Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Development and Validation of a Self-Assessment Tool for an Integrative Model of Health Promotion in Hospitals: Taiwan's Experience.

The Health Promotion Administration of Taiwan launched an integrative certification initiative in 2016 to streamline a plural system of certifications of health promotion in hospitals. It endeavored to replace original certifications, thereby establishing the proposal of a self-assessment instrument to aid in this integration. This study aimed to verify the robustness of this self-assessment tool by conducting exploratory factor analyses through stratification, reliability tests, content and construct validity tests, and specialist evaluations, which were convened to judge the comprehensibility, applicability, and importance of the standards and measures of this tool. A stratified random sampling of 46 hospitals was performed to confirm the validity of this tool. The tool rendered a floor effect of 0% and a ceiling effect of 13%. A valid factor structure and internal consistency (α ranged from 0.88 to 0.96) in each standard were verified. Hospitals with previous certificates or with 300+ beds achieved high compliance scores. A majority of experts agreed that the sub-standards were comprehensible (≥80%), applicable (≥70%), and important (≥70%). Finally, we conclude that the self-assessment tool is valid and can serve as a reference for other countries with hospitals committed to health promotion in hospital settings.

Correction: Avoiding Biased-Feeding in the Scheduling of Collaborative Multipath TCP.

[This corrects the article DOI: 10.1371/journal.pone.0161213.].

Avoiding Biased-Feeding in the Scheduling of Collaborative Multipath TCP.

Smartphones have become the major communication and portable computing devices that access the Internet through Wi-Fi or mobile networks. Unfortunately, users without a mobile data subscription can only access the Internet at limited locations, such as hotspots. In this paper, we propose a collaborative bandwidth sharing protocol (CBSP) built on top of MultiPath TCP (MPTCP). CBSP enables users to buy bandwidth on demand from neighbors (called Helpers) and uses virtual interfaces to bind the subflows of MPTCP to avoid modifying the implementation of MPTCP. However, although MPTCP provides the required multi-homing functionality for bandwidth sharing, the current packet scheduling in collaborative MPTCP (e.g., Co-MPTCP) leads to the so-called biased-feeding problem. In this problem, the fastest link might always be selected to send packets whenever it has available cwnd, which results in other links not being fully utilized. In this work, we set out to design an algorithm, called Scheduled Window-based Transmission Control (SWTC), to improve the performance of packet scheduling in MPTCP, and we perform extensive simulations to evaluate its performance.

Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola.

Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the ß-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus-M. fructicola interactions.

Overexpression of a redox-regulated cutinase gene, MfCUT1, increases virulence of the brown rot pathogen Monilinia fructicola on Prunus spp.

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H(2)O(2) enhanced MfCUT1 gene expression. A beta-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.