Biological Control of Fusarium oxysporum, the Causal Agent of Fusarium Basal Rot in Onion by Bacillus spp.

Fusarium oxysporum is the main pathogen causing Fusarium basal rot in onion (Allium cepa L.), which incurs significant yield losses before and after harvest. Among management strategies, biological control is an environmentally safe and sustainable alternative to chemical control. In this study, we isolated and screened bacteria for antifungal activity against the basal rot pathogen F. oxysporum. Isolates 23-045, 23-046, 23-052, 23-055, and 23-056 significantly inhibited F. oxysporum mycelial growth and conidial germination. Isolates 23-045, 23-046, 23-052, and 23-056 suppressed the development of Fusarium basal rot in both onion seedlings and bulbs in pot and spray inoculation assays. Isolate 23-055 was effective in onion seedlings but exhibited weak inhibitory effect on onion bulbs. Based on analyses of the 16S rRNA and rpoB gene sequences together with morphological analysis, isolates 23-045, 23-046, 23-052, and 23-055 were identified as Bacillus thuringiensis, and isolate 23-056 as Bacillus toyonensis. All five bacterial isolates exhibited cellulolytic, proteolytic, and phosphate-solubilizing activity, which may contribute to their antagonistic activity against onion basal rot disease. Taken together B. thuringiensis 23-045, 23-046, 23-052, and 23-055 and B. toyonensis 23-056 have potential for the biological control of Fusarium basal rot in onion.

Identification of Fusarium Basal Rot Pathogens of Onion and Evaluation of Fungicides against the Pathogens.

Onion (Allium cepa L.) is an economically important vegetable crop worldwide. However, various fungal diseases, including Fusarium basal rot (FBR), neck rot, and white rot, reduce onion production or bulb storage life. FBR caused by Fusarium species is among the most destructive onion diseases. In this study, we identified Fusarium species associated with FBR in Jeolla and Gyeongsang Provinces in South Korea and evaluated fungicides against the pathogens. Our morphological and molecular analyses showed that FBR in onions is associated with Fusarium commune, Fusarium oxysporum, and Fusarium proliferatum. We selected seven fungicides (fludioxonil, hexaconazole, mandestrobin, penthiopyrad, prochloraz-manganese, pydiflumetofen, and tebuconazole) and evaluated their inhibitory effects on mycelial growth of the pathogens at three different concentrations (0.01, 0.1, and 1 mg/mL). We found that prochloraz-manganese was highly effective, inhibiting 100% of the mycelial growth of the pathogens at all concentrations, followed by tebuconazole. Fludioxonil showed < 50% inhibition at 1 mg/mL for the tested isolates.

Factors Affecting the Length of Stay in the Emergency Department in Psychiatric Emergency Patients in the COVID-19 Pandemic Context.

To reduce overcrowding in emergency departments (ED), which is a serious international problem, it is important to reduce the length of ED stay (ED LOS) of emergency patients. In particular, due to the COVID 19 pandemic, psychiatric emergency patients spent much longer in ED. This study was conducted to identify the characteristics of psychiatric emergency patients who visited the ED during the COVID-19 pandemic and to identify factors affecting ED LOS. This retrospective study was conducted on adult patients aged 19 years or older who visited a psychiatric emergency center operated by an ED from 1 May 2020 to 31 April 2021 because of the COVID-19 pandemic. In this study, the average ED LOS of psychiatric emergency patients was 7.8 h. Factors affecting ED LOS for over 12 h were isolation (OR = 2.39, CI = 1.409-4.052), unaccompanied police officers (OR = 2.106, CI = 1.338-3.316), night-time visits (OR = 2.127, CI = 1.357-3.332), use of sedatives (OR = 1.671, CI = 1.030-2.713), and restraints (OR = 1.968, CI = 1.172-4.895). The ED LOS of psychiatric emergency patients is longer than that of general emergency patients, and a long ED LOS causes ED overcrowding. To reduce the ED LOS of psychiatric emergency patients, they must be accompanied by a police officer when visiting the ED, and the treatment process should be reorganized so that a psychiatrist can promptly intervene. Furthermore, it is necessary to reorganize the isolation guidelines and admission criteria for mental emergency patients.

Quantitative proteomic analysis of human serum using tandem mass tags to predict cardiovascular risks in patients with psoriasis.

Although biomarker candidates associated with psoriasis have been suggested, those for predicting the risk of cardiovascular disease (CVD) early in patients with psoriasis are lacking. We aimed to identify candidate biomarkers that can predict the occurrence of CVD in psoriasis patients. We pursued quantitative proteomic analysis of serum samples composed of three groups: psoriasis patients with and those without CVD risk factors, and healthy controls. Age/Sex-matched serum samples were selected and labeled with 16-plex tandem mass tag (TMT) and analyzed using liquid chromatography-mass spectrometry and subsequent verification with ELISA. Of the 184 proteins that showed statistical significance (P-value < 0.05) among the three groups according to TMT-based quantitative analysis, 98 proteins showed significant differences (> 2.0-fold) between the psoriasis groups with and without CVD risk factors. Verification by ELISA revealed that caldesmon (CALD1), myeloid cell nuclear differentiation antigen (MNDA), and zyxin (ZYX) levels were significantly increased in the psoriasis group with CVD risk factors. Further network analysis identified pathways including integrin signaling, which could be related to platelet aggregation, and actin cytoskeleton signaling. Three novel candidates (MNDA, ZYX, and CALD1) could be potential biomarkers for predicting CVD risks in psoriasis patients. We expect these biomarker candidates can be used to predict CVD risk in psoriasis patients in clinical settings although further studies including large validation are needed.

Impact of COVID-19 Pandemic on Management and Outcomes in Patients with Septic Shock in the Emergency Department.

This study aimed to determine the impact of modifications in emergency department (ED) practices caused by the coronavirus disease 2019 (COVID-19) pandemic on the clinical outcomes and management of patients with septic shock. We performed a retrospective study. Patients with septic shock who presented to the ED between 1 January 2018 and 19 January 2020 were allocated to the pre-COVID-19 group, whereas those who presented between 20 January 2020 and 31 December 2020 were assigned to the post-COVID-19 group. We used propensity score matching to compare the sepsis-related interventions and clinical outcomes. The primary outcome measure was in-hospital mortality. Of the 3697 patients included, 2254 were classified as pre-COVID-19 and 1143 as post-COVID-19. A total of 1140 propensity score-matched pairings were created. Overall, the in-hospital mortality rate was 25.5%, with no statistical difference between the pre- and post-COVID-19 groups (p = 0.92). In a matched cohort, the post-COVID-19 group had delayed lactate measurement, blood culture test, and infection source control (all p < 0.05). There was no significant difference in time to antibiotics (p = 0.19) or vasopressor administration (p = 0.09) between the groups. Although sepsis-related interventions were delayed during the COVID-19 pandemic, there was no significant difference in the in-hospital mortality between the pre- and post-COVID-19 groups.

The CsSTE50 Adaptor Protein in Mitogen-Activated Protein Kinase Cascades Is Essential for Pepper Anthracnose Disease of Colletotrichum scovillei.

Anthracnose, caused by the ascomycete fungus Colletotrichum scovillei, is a destructive disease in pepper. The fungus germinates and develops an infection structure called an appressorium on the plant surface. Several signaling cascades, including cAMP-mediated signaling and mitogen-activated protein kinase (MAPK) cascades, are involved in fungal development and pathogenicity in plant pathogenic fungi, but this has not been well studied in the fruit-infecting fungus C. scovillei. Ste50 is an adaptor protein interacting with multiple upstream components to activate the MAPK cascades. Here, we characterized the CsSTE50 gene of C. scovillei, a homolog of Magnaporthe oryzae MST50 that functions in MAPK cascades, by gene knockout. The knockout mutant ΔCsste50 had pleiotropic phenotypes in development and pathogenicity. Compared with the wild-type, the mutants grew faster and produced more conidia on regular agar but were more sensitive to osmotic stress. On artificial and plant surfaces, the conidia of the mutant showed significantly reduced germination and failed to form appressoria. The mutant was completely non-pathogenic on pepper fruits with or without wounds, indicating that pre-penetration and invasive growth were both defective in the mutant. Our results show that the adaptor protein CsSTE50 plays a role in vegetative growth, conidiation, germination, appressorium formation, and pathogenicity in C. scovillei.

NADPH Oxidases Are Required for Appressorium-Mediated Penetration in Colletotrichum scovillei-Pepper Fruit Pathosystem.

NADPH oxidase (Nox) complexes are known to play essential roles in differentiation and proliferation of many filamentous fungi. However, the functions of Noxs have not been elucidated in Colletotrichum species. Therefore, we set out to characterize the roles of Nox enzymes and their regulators in Colletotrichum scovillei, which causes serious anthracnose disease on pepper fruits in temperate and subtropical and temperate region. In this study, we generated targeted deletion mutants for CsNox1, CsNox2, CsNoxR, and CsNoxD via homologous recombination. All deletion mutants were normal in mycelial growth, conidiation, conidial germination, and appressorium formation, suggesting that CsNox1, CsNox2, CsNoxR, and CsNoxD are not involved in those developmental processes. Notably, conidia of ΔCsnox2 and ΔCsnoxr, other than ΔCsnox1 and ΔCsnoxd, failed to cause anthracnose on intact pepper fruits. However, they still caused normal disease on wounded pepper fruits, suggesting that Csnox2 and CsnoxR are essential for penetration-related morphogenesis in C. scovillei. Further observation proved that ΔCsnox2 and ΔCsnoxr were unable to form penetration peg, while they fully developed appressoria, revealing that defect of anthracnose development by ΔCsnox2 and ΔCsnoxr resulted from failure in penetration peg formation. Our results suggest that CsNox2 and CsNoxR are critical for appressorium- mediated penetration in C. scovillei-pepper fruit pathosystem, which provides insight into understanding roles of Nox genes in anthracnose disease development.

CsPOM1, a DYRK Family Kinase, Plays Diverse Roles in Fungal Development, Virulence, and Stress Tolerance in the Anthracnose Pathogen Colletotrichum scovillei.

Colletotrichum scovillei is the major anthracnose fungus of sweet pepper and chili pepper (Capsicum annuum L.), causing significant losses in the yield and quality of the pepper fruits. Molecular mechanisms governing development and pathogenicity have been widely studied in many foliar fungal pathogens, but the information on fruit diseases is still limited. In this study, we determined the functional roles of the dual-specificity tyrosine phosphorylation-regulated kinase CsPOM1 in C. scovillei. Knockout mutant for CsPOM1 gene was obtained via homology-dependent gene replacement. The ΔCspom1 mutant exhibited a reduction in vegetative growth on osmotic stress, surface hydrophobicity, and conidiation compared with wild-type. Conidia of the ΔCspom1 mutant were already two-celled before inoculation on an induction surface, indicating that CsPOM1 negatively regulates conidial cell division. The ΔCspom1 mutant, similar to wild-type, formed appressoria on the plant surface, but was significantly reduced on hydrophobic coverslips, probably due to a defect in the recognition of surface hydrophobicity. Treatment of conidia with cutin monomers restored appressorium formation on hydrophobic coverslips in the ΔCspom1 mutant. On pepper fruits, the ΔCspom1 mutant exhibited delayed penetration and invasive growth, leading to significantly reduced virulence. Collectively, the results showed that CsPOM1 is important for stress tolerance, conidiation, surface hydrophobicity, appressorium formation, and virulence in C. scovillei.

Mitogen-Activated Protein Kinase CsPMK1 Is Essential for Pepper Fruit Anthracnose by Colletotrichum scovillei.

The phytopathogenic fungus Colletotrichum scovillei, belonging to the Colletotrichum acutatum species complex, causes severe anthracnose disease on several fruits, including chili pepper (Capsicum annuum). However, the molecular mechanisms underlying the development and pathogenicity of Colletotrichum scovillei are unclear. The conserved Fus3/Kss1-related MAPK regulates fungal development and pathogenicity. Here, the role of CsPMK1, orthologous to Fus3/Kss1, was characterized by phenotypic comparison of a target deletion mutant (ΔCspmk1). The mycelial growth and conidiation of ΔCspmk1 were normal compared to that of the wild type. ΔCspmk1 produced morphologically abnormal conidia, which were delayed in conidial germination. Germinated conidia of ΔCspmk1 failed to develop appressoria on inductive surfaces of hydrophobic coverslips and host plants. ΔCspmk1 was completely defective in infectious growth, which may result from failure to suppress host immunity. Furthermore, ΔCspmk1 was impaired in nuclear division and lipid mobilization during appressorium formation, in response to a hydrophobic surface. CsPMK1 was found to interact with CsHOX7, a homeobox transcription factor essential for appressorium formation, via a yeast two-hybridization analysis. Taken together, these findings suggest that CsPMK1 is required for fungal development, stress adaptation, and pathogenicity of C. scovillei.

Relationship between time of emergency department admission and adherence to the Surviving Sepsis Campaign bundle in patients with septic shock.

BACKGROUND: Nighttime hospital admission is often associated with increased mortality risk in various diseases. This study investigated compliance rates with the Surviving Sepsis Campaign (SSC) 3-h bundle for daytime and nighttime emergency department (ED) admissions and the clinical impact of compliance on mortality in patients with septic shock. METHODS: We conducted an observational study using data from a prospective, multicenter registry for septic shock provided by the Korean Shock Society from 11 institutions from November 2015 to December 2017. The outcome was the compliance rate with the SSC 3-h bundle according to the time of arrival in the ED. RESULTS: A total of 2049 patients were enrolled. Compared with daytime admission, nighttime admission was associated with higher compliance with the administration of antibiotics within 3 h (adjusted odds ratio (adjOR), 1.326; 95% confidence interval (95% CI), 1.088-1.617, p = 0.005) and with the complete SSC bundle (adjOR, 1.368; 95% CI, 1.115-1.678; p = 0.003), likely to result from the increased volume of all patients and sepsis patients admitted during daytime hours. The hazard ratios of the completion of SSC bundle for 28-day mortality and in-hospital mortality were 0.750 (95% CI 0.590-0.952, p = 0.018) and 0.714 (95% CI 0.564-0.904, p = 0.005), respectively. CONCLUSION: Septic shock patients admitted to the ED during the daytime exhibited lower sepsis bundle compliance than those admitted at night. Both the higher number of admitted patients and the higher patients to medical staff ratio during daytime may be factors that are responsible for lowering the compliance.

The Small GTPase CsRAC1 Is Important for Fungal Development and Pepper Anthracnose in Colletotrichum scovillei.

The pepper anthracnose fungus, Colletotrichum scovillei, causes severe losses of pepper fruit production in the tropical and temperate zones. RAC1 is a highly conserved small GTP-binding protein in the Rho GTPase family. This protein has been demonstrated to play a role in fungal development, and pathogenicity in several plant pathogenic fungi. However, the functional roles of RAC1 are not characterized in C. scovillei causing anthracnose on pepper fruits. Here, we generated a deletion mutant (ΔCsrac1) via homologous recombination to investigate the functional roles of CsRAC1. The ΔCsrac1 showed pleiotropic defects in fungal growth and developments, including vegetative growth, conidiogenesis, conidial germination and appressorium formation, compared to wild-type. Although ΔCsrac1 was able to develop appressoria, it failed to differentiate appressorium pegs. However, ΔCsrac1 still caused anthracnose disease with significantly reduced rate on wounded pepper fruits. Further analyses revealed that ΔCsrac1 was defective in tolerance to oxidative stress and suppression of host-defense genes. Taken together, our results suggest that CsRAC1 plays essential roles in fungal development and pathogenicity in C. scovillei-pepper fruit pathosystem.

Pex7 selectively imports PTS2 target proteins to peroxisomes and is required for anthracnose disease development in Colletotrichum scovillei.

Pex7 is a shuttling receptor that imports matrix proteins with a type 2 peroxisomal targeting signal (PTS2) to peroxisomes. The Pex7-mediated PTS2 protein import contributes to crucial metabolic processes such as the fatty acid ß-oxidation and glucose metabolism in a number of fungi, but cellular roles of Pex7 between the import of PTS2 target proteins and metabolic processes have not been fully understood. In this study, we investigated the functional roles of CsPex7, a homolog of the yeast Pex7, by targeted gene deletion in the pepper anthracnose fungus Colletotrichum scovillei. CsPex7 was required for carbon source utilization, scavenging of reactive oxygen species, conidial production, and disease development in C. scovillei. The expression of fluorescently tagged PTS2 signal of hexokinases and 3-ketoacyl-CoA thiolases showed that peroxisomal localization of the hexokinase CsGlk1 PTS2 is dependent on CsPex7, but those of the 3-ketoacyl-CoA thiolases are independent on CsPex7. In addition, GFP-tagged CsPex7 proteins were intensely localized to the peroxisomes on glucose-containing media, indicating a role of CsPex7 in glucose utilization. Collectively, these findings indicate that CsPex7 selectively recognizes specific PTS2 signal for import of PTS2-containing proteins to peroxisomes, thereby mediating peroxisomal targeting efficiency of PTS2-containing proteins in C. scovillei. On pepper fruits, the ΔCspex7 mutant exhibited significantly reduced virulence, in which excessive accumulation of hydrogen peroxide was observed in the pepper cells. We think the reduced virulence results from the abnormality in hydrogen peroxide metabolism of the ΔCspex7 mutant. Our findings provide insight into the cellular roles of CsPex7 in PTS2 protein import system.

Homeobox Transcription Factors Are Required for Fungal Development and the Suppression of Host Defense Mechanisms in the Colletotrichum scovillei-Pepper Pathosystem.

Colletotrichum scovillei, an ascomycete phytopathogenic fungus, is the main causal agent of serious yield losses of economic crops worldwide. The fungus causes anthracnose disease on several fruits, including peppers. However, little is known regarding the underlying molecular mechanisms involved in the development of anthracnose caused by this fungus. In an initial step toward understanding the development of anthracnose on pepper fruits, we retrieved 624 transcription factors (TFs) from the whole genome of C. scovillei and comparatively analyzed the entire repertoire of TFs among phytopathogenic fungi. Evolution and proliferation of members of the homeobox-like superfamily, including homeobox (HOX) TFs that regulate the development of eukaryotic organisms, were demonstrated in the genus Colletotrichum. C. scovillei was found to contain 10 HOX TF genes (CsHOX1 to CsHOX10), which were functionally characterized using deletion mutants of each CsHOX gene. Notably, CsHOX1 was identified as a pathogenicity factor required for the suppression of host defense mechanisms, which represents a new role for HOX TFs in pathogenic fungi. CsHOX2 and CsHOX7 were found to play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study provides a molecular basis for understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid in the development of novel approaches for disease management. IMPORTANCE The ascomycete phytopathogenic fungus, Colletotrichum scovillei, causes serious yield loss on peppers. However, little is known about molecular mechanisms involved in the development of anthracnose caused by this fungus. We analyzed whole-genome sequences of C. scovillei and isolated 624 putative TFs, revealing the existence of 10 homeobox (HOX) transcription factor (TF) genes. We found that CsHOX1 is a pathogenicity factor required for the suppression of host defense mechanism, which represents a new role for HOX TFs in pathogenic fungi. We also found that CsHOX2 and CsHOX7 play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study contributes to understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid for initiating novel approaches for disease management.

Association of Health Insurance Status with Outcomes of Sepsis in Adult Patients: A Retrospective Cohort Study.

(1) Background: Sepsis is a life-threatening disease, and various demographic and socioeconomic factors affect outcomes in sepsis. However, little is known regarding the potential association between health insurance status and outcomes of sepsis in Korea. We evaluated the association of health insurance and clinical outcomes in patients with sepsis. (2) Methods: Prospective cohort data of adult patients with sepsis and septic shock from March 2016 to December 2018 in three hospitals were retrospectively analyzed. We categorized patients into two groups according to their health insurance status: National Health Insurance (NHI) and Medical Aid (MA). The primary end point was in-hospital mortality. The multivariate logistic regression model and propensity score matching were used. (3) Results: Of a total of 2526 eligible patients, 2329 (92.2%) were covered by NHI, and 197 (7.8%) were covered by MA. The MA group had fewer males, more chronic kidney disease, more multiple sources of infection, and more patients with initial lactate > 2 mmol/L. In-hospital, 28-day, and 90-day mortality were not significantly different between the two groups and in-hospital mortality was not different in the subgroup analysis. Furthermore, health insurance status was not independently associated with in-hospital mortality in multivariate analysis and was not associated with survival outcomes in the propensity score-matched cohort. (4) Conclusions: Our propensity score-matched cohort analysis demonstrated that there was no significant difference in in-hospital mortality by health insurance status in patients with sepsis.

Antagonistic and Plant Growth-Promoting Effects of Bacillus velezensis BS1 Isolated from Rhizosphere Soil in a Pepper Field.

Pepper (Capsicum annuum L.) is an important agricultural crop worldwide. Recently, Colletotrichum scovillei, a member of the C. acutatum species complex, was reported to be the dominant pathogen causing pepper anthracnose disease in South Korea. In the present study, we isolated bacterial strains from rhizosphere soil in a pepper field in Gangwon Province, Korea, and assessed their antifungal ability against C. scovillei strain KC05. Among these strains, a strain named BS1 significantly inhibited mycelial growth, appressorium formation, and disease development of C. scovillei. By combined sequence analysis using 16S rRNA and partial gyrA sequences, strain BS1 was identified as Bacillus velezensis, a member of the B. subtilis species complex. BS1 produced hydrolytic enzymes (cellulase and protease) and iron-chelating siderophores. It also promoted chili pepper (cv. Nockwang) seedling growth compared with untreated plants. The study concluded that B. velezensis BS1 has good potential as a biocontrol agent of anthracnose disease in chili pepper caused by C. scovillei.

Selective Identification of α-Galactosyl Epitopes in N-Glycoproteins Using Characteristic Fragment Ions from Higher-Energy Collisional Dissociation.

The α-galactosyl epitope is a terminal N-glycan moiety of glycoproteins found in mammals except in humans, and thus, it is recognized as an antigen that provokes an immunogenic response in humans. Accordingly, it is necessary to analyze the α-galactosyl structure in biopharmaceuticals or organ transplants. Due to an identical glycan composition and molecular mass between α-galactosyl N-glycans and hybrid/high-mannose-type N-glycans, it is challenging to characterize α-galactosyl epitopes in N-glycoproteins using mass spectrometry. Here, we describe a method to identify α-galactosyl N-glycopeptides in mice glycoproteins using liquid chromatography with tandem mass spectrometry with higher-energy collisional dissociation (HCD). The first measure was an absence of [YHM] ion peaks in the HCD spectra, which was exclusively observed in hybrid and/or high-mannose-type N-glycopeptides. The second complementary criterion was the ratio of an m/z 528.19 (Hex2HexNAc1) ion to m/z 366.14 (Hex1HexNAc1) ion (Im/z528/Im/z366). The measure of [Im/z528/Im/z366 > 0.3] enabled a clear-cut determination of α-galactosyl N-glycopeptides with high accuracy. In Ggta1 knockout mice, we could not find any α-galactosyl N-glycoproteins identified in WT mice plasma. Using this method, we could screen for α-galactosyl N-glycoproteins from mice spleen, lungs, and plasma samples in a highly sensitive and specific manner. Conclusively, we suggest that this method will provide a robust analytical tool for determination of α-galactosyl epitopes in pharmaceuticals and complex biological samples.

Classification of Mucin-Type O-Glycopeptides Using Higher-Energy Collisional Dissociation in Mass Spectrometry.

Changes in mucin-type O-glycosylation of human proteins affect protein function, immune response, and cancer progression. Since O-glycoproteins are characterized by the microheterogeneity of diverse O-glycans with no conserved sequence and the macroheterogeneity of multiple glycosylation sites on serine and/or threonine in human proteins, the assessment of different mucin types, such as Tn-antigen, core 1, and core 2, and their extended core types in O-glycopeptides, is extremely challenging. Here, we present an O-GlycoProteome Analyzer (O-GPA) that automatically classifies mucin-type O-glycosylation using higher-energy collisional dissociation (HCD) in mass spectrometry. First, we estimated the number of GlcNAc residues using the intensity ratio of GlcNAc-specific fragment ions (HexNAc-CH6O3 and HexNAc-2H2O) over GalNAc-specific fragment ions (HexNAc-C2H6O3 and HexNAc-C2H4O2) in the HCD spectrum. Furthermore, we classified the different mucin types of O-glycopeptides from characteristic B2 (HexNAc2) or Y2α (PEP + HexNAc2), and Y2ß (PEP + HexNAcHex) fragment ions, along with the number of GlcNAc. Furthermore, O-GPA automatically determined single or multiple O-glycosylation, regardless of the mucin types. The mucin type of O-glycopeptides from human urine and plasma was confirmed with an overall accuracy of 96%. We found 97 core 1, 56 core 2, 13 extended core 1, and 12 extended core 2 glycopeptides from urine; and 22 core 1, 13 core 2, 7 extended core 1, 1 extended core 2, and 1 Tn-antigen from plasma. Our strategy can be used to successfully characterize specific mucin types of O-glycoproteins in human biological samples.

MoRBP9 Encoding a Ran-Binding Protein Microtubule-Organizing Center Is Required for Asexual Reproduction and Infection in the Rice Blast Pathogen Magnaporthe oryzae.

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach.

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH4Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 µg/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.