X. duriense, X. iznajarense sp. nov., X. mengibarense sp. nov., X. opisthohysterum and X. pseudocoxi were obtained for these species in this study. On the other hand, sequences from X. adenohystherum, X. cohni, X. conurum, X. hispanum, X. hispidum, X. incertum, X. index, X. italiae, X. nuragicum, X. parapachydermum, X. turcicum and X. turdetanense matched well with former sequences deposited in GenBank, and spread out the molecular diversity of these species to the newly studied areas.The amplification of D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial 18S rRNA yielded a single fragment of approximately 800 bp, 1000 bp, and 1800 bp, respectively, based on gel electrophoresis. Sequences from other species of Xiphinema non-americanum-group species inferred from analyses of D2-D3 expansion segments of 28S, ITS1, and the partial 18S rDNA gene sequences using BI are given in Figs X. non-americanum-group spp. based in a multiple edited alignment including 103 sequences and 753 total characters showed two clearly separated (PP = 1.00) major clades  with X. cohni  and X. hispanum , this clade was related (PP = 1.00) with another subclade which was formed by X. iznajarense sp. nov. (KX244891-KX244892), X. adenohystherum , X. hispidum  and X. gersoni (KC567180). Finally, X. mengibarense sp. nov. formed a low-supported subclade (PP = 0.76) with X. italiae , X. pyrenaicum (GU725073), and X. meridianum Heyns, 1971 [X. bakeri and X. index which belong to Groups 7 and 8, respectively. This clade grouped sequences from the new species X. andalusiense sp. nov. (KX244884-KX244888) and the new accessions from X. cadavalense (KX244900), X. conurum (KX244902), and X. pseudocoxi (KX244915-KX244916). Xiphinema andalusiense sp. nov. (KX244884-KX244888) from wild olive occupied a superior position within the clade II forming a well-supported subclade (PP = 1.00) with X. cadavalense (KX244900) from cultivated olive, X. baetica  and X. macrodora . Finally, X. pseudocoxi (KX244915-KX244916) was phylogenetically related to X. globosum (GU549474) forming a well-supported clade (PP = 0.99).Phylogenetic relationships among r clades . Clade IX. non-americanum-group species, was 1113 characters after discarding ambiguously aligned regions from the alignment. Two new accessions were used as outgroup, X. duriense (KX244935) and X. opisthohysterum (KX244938). The 50% majority rule consensus BI tree of X. non-americanum-group spp. showed two major clades (PP = 1.00) similar to those obtained for D2-D3 region , X. pseudocoxi (KX244939-KX244940) and X. cadavalense (KX244932). Xiphinema andalusiense sp. nov. (KX244921-KX244925) and X. cadavalense (KX244932) clustered with X. baetica (KC567156-KC567157) and X. macrodora (KU171048) in a well-supported subclade (PP = 1.00), these results agree with the results obtained with D2-D3 region. Xiphinema pseudocoxi and X. globosum were also phylogenetically related to this marker and they were placed in a well-supported subclade (PP = 1.00) which was related (PP = 0.96) at the same time with X. turdetanense (KC567163). Clade II grouped thirteen species from different morphospecies Groups 1, 4, 5 and 7, including X. celtiense sp. nov., X. iznajarense sp. nov. and X. mengibarense sp. nov. Xiphinema iznajarense sp. nov. (KX244928-KX244929), and X. celtiense sp. nov. (KX244926-KX244927) clustered together with X. cohni (KX244933), X. adenohystherum (GU725063), X. hispanum (GU725061) and X. hispidum (HM921367) as occurred in the D2-D3 tree. Finally, X. mengibarense sp. nov. (KX244930-KX244931) formed a low-supported subclade with X. barense Lamberti, Roca, Agostinelli & Bleve-Zacheo, 1986 [X. pyrenaicum (GU725060) although this relation also was poorly supported. The new accessions for X. duriense (KX244935) and X. opisthohysterum (KX244938) clustered together with X. pachtaicum (AY430178) as an outgroup, all of them from the X. americanum-group  clustered with X. cadavalense, X. macrodora and X. baetica within the same well-supported subclade (PP = 1.00). Phylogenetic inferences based on 18S also suggest that X. pseudocoxi and X. globosum are related species, although this relation was poorly supported (X. iznajarense sp. nov. (KX244944), X. celtiense sp. nov. (KX244943) and X. mengibarense sp. nov. (KX244945) clustered in this case with X. cohni (KC567151), X. hispanum (GU725083), X. adenohystherum (GU725084), X. italiae , X. barense (KM199695), X. gersoni (KC567154), X. sphaerocephalum (GU725082), and X. pyrenaicum (GU725085) within a well-supported subclade (PP = 1.00).The 50% majority rule BI tree of a multiple alignment including 60 18S sequences and 1647 bp long showed several major clades . Additioupported . FinallyXiphinema associated with wild and cultivated olives in southern Spain, as well as their distribution and molecular phylogeny. This was conducted in an extensive and systematic nematological survey that included 211 locations and 453 sampling sites. We found 385 Spanish populations of Xiphinema spp. infesting olive soils. We described four new Xiphinema species, enlarging the diversity of Xiphinema species in the Iberian Peninsula which is in agreement with previous data obtained for the phylogeny and biogeography of the genus Xiphinema and Longidorus in the Euro-Mediterranean region [Xiphinema based on nuclear rDNA markers.This study aimed to get knowledge and a better understanding on the occurrence, abundance and biodiversity of dagger nematodes of the genus n region , 101. ToXiphinema is one of the most diverse PPN associated with olive, with twenty species  reported in various countries of the Mediterranean Basin [Xiphinema species detected in olive worldwide, including four new species from the X. non-americanum-group. All these species were new records for olive with the exception of X. pachtaicum, X. index, X. italiae, X. nuragicum and X. turcicum [Xiphinema spp. observed in both olive types, our study showed a great species diversity, that was mainly associated with the X. non-americanum-group species  [X. non-americanum-group, i.e. X. italiae found in all provinces, X. nuragicum in 7 out of 8 provinces, and X. coxi europaeum in 5 out of 8 provinces. In this sense, the presence of a high number of frequent species belonging to X. non-americanum-group  explains the higher value observed in Hill´s 2 (Dominance diversity) index with respect to X. americanum-group  . Other sXiphinema cause damage to olive by feeding on unmodified plant root cells and causing cell necrosis and galling in root apex [X. diversicaudatum and X. vuittenezi [Xiphinema spp. were responsible for 5 to 10% of loss production resulting in an estimated $39 million loss [Xiphinema spp. in both olive types studied  because of their sensitivity to fast desiccation, large body size, and the absence of survival-resistance forms”. Unfortunately, little is known about the ecological requirements of Xiphinema nematodes and further research is needed [Longidorus spp. showed evidence of some geographic species associations in Andalusia [Xiphinema spp. in southern Spain and other wider areas.Overall, nematode diversity decreases rapidly to agricultural management including plant-parasitic nematodes . Our resin areas . Althoug species , the geoe et al.  for Longs needed . Some prndalusia . ConsequX. cadavalense, X. pseudocoxi, and X. conurum) and for ITS1 . D2-D3 expansion region was more useful for establishing phylogenetic relationships among Xiphinema species than ITS1 or 18S. Phylogenetic analyses based on D2-D3, ITS1, and partial 18S using BI resulted in a consistent position for the newly described species of X. non-americanum-group species from Spain, which grouped in two separated clades, and mostly agree with the clustering obtained by other authors [X. andalusiense sp. nov., X. baetica, and X. cadavalense. In the case of X. andalusiense sp. nov. vs X. baetica, only lower a and c’ ratios, the absence of spines in the uterus, the absence of males and different ribosomal genes could separate X. baetica from X. andalusiense sp. nov. These species probably evolved in the Iberian Peninsula as they occur only there. The Iberian Peninsula has been suggested as a possible center of recent speciation for PPN nematode genera such as Longidorus, Trichodorus or Rotylenchus species [Xiphinema celtiense sp. nov., X. iznajarense sp. nov. and X. mengibarense sp. nov. could be clearly separated morphologically and molecularly from the other Xiphinema species. The majority of the species showed congruence in the phylogenetic relationships within D2-D3, ITS1, and partial 18S using the DNA from the same individual and these markers matched very well with the sequences deposited in the GenBank. This result is in contrast with the close related genus Longidorus found in a similar sampling scheme and localities in which the diversity of species was lower and all the species occupies two major positions in the phylogenetic clade [Sequences of nuclear ribosomal RNA genes, particularly D2-D3 and ITS1, are useful molecular markers for providing accurate species identification of Longidoridae , 30, 127 authors , 18. The species . Xiphineic clade .X. americanum-group and the non-americanum group species. This research provides molecular markers for precise and unequivocal diagnosis of some species of Xiphinema in order to differentiate virus vector or quarantine species. Furthermore, it reflects that similar intensive and extensive integrative studies on Xiphinema species based on widest areas may help to elucidate the evolutionary origin of Xiphinema species. In this sense, further studies based on widespread species (i.e. X. pachtaicum) could also help to clarify if the main speciation occurred in Africa leading to many apomictic species in tropical and subtropical environments as hypothesised by Coomans [In summary, this study provides new insights into the diversity of this genus associated with the olive in Mediterranean conditions with important differences related to the species within the  Coomans , or in SS1 Table(DOCX)Click here for additional data file.


Alchornea cordifolia and Canthium subcordatum were obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The essential oils were subjected to in vitro antibacterial, antifungal and cytotoxic activity screening. Thirty-eight compounds comprising 97.7% of A. cordifolia oil and forty-six constituents representing 98.2% of C. subcordatum oil were identified. The major components in A. cordifolia oil were methyl salicylate (25.3%), citronellol (21.4%), α-phellandrene (7.4%), terpinolene (5.7%) and 1,8-cineole (5.5%). Benzaldehyde (28.0%), β-caryophyllene (15.5%), -α-farnesene (5.3%) and methyl salicylate (4.5%) were the quantitatively significant constituents in C. subcordatum fruit essential oil. A. cordifolia essential oil demonstrated potent in vitro antibacterial activity against Staphylococcus aureus (MIC = 78 μg/mL) and marginal antifungal activity against Aspergillus niger (MIC = 156 μg/mL). C. subcordatum showed antibacterial activity against Bacillus cereus and S. aureus (MIC = 156 μg/mL) and notable antifungal activity against A. niger (MIC = 39 μg/mL). However, no appreciable cytotoxic effects on human breast carcinoma cells (Hs 578T) and human prostate carcinoma cells (PC-3) were observed for either essential oil. The antimicrobial activities of A. cordifolia and C. subcordatum fruit essential oils are a function of their distinct chemical profiles; their volatiles and biological activities are reported for the first time.Bacterial resistance has been increasingly reported worldwide and is one of the major causes of failure in the treatment of infectious diseases. Natural-based products, including plant secondary metabolites , can be exploited to ameliorate the problem of microbial resistance. The fruit essential oils of  Antimicrobial resistance is one of the most serious public health threats that results mostly from the selective pressure exerted by antibiotic use and abuse ,2. DurinAlchornea cordifolia (Schumach. and Thonn.) Müll. Arg. (Euphorbiaceae) is a shrub found along the coastal regions of West Africa. It has multipurpose utilization as fodder, food and medicine. The leaves, roots and stem bark extracts are used extensively in traditional medicine in the preparation of drugs for urinary, respiratory and gastro intestinal disorders [A. cordifolia has been a subject of scientific studies: anti-microbial, antioxidant and anticancer activities, etc. [A. cordifolia has been reported [A. cordifolia has demonstrated antibacterial activity against 21 bacterial strains tested and showed the highest levels of antibacterial activity with MICs against methicillin-resistant Staphylococcus aureus (MRSA) in the range of 1.6–3.1 mg/mL and MBCs in the range of 6.3–12.5 mg/mL among 24 other plant species studied [A. cordifolia leaf [isorders . A slurrisorders . The leaisorders . Hithertes, etc. ,11,12,13reported . The aqu studied . Phyto-clia leaf ,17,18,19Canthium subcordatum DC.  is a tree that grows in central and western Africa and reaches a height of more than 10 m [C. subcordatum [S,9R)-roseoside [C. subcordatum and their structures deduced. GC-MS analysis and anticancer activity of ethanol leaf extract of C. parviflorum have also been reported [A. cordifolia and C. subcordatum.han 10 m . Its roohan 10 m  and the han 10 m . Recentlcordatum . A numbeoseoside  and shanoseoside  have beereported ,27. As pA. cordifolia and C. subcordatum were collected in the month of July 2004, from the campus of the University of Ibadan, Nigeria. Plant samples were authenticated by F. Usang of the Herbarium Headquarters, Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where voucher specimens  were deposited.The mature fresh fruits of The fruit essential oils were obtained by hydrodistillation (4 h) of the pulverized air-dried plant samples (500 g) in an all glass Clevenger-type apparatus following the British Pharmacopoeia specifications . The fruhttp://www.agilent.com/en-us/products/software-informatics/massspec-workstations/gc-msd-chemstation-software). The GC column was an HP-5ms fused silica capillary with a (5% phenyl)-methyl polysiloxane stationary phase (30 m × 0.25 μm film thickness). The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mL/min. Inlet temperature was 200 °C and MSD detector temperature was 280 °C. The GC oven temperature program was used as follows: 40 °C initial temperature, held for 10 min; increased at 3 °C/min to 200 °C; increased 2 °C/min to 220 °C. The sample was dissolved in CH2Cl2, and 1 μL was injected using a splitless injection technique.The essential oils were subjected to GC-MS analysis on an Agilent system consisting of a model 6890 gas chromatograph, a model 5973 mass selective detector (MSD) , and an Agilent ChemStation data system  and by comparison of their mass spectral fragmentation patterns  and with the literature .A. cordifolia and C. subcordatum essential oils were screened for antibacterial activity against Bacillus cereus (ATCC No. 14579), Staphylococcus aureus (ATCC No. 29213), Pseudomonas aeruginosa (ATCC No. 27853), and Escherichia coli (ATCC No. 10798). Minimum inhibitory concentrations (MICs) were determined using the micro broth dilution technique [w/w solutions of samples in DMSO plus 50 μL CAMHB. The sample solutions were serially diluted (1:1) in CAMHB in 96-well plates to give concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 19.5 μg/mL. Organisms at a concentration of approximately 1.5 × 108 colony-forming units (CFU)/mL were added to each well. Plates were incubated at 37 °C for 24 h; the minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control; DMSO was used as a negative control .echnique . Dilutioi.e., serial dilution, concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 19.5 μg/mL) using Candida albicans (ATCC No. 10231) in a yeast-nitrogen base growth medium with approximately 7.5 × 107 CFU/mL. Amphotericin B was used as the positive control. An additional test for antifungal activity against Aspergillus niger (ATCC No. 16888) was determined as above using yeast mold (YM) broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control.Antifungal activity was determined, as described above for bacteria (2 environment at 37 °C in Dulbecco’s modified Eagle medium (DMEM) with 4500 mg glucose per liter of medium, supplemented with 10% fetal bovine serum, 10 μg bovine insulin, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO3, pH 7.35.Human Hs578T breast ductal carcinoma cells (ATCC No. HTB-129)  were gro2 environment at 37 °C in RPMI-1640 medium with l-glutamine, supplemented with 10% fetal bovine serum, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium and buffered with 15 mM Hepes and 23.6 mM NaHCO3, pH 7.30.Human PC-3 prostatic carcinoma cells (ATCC No. CRL-1435)  were gro5 cells per well and PC-3 cells at 1.9 × 104 cells per well. The volume in each well was 100 μL for both cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 μL growth medium containing either 2.5 or 1.0 μL of dimethylsulfoxide (DMSO) solution of oils (1% w/w in DMSO), giving a final concentration of 250 or 100 μg/mL, respectively, for each oil. Hs 578T cells were tested with final concentrations at 250 μg/mL and PC-3 at final concentration of 100 μg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 or 10 μL DMSO/mL) were used. Tingenone (250 or 100 μg/mL) was used as a positive control [®AQueous Non-Radioactive Cell Proliferation assay was performed [oil/% killDMSO) were calculated.Hs 578T cells were plated into 96-well cell culture plates at 1.0 × 10 control . After terformed . After cA. cordifolia and C. subcordatum fruits, according to their elution order on HP-5ms capillary column are presented in w/w) yield. Thirty-eight compounds comprising 97.7% of A. cordifolia oil and forty-six constituents representing 98.2% of C. subcordatum oil were identified. The fruit essential oil of A. cordifolia consisted of oxygenated monoterpenoids and aromatic esters (52.9% and 26.5%), monoterpene and sesquiterpene hydrocarbons (17.2% and 14.6%) and low amounts of aliphatic alcohol and aldehyde (3.7%). The major components identified in this sample include methyl salicylate (25.3%), citronellol (21.4%), α-phellandrene (7.4%), terpinolene (5.7%) and 1,8-cineole (5.5%). Other minor constituents detected in considerable quantities were p-cymene (3.6%), α-humulene (3.0%), β-caryophyllene (2.8%) (E)-β-damascenone (2.0%) and 1-octen-3-ol (2.0%). Two uncommon essential oil constituents were identified as (Z)-rose oxide and geosmin in A. cordifolia essential oil. (Z)-Rose-oxide (isobutenyl-4-methyl tetrahydropyran) is a perfumery ingredient and a constituent  of Pelargonium essential oils and secretions of Aromia moschata [Actinomycetes, Streptomyces riseus and Streptomyces odourifer) are reported to be present on some fruits, grapes for example, and are known for their ability to produce geosmin during metabolism [et al. [A. cordifolia reported by Okoye et al. [E)-α-bergamotene (4.54%). Twenty-five constituents consisting 90.3% of the composition of the leaf essential oil were identified in 0.13% w/w yield.The relative concentrations of the volatile components in moschata . This cymoschata . Geosmintabolism . Accordi [et al. , geosmin,EE)-α-farnesene (5.3%) and methyl salicylate (4.5%) were the quantitatively significant constituents in C. subcordatum fruit essential oil. C. subcordatum fruit oil is comprised of 41.3% hydrocarbons and 56.9% oxygen-containing compounds. The five classes of organic compounds identified and reported are twenty-four hydrocarbons (41.3%), twelve alcohols (18.6%), three aldehydes (28.7%), four aromatic esters (5.8%), one ketone and one ether (3.8%). The fruit volatile oil is dominated by sesquiterpenoid compounds (50.8%), followed by aromatic compounds (33.8%), monoterpenoid compounds (7.3%), simple aldehydes and alcohols (6.3%). The monoterpenoid profile consisted of two monoterpene hydrocarbons (2.8%) and six oxygenated monoterpenes (4.5%). Twenty-two sesquiterpene hydrocarbons and seven oxygenated sesquiterpenes made up the sesquiterpenoid profile of the oil. Two unusual sesquiterpenoids, α-calacorene and longiborneol were identified in C. subcordatum fruit oil. The sesquiterpene alcohol, longiborneol is a constituent of Juniperus, Pinus, Cupressus, Dacrydium species and Cedrus deodara. It is reported to be a plant growth regulator (inhibits cress root growth and promotes wheat germination) [Canthium species. The high concentration of benzaldehyde in essential oil samples has been reported by other workers. Lei et al. [Cerasus subhirtella and C. serrulata contain 31.2% and 42.1% benzaldehyde, respectively as major constituents. Benzaldehyde (96.96%) was also indicated as a major component in the leaf essential oil of Prunus myrtifolia [Benzaldehyde (28.0%), β-caryophyllene (15.5%), (ination) . A literi et al.  showed trtifolia .A. cordifolia and C. subcordatum showed promising antimicrobial activity  and moderate antifungal activity against A. niger (MIC = 156 μg/mL); C. subcordatum oil exhibited moderate antibacterial activity against B. cereus and S. aureus (MIC = 156 μg/mL) and good antifungal activity (MIC = 39 μg/mL) against A. niger. It has been documented that Gram-positive bacteria are more sensitive to chemical compounds than Gram-negative bacteria due to differences in the structures of their cell walls; Gram-negative bacteria are less susceptible to hydrophobic small molecules such as essential oil components due to hydrophilic lipopolysaccharides in their outer membrane [A. cordifolia essential oil, Laportea aestuans essential oil, rich in methyl salicylate (54.50%), has been shown to exhibit antimicrobial activity against S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans at 200 mg/mL compared to the standard drug; however, it was more active against the fungi Rhizopus stolon and A. niger at 25 mg/mL [Pelargonium graveolens essential oil, dominant in citronellol (26.7%), inter alia, and citronellol, also demonstrated strong inhibitory activity against S. aureus and E. coli [P. myrtifolia and apricot, Prunus armeniaca, seed essential oils) exhibited antimicrobial activity against S. aureus, S. epidermidis, B. subtilis, P. aeruginosa, E. coli and C. albicans [Stachys cretica essential oil  and β-caryophyllene are reported to exhibit strong antimicrobial activity, particularly against P. aeruginosa and B. subtilis [The antibacterial and antifungal activities of the fruit volatile oils of activity . Alchornmembrane . This is25 mg/mL ; Pelargo E. coli ,44. Simity MIC =  μg/mL agalbicans . Additiosubtilis .A. cordifolia and C. subcordatum fruit essential oils were screened for in vitro cytotoxic activity against Hs 578T human breast adenocarcinoma and PC-3 human prostatic carcinoma cells. Neither oil showed activity, however, with 0% kill at the concentrations tested.Both A. cordifolia and C. subcordatum essential oils are a function of their distinct chemical profiles. The fruit essential oil of A. cordifolia, rich in methyl salicylate and citronellol, showed antibacterial activity against S. aureus and antifungal activity against A. niger. The antimicrobial activity can be attributed to these two major components, which have shown antimicrobial activities [C. subcordatum fruit oil was active against B. cereus, S. aureus, and A. niger, but the activity is not likely due to the major component benzaldehyde, which is generally not antimicrobial [A. cordifolia and C. subcordatum essential oils are consistent with traditional uses of these medicinal plants.The antimicrobial activities of tivities ,47,48. Cicrobial , but rat


Triticosecale Wittmack) is an important feed crop which suffers severe yield, grade and end-use quality losses due to Fusarium head blight (FHB). Development of resistant triticale cultivars is hindered by lack of effective genetic resistance sources. To dissect FHB resistance, a doubled haploid spring triticale population produced from the cross TMP16315/AC Ultima using a microspore culture method, was phenotyped for FHB incidence, severity, visual rating index (VRI), deoxynivalenol (DON) and some associated traits  followed by single nucleotide polymorphism (SNP) genotyping. A high-density map consisting of 5274 SNPs, mapped on all 21 chromosomes with a map density of 0.48 cM/SNP, was constructed. Together, 17 major quantitative trait loci were identified for FHB on chromosomes 1A, 2B, 3A, 4A, 4R, 5A, 5R and 6B; two of incidence loci (on 2B and 5R) also co-located with loci for severity and VRI, and two other loci of VRI (on 1A and 4R) with DON accumulation. Major and minor loci were also identified for all other traits in addition to many epistasis loci. This study provides new insight into the genetic basis of FHB resistance and their association with other traits in triticale.Triticale (x Triticosecale Wittmack; 2n = 6 × = 42) crop suffers severe losses in yield, grade and end-use quality due to Fusarium head blight (FHB) caused by Fusarium graminearum, Fusarium avenaceum, Fusarium poae and other spp. of genus Fusarium , five main effect QTL for Type-II resistance , seven main effect QTL for VRI  and seven main effect QTL for Type-III resistance  (R2) and were considered major QTL  ; Table 4ajor QTL . The totIn addition to the above main effect QTL, epistasis QTL were also identified for DS and VRI on chrs 1A, 2A, 3A and 3B ; Table 5QErg.lrdc-4A), 5R (QErg.lrdc-5R) and 7A (QErg.lrdc-7A)) (R2) and were considered major QTL ) ; Table 6ajor QTL ; Table 6In addition to above main effect QTL, epistasis QTL were also identified for reduced ERG incidence on chrs 6B and 6R ; Table 7QGpc.lrdc-2A), 2B (QGpc.lrdc-2B), 4R (QGpc.lrdc-4R), 5R (QGpc.lrdc-5R), 6R (QGpc.lrdc-6R) and 7B (QGpc.lrdc-7B)) (R2) and were considered major QTL ) ; Table 6QTwt.lrdc.1R), 4R (QTwt.lrdc.4R), 5A  and 5R (QTwt.lrdc.5R)) (R2) and were considered major QTL ) ; Table 6ajor QTL ; Table 7QYld.lrdc-4A), 4R (QYld.lrdc-4R.2), 6A (QYld.lrdc-6A) and 6R (QYld.lrdc-6R)) (R2) and were considered major QTL ) ; Table 6ajor QTL ; Table 7QPht.lrdc-5A), 5R (QPht.lrdc-5R), 6R (QPht.lrdc-6R) and 7R (QPht.lrdc-7R)) (QLdg.lrdc-2R), 4R , 6R (QLdg.lrdc-6R), 7B (QLdg.lrdc-7B) and 7R (QLdg.lrdc-7R)) (R2) explained by these QTL ranged from 6.0 to 38.0%. One of the above identified QTL on chr 5R (QPht.lrdc-5R) for reduced PHT was commonly shared with reduced Type-II and -III resistance and interestingly favorable allele for these, reduced PHT, DS and DON content, were contributed by TMP16315 ) ; Table 6rdc-7R)) ; Table 6Additionally, epistasis QTL were identified for PHT  but not for LDG ; Table 7QFhb.lrdc-1A) and DON (QDon.lrdc-1A), also harbored QTL for TWT (QTwt.lrdc-1A) (QFhs.lrdc-2A) harbored a QTL for PHT (QPht.lrdc-2A), a 2B region for DI (QFhi.lrdc-2B), DS (QFhs.lrdc-2B) and VRI (QFhb.lrdc-2B) harbored QTL for GPC (QGpc.lrdc-2B), a 4R region for VRI (QFhb.lrdc-4R) and DON  harbored QTL for TWT (QTwt.lrdc.4R) and LDG (QLdg.lrdc-4R), another 4R region for GPC (QGpc.lrdc-4R) harbored QTL for YLD (QYld.lrdc-4R.2), a 5A region for DON (QDon.lrdc-5A) harbored QTL for PHT (QPht.lrdc-5A), a 5R region for DI (QFhi.lrdc-5R), DS (QFhs.lrdc-5R.1) and VRI (QFhb.lrdc-5R.1) harbored QTL for TWT (QTwt.lrdc-5R), another 5R region for DON (QDon.lrdc-5R) and PHT (QPht.lrdc-5R) also harbored QTL for DS (QFhs.lrdc-5R.2) and a 6R region for GPC (QGpc.lrdc-6R) and YLD (QYld.lrdc-6R) harbored QTL for PHT (QPht.lrdc-6R) and LDG (QLdg.lrdc-6R)  QTL listed above, fifteen more  QTL were also co-localized  with other QTL. One region on chr 1A, which harbored QTL for VRI (lrdc-1A) . Similarlrdc-6R) .Fusarium head blight (then scab) in England in 1884, many scientific studies have been conducted in wheat, barley and other cereals. The genetic factors, their chromosomal locations and sources  for FHB resistance have been identified and well-studied in wheat [After the first report of in wheat . Howeverin wheat . Previouin wheat , predomiin wheat  and possin wheat ,12. In ain wheat ,55, diffin wheat  and theiin wheat  was subjParents to make this DH population were selected for commercial breeding program and neither of the parents was highly susceptible for FHB, ERG and both parents shared many other favorable characters . This inSecale cereale L. genome, which is a mosaic of various progenitor genomes [Gene associated SNPs, high-throughput genotype calling and low cost per data point of recently developed SNP assays for wheat (Infinium iSelect 90K SNP assay)  and rye  genomes . Around  genomes  also obs genomes  to 5.2 c genomes , however genomes  to 4907. genomes  cM. On t genomes  also obs genomes  construc genomes . We also genomes  and can  genomes  or previ genomes  and use R2) (R2) another FHB/DON resistance QTL. Interestingly 1A QTL region  was never identified in any of the previous triticale studies. This region also harbored an important QTL for TWT  and was derived from AC Ultima, where most of the other important QTL were derived from TMP16315. This 5A QTL seems to be common with one of the three  QTL of widely used Chinese cv. Sumai-3. However, phenotypic data indicate that AC Ultima possess low level of resistance in comparison to Sumai-3. This is obvious since AC Ultima may not have 3B and 6B QTL. However, effect of 5A QTL on FHB resistance can be validated in multiple environments for its further use in breeding programs. These observations suggest that while TMP16315 possess quantitative inheritance of minor additive genes/QTLs, AC Ultima carries a major QTL for FHB resistance along with some minor QTL. Since both parents, AC Ultima and TMP16315, contribute favorable QTL alleles, they can be utilized to pyramid and obtain a high level of FHB resistance in triticale. In this study, a few of the DH lines showed significantly better phenotype for FHB resistance than both parents. Phenotypic data largely showed continuous variation for FHB-DI, -DS, -VRI and DON traits . These oR2)  which inR2)  though QQPht.lrdc-5R); most likely this region was flanked by markers Xiac130 and Xiac128 in Kalih et al. [rPt509721 and wPtm8731 in Kalih et al. [QErg.lrdc-7A which had an additive effect of 34.0 and LOD score 8.1 (QPht.lrdc-5R) on chr 5R, which have also been most likely identified in earlier studies, Kalih et al. [Previous studies showed negative relationship of FHB resistance with plant height ,19,20,22h et al. . Howevercore 8.1 . Though,core 8.1 . Similarh et al. . Since, The understanding of genetic architecture of FHB resistance and some related traits like ERG, GPC, TWT, YLD, PHT and LDG in spring triticale is important for developing superior cultivars. Therefore, for the first time, a high-density spring triticale SNP genetic map was generated and several new major and minor effects and epistasis QTL were mapped for FHB resistance and other related traits. These QTL includes both specific (such as a 3A QTL for reduced DON content) and common/pleotropic QTL . On the other hand, both parents contributed favorable QTL alleles  and identified QTL expressed either across environments  or specifically in an environment (such as 2B QTL for VRI and 1A QTL for DON). Most of the identified FHB resistance QTL were also co-localized with one or more agronomic traits such as a QTL on interstitial region of chr 5R for reduced DS, VRI and DON was found pleotropic for reduced PHT. At many pleotropic QTL regions, such as those mentioned above, the favorable alleles were contributed by same parent and these QTL can efficiently be used for marker-assisted selection (MAS) without any disadvantageous results to improve FHB resistance and other agronomic traits simultaneously. The high-density spring triticale SNP map also promises a starting platform for the fine mapping and map-based cloning of major stable QTL identified during the present study.