Action mechanisms and biocontrol of Purpureocillium lilacinum against green mould caused by Penicillium digitatum in orange fruit.

AIMS: The present study evaluated, for the first time, the inhibitory effects of the filtrate of Purpureocillium lilacinum against Penicillium digitatum. METHODS AND RESULTS: No direct contact between P. lilacinum and P. digitatum was observed during the dual culture test and the inhibition zone was 6·1 mm. The filtrate of P. lilacinum completely inhibited P. digitatum growth and spore germination at the concentration of 64%. The filtrate increased the permeability of the cell membrane and the content of MDA in P. digitatum. The ergosterol content in P. digitatum was strongly inhibited at 32% by 81·1%. The green mould incidence and severity in filtrate-treated fruit at 64% were 71·7 and 80·7% lower than in the control, respectively. The filtrate enhanced the activity of PAL, PPO and POD enzymes in orange fruit. The POD and PAL gene expression levels were significantly upregulated in the fruit treated with the filtrate. CONCLUSIONS: This study indicated that the antifungal mechanism of P. lilacinum filtrate against P. digitatum is mainly by the damage of the fungal cell membrane and its components. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the pioneer evidence on the application of P. lilacinum filtrate as a novel biocontrol agent for orange green mould.

Epilobium angustifolium L. Essential Oil-Biological Activity and Enhancement of the Skin Penetration of Drugs-In Vitro Study.

Epilobium angustifolium L. is a popular medicinal plant found in many regions of the world. This plant contains small amounts of essential oil whose composition and properties have not been extensively investigated. There are few reports in the literature on the antioxidant and antifungal properties of this essential oil and the possibility of applying it as a potential promoter of the skin penetration of drugs. The essential oil was obtained by distillation using a Clavenger type apparatus. The chemical composition was analyzed by the GC-MS method. The major active compounds of E. angustifolium L. essential oil (EOEa) were terpenes, including α-caryophyllene oxide, eucalyptol, ß-linalool, camphor, (S)-carvone, and ß-caryophyllene. The analyzed essential oil was also characterized by antioxidant activity amounting to 78% RSA (Radical Scavenging Activity). Antifungal activity against the strains Aspergillus niger, A. ochraceus, A. parasiticum, and Penicillium cyclopium was also determined. The largest inhibition zone was observed for strains from the Aspergillus group. The EOEa enhanced the percutaneous penetration of ibuprofen and lidocaine. After a 24 h test, the content of terpene in the skin and the acceptor fluid was examined. It has been shown that the main compounds contained in the essential oil do not penetrate through the skin, but accumulate in it. Additionally, FTIR-ATR analysis showed a disturbance of the stratum corneum (SC) lipids caused by the essential oil application. Due to its rich composition and high biological activity, EOEa may be a potential candidate to be applied, for example, in the pharmaceutical or cosmetic industries. Moreover, due to the reaction of the essential oil components with SC lipids, the EOEa could be an effective permeation enhancer of topically applied hydrophilic and lipophilic drugs.

Genetic control of α-farnesene production in apple fruit and its role in fungal pathogenesis.

Terpenes are important compounds in plant trophic interactions. A meta-analysis of GC-MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)-α-farnesene. Four quantitative trait loci (QTLs) for α-farnesene production in ripe fruit were identified in a segregating 'Royal Gala' (RG) × 'Granny Smith' (GS) population with one major QTL on linkage group 10 co-locating with the MdAFS1 (α-farnesene synthase-1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC-MS analysis of headspace and solvent-extracted terpenes showing that cold-treated GS apples produced higher levels of (E,E)-α-farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)-α-farnesene. To evaluate the role of (E,E)-α-farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post-harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)-α-farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post-inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)-α-farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.

Sugar-regulated susceptibility of tomato fruit to Colletotrichum and Penicillium requires differential mechanisms of pathogenicity and fruit responses.

Colletotrichum gloeosporioides and Penicillium expansum cause postharvest diseases in tropical and deciduous fruit. During colonization, C. gloeosporioides and P. expansum secrete ammonia in hosts with low sugar content (LowSC) and gluconic acid in hosts with high sugar content (HighSC), respectively, as a mechanism to modulate enhanced pathogenicity. We studied the pathogens interactions with tomato lines of similar genetic background but differing in their sugar content. Colletotrichum gloeosporioides showed enhanced colonization of the LowSC line with differential expression response of 15% of its genes including enhanced relative expression of glycosyl hydrolases, glucanase and MFS-transporter genes. Enhanced colonization of P. expansum occurred in the HighSC line, accompanied by an increase in carbohydrate metabolic processes mainly phosphoenolpyruvate carboxykinase, and only 4% of differentially expressed genes. Gene response of the two host lines strongly differed depending on the sugar level. Limited colonization of HighSC line by C. gloeosporioides was accompanied by a marked alteration of gene expression compared the LowSC response to the same pathogen; while colonization by P. expansum resulted in a similar response of the two different hosts. We suggest that this differential pattern of fungal/host responses may be the basis for the differential of host range of both pathogens in nature.

Transcriptome analysis reveals underlying immune response mechanism of fungal (Penicillium oxalicum) disease in Gastrodia elata Bl. f. glauca S. chow (Orchidaceae).

BACKGROUND: Gastrodia elata Bl. f. glauca S. Chow is a medicinal plant. G. elata f. glauca is unavoidably infected by pathogens in their growth process. In previous work, we have successfully isolated and identified Penicillium oxalicum from fungal diseased tubers of G. elata f. glauca. As a widespread epidemic, this fungal disease seriously affected the yield and quality of G. elata f. glauca. We speculate that the healthy G. elata F. glauca might carry resistance genes, which can resist against fungal disease. In this study, healthy and fungal diseased mature tubers of G. elata f. glauca from Changbai Mountain area were used as experimental materials to help us find potential resistance genes against the fungal disease. RESULTS: A total of 7540 differentially expressed Unigenes (DEGs) were identified (FDR < 0.01, log2FC > 2). The current study screened 10 potential resistance genes. They were attached to transcription factors (TFs) in plant hormone signal transduction pathway and plant pathogen interaction pathway, including WRKY22, GH3, TIFY/JAZ, ERF1, WRKY33, TGA. In addition, four of these genes were closely related to jasmonic acid signaling pathway. CONCLUSIONS: The immune response mechanism of fungal disease in G. elata f. glauca is a complex biological process, involving plant hormones such as ethylene, jasmonic acid, salicylic acid and disease-resistant transcription factors such as WRKY, TGA.

Multiple transcriptomic analyses and characterization of pathogen-related core effectors and LysM family members reveal their differential roles in fungal growth and pathogenicity in Penicillium expansum.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.

Biocontrol ability and action mechanisms of Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 against Penicillium digitatum DSM2750 and Penicillium expansum DSM62841 causing postharvest diseases.

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

Novel Penicillium species causing disseminated disease in a Labrador Retriever dog.

This report describes the phenotypic characteristics of a novel Penicillium species, Penicillium labradorum, isolated from a 3-year-old male, castrated, Labrador retriever with disseminated fungal disease. The dog's presenting clinical signs included lethargy, lymphadenopathy, tachypnea, moderate pitting edema, and nonweight bearing lameness associated with the right hind limb. Fine-needle aspirate biopsies from the sublumbar and prescapular lymph nodes were initially examined. The cytologic findings were consistent with pyogranulomatous inflammation with abundant extracellular and phagocytized fungal fragments and hyphae. Based on the morphology of the organisms and lack of endogenous pigment, hyalohyphomycosis was considered most likely, with Fusarium, Penicillium, and Paecilomyces species being considerations. Fungal isolates were obtained via culture of samples from the lymph nodes, and molecular identification testing originally identified an undescribed Penicillium species belonging to the Penicillium section Exilicaulis. BLAST searches and phylogenetic analyses performed approximately 1 year and 9 months after the isolation date revealed an isolate within the Penicillium parvum clade in the Penicillium section Exilicaulis but phylogenetically distant from the other species in the section, thus representing a new species, Penicillium labradorum. Antifungal susceptibility testing was also performed on the isolate and low minimum inhibitory concentrations were observed with terbinafine, voriconazole, and posaconazole, while in vitro resistance was observed with fluconazole. The dog had been previously treated with fluconazole, itraconazole, amphotericin B lipid complex, voriconazole, and terbinafine. Approximately 587 days after the initial diagnosis, the dog was euthanized due to worsening of clinical signs and concerns for quality of life.

Breakthrough invasive fungal infections: Who is at risk?

The epidemiology of invasive fungal infections (IFIs) in immunocompromised individuals has changed over the last few decades, partially due to the increased use of antifungal agents to prevent IFIs. Although this strategy has resulted in an overall reduction in IFIs, a subset of patients develop breakthrough IFIs with substantial morbidity and mortality in this population. Here, we review the most significant risk factors for breakthrough IFIs in haematology patients, solid organ transplant recipients, and patients in the intensive care unit, focusing particularly on host factors, and highlight areas that require future investigation.

Regulation of Secondary Metabolism in the Penicillium Genus.

Penicillium, one of the most common fungi occurring in a diverse range of habitats, has a worldwide distribution and a large economic impact on human health. Hundreds of the species belonging to this genus cause disastrous decay in food crops and are able to produce a varied range of secondary metabolites, from which we can distinguish harmful mycotoxins. Some Penicillium species are considered to be important producers of patulin and ochratoxin A, two well-known mycotoxins. The production of these mycotoxins and other secondary metabolites is controlled and regulated by different mechanisms. The aim of this review is to highlight the different levels of regulation of secondary metabolites in the Penicillium genus.

Unravelling the Antifungal Effect of Red Thyme Oil (Thymus vulgaris L.) Compounds in Vapor Phase.

The aim of this work was to evaluate the antifungal activity in vapor phase of thymol, p-cymene, and γ-terpinene, the red thyme essential oil compounds (RTOCs). The Minimum Inhibitory Concentration (MIC) of RTOCs was determined against postharvest spoilage fungi of the genera Botrytis, Penicillium, Alternaria, and Monilinia, by measuring the reduction of the fungal biomass after exposure for 72 h at 25 °C. Thymol showed the lowest MIC (7.0 µg/L), followed by γ-terpinene (28.4 µg/L) and p-cymene (40.0 µg/L). In the case of P. digitatum ITEM 9569, resistant to commercial RTO, a better evaluation of interactions among RTOCs was performed using the checkerboard assay and the calculation of the Fractional Inhibitory Concentration Index (FICI). During incubation, changes in the RTOCs concentration were measured by GC-MS analysis. A synergistic effect between thymol (0.013 ± 0.003 L/L) and γ-terpinene (0.990 ± 0.030 L/L) (FICI = 0.50) in binary combinations, and between p-cymene (0.700 ± 0.010 L/L) and γ-terpinene (0.290 ± 0.010 L/L) in presence of thymol (0.008 ± 0.001 L/L) (FICI = 0.19), in ternary combinations was found. The synergistic effect against the strain P. digitatum ITEM 9569 suggests that different combinations among RTOCs could be defined to control fungal strains causing different food spoilage phenomena.

Molecular basis and regulation of pathogenicity and patulin biosynthesis in Penicillium expansum.

Penicillium expansum is a necrotrophic plant pathogen with a wide range of fruit hosts. It causes blue mold rot during fruit storage, transport, and sale, resulting in huge economic losses to the fruit industry. Moreover, this pathogen is also the main producer of patulin, a toxic secondary metabolite that contaminates fruit and fruit-derived products and impairs human health. Therefore, understanding molecular basis of the pathogenicity and patulin biosynthesis of the fungal pathogen has important scientific significance and also plays an important guiding role in the research and development of new control technologies. Here, we comprehensively summarize the recent research progress, particularly regarding the molecular aspects of pathogenicity, patulin biosynthesis, and the related regulatory mechanisms, as well as control technologies for blue mold rot in the fruit industry.

Population Pharmacodynamics of Amphotericin B Deoxycholate for Disseminated Infection Caused by Talaromyces marneffei.

Amphotericin B deoxycholate (DAmB) is a first-line agent for the initial treatment of talaromycosis. However, little is known about the population pharmacokinetics and pharmacodynamics of DAmB for talaromycosis. Pharmacokinetic data were obtained from 78 patients; among them, 55 patients had serial fungal CFU counts in blood also available for analysis. A population pharmacokinetic-pharmacodynamic model was fitted to the data. The relationships between the area under the concentration-time curve (AUC)/MIC and the time to blood culture sterilization and the time to death were investigated. There was only modest pharmacokinetic variability in the average AUC, with a mean ± standard deviation of 11.51 ± 3.39 mg·h/liter. The maximal rate of drug-induced kill was 0.133 log10 CFU/ml/h, and the plasma concentration of the DAmB that induced the half-maximal rate of kill was 0.02 mg/liter. Fifty percent of patients sterilized their bloodstreams by 83.16 h (range, 13 to 264 h). A higher initial fungal burden was associated with a longer time to sterilization (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.36 to 0.70; P < 0.001). There was a weak relationship between AUC/MIC and the time to sterilization, although this did not reach statistical significance (HR, 1.03; 95% CI, 1.00 to 1.06, P = 0.091). Furthermore, there was no relationship between the AUC/MIC and time to death (HR, 0.97; 95% CI, 0.88 to 1.08; P = 0.607) or early fungicidal activity {slope = log[(0.500 - 0.003·(AUC/MIC)]; P = 0.319} adjusted for the initial fungal burden. The population pharmacokinetics of DAmB are surprisingly consistent. The time to sterilization of the bloodstream may be a useful pharmacodynamic endpoint for future studies. (This study has been registered at the ISRCTN registry under no. ISRCTN59144167.).

Differential roles, crosstalk and response to the Antifungal Protein AfpB in the three Mitogen-Activated Protein Kinases (MAPK) pathways of the citrus postharvest pathogen Penicillium digitatum.

Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.

Insights into stress responses in mandarins triggered by Bacillus subtilis cyclic lipopeptides and exogenous plant hormones upon Penicillium digitatum infection.

KEY MESSAGE: Bacillus subtilis CLP extract activates defense gene expression and increases the unique protein production involving in pathways of ISR, SAR, ubiquitin-proteasome system, and glycolysis for stress responses in flavedo tissues. Cyclic lipopeptides (CLPs) of Bacillus subtilis ABS-S14 had ability to activate plant defensive pathways, increase resistance and control green mold rot caused by Penicillium digitatum in mandarin fruit. The current study investigated transcriptional and proteomic data to highlight the unique induction effect of CLPs produced by B. subtilis ABS-S14 on the defense mechanism of mandarins in response to P. digitatum attack, and their differences from those following the exogenous plant hormone application. The proteomic patterns of the flavedo tissues as affected by Bacillus CLP extract, salicylic acid (SA), methyl jasmonate (MeJA), and ethephon (Et) were explored. qPCR analysis revealed the great effects of CLP extract in enhancing the transcription of PAL, ACS1, GLU, POD, and PR1. Tryptic peptides by LC-MS analysis between treatments with and without fungal infection were compared. B. subtilis CLP extract empowered the plant's immune response to wound stress by the significant production of calmodulin-binding receptor-like cytoplasmic kinase 2, molybdenum cofactor sulfurase, and NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase. Ubiquitin carrier protein abundance was developed only in the treated flavedo with CLP extract coupled with P. digitatum infection. The gene expression and overall proteome findings involving pathways of ubiquitin proteasome system, ISR, SAR, and energy production provide a new insight into the molecular mechanisms of the antagonist B. subtilis ABS-S14 inducing resistance against green mold in mandarins.

Development of a Bioactive Sauce Based on Oriental Mustard Flour with Antifungal Properties for Pita Bread Shelf Life Improvement.

Ochratoxin A (OTA) is a mycotoxin produced in the secondary metabolism of fungus belonging to the genus Aspergillus and Penicillium. In this study, the employment of oriental mustard flour (OMF) as an ingredient in a packaged sauce was evaluated for the generation in situ of the antimicrobial compound allyl isothiocyanate (AITC) in order to preserve pita bread contaminated with Penicillium verrucosum VTT D-01847, an OTA producer, in an active packaging system. Four different concentrations (8, 16, 33 and 50 mg/g) were tested. Mycelium formation, mycotoxin production, AITC absorbed by the food matrix, and volatilization kinetics were studied for each concentration. The results obtained were compared with bread treated with the commercial additive calcium propionate (E-282). The results showed a shelf life increase of two and three days with the employment of 33 and 50 mg/g of OMF, with a significant reduction of the fungal population (3.1 and 5.7 logs, respectively) in comparison with the control experiment. The use of 16 and 33 mg/g of OMF in the sauce formulation decreased the concentration of OTA in the bread samples while no OTA production was detected employing 50 mg/g of OMF.

The pH-responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum.

The PacC (loss or reduction in phosphatase activity at acid but not at alkaline pH [Pac]) transcription factor regulates environmental adaptation, secondary metabolism and virulence in many fungal pathogens. Here, we report the functions of PacC in Penicillium expansum, a postharvest pathogenic fungus in horticultural crops, and ascertain that the gene expression and proteolytic processing of PePacC are strictly pH-dependent. Loss of PePacC resulted in an obvious decrease in growth and conidiation of P. expansum cultured in both acidic and alkaline conditions. The ΔPePacC mutant lost the ability of patulin production at pH values above 6.0 because expressions of all the genes in patulin cluster were significantly down-regulated. Additionally, virulence of the ΔPePacC mutant was obviously reduced in pear and apple fruits. Proteome analysis revealed that PePacC could function as an activator or repressor for different target proteins, including calreticulin (PeCRT) and sulfate adenylyltransferase (PeSAT), which were further proved to be involved in virulence of P. expansum. Our results demonstrate important roles for PePacC in patulin biosynthesis via limiting expressions of the genes in the cluster, and in pathogenesis via mediating a known virulence factor glucose oxidase (PeGOD) and new virulence factors, such as PeCRT and PeSAT.

Rapid and precise diagnosis of disseminated T.marneffei infection assisted by high-throughput sequencing of multifarious specimens in a HIV-negative patient: a case report.

BACKGROUND: Talaromyces marneffei, is an opportunistic pathogenic fungus that is most commonly reported in Southeast Asia and disseminated T.marneffei infection predominantly occurs in patients with immunodeficiency. With a potential to invade multiple organs, it can be fatal for patients if diagnosis and treatment are delayed. In current clinical practice, the diagnosis of T.marneffei infection relies heavily on tissue culture and histologic analysis, which may suffer from limited positive rate and is sometimes time consuming. The rapid and accurate diagnosis of disseminated T.marneffei infection remains challenging. CASE PRESENTATION: A 22-year-old man gradually developed fever, cough, lower extremities weakness, jaundice and rash, for which a 3-month extensive investigation failed to reach a diagnosis. After admitted into our hospital, laboratory and radiological tests revealed multiple lesions in the patient's brain, spinal cord, and lungs. We performed next generation sequencing on the patient's skin tissue, bone marrow, blood and cerebrospinal fluid, which all identified numerous Talaromyces marneffei nucleotide sequences and leaded to the rapid diagnosis and treatment of disseminated T.marneffei infection. CONCLUSIONS: This case underline the clinical significance of T.marneffei as a possible pathogen in immune-competent patients. This successful application of the next generation sequencing assisting the rapid diagnosis of disseminated T.marneffei infection provides a new perspective in the clinical approach to the systematic fungi infections and highlights the potential of this technique in rapid etiological diagnosis.

Antagonistic Activity and the Mechanism of Bacillus amyloliquefaciens DH-4 Against Citrus Green Mold.

Citrus fruit usually suffer significant losses during the storage and transportation stages. Green mold, a postharvest rot of citrus fruit caused by Penicillium digitatum, is one of the most serious fungal diseases. In this study, the antagonist strain DH-4 was identified as Bacillus amyloliquefaciens according to morphological observation and 16S ribosomal DNA analysis. In addition, it showed broad antifungal activity, especially the suppression of Penicillium spp. The culture filtrate of strain DH-4 exhibited apparent activity against P. digitatum in vitro and in vivo. In storage, the culture filtrate with DH-4 in it showed a better antiseptic effect. The antifungal substances in the culture filtrate, produced by strain DH-4, displayed stable activity in various extreme conditions. In addition, the antifungal substances in the culture filtrate were identified as macrolactin, bacillaene, iturins, fengycin, and surfactin by ultraperformance liquid chromatography (UPLC) electrospray ionization mass spectrometry analysis. The UPLC fractions containing these antifungal compounds were basically heat tolerant and all responsible for the antagonistic activity against P. digitatum. Transmission electron microscope observation indicated that the antifungal substances might cause abnormalities in the P. digitatum cellular ultrastructure, which could be the possible mode of action of B. amyloliquefaciens against P. digitatum. In addition, it was confirmed via scanning electron microscope analysis that the main way it inhibited P. digitatum was by secreting antimicrobial compounds without direct interaction. This study contributes to the understanding of the mechanism of B. amyloliquefaciens against citrus green mold as well as providing a potential application for the biocontrol of postharvest rot diseases in citrus fruit.

Unravelling the contribution of the Penicillium expansum PeSte12 transcription factor to virulence during apple fruit infection.

Blue mould disease caused by Penicillium expansum infection is one of the most important diseases of pome fruit accounting for important economic losses. In the present study, the PeSte12 transcription factor gene was identified, and deletant mutants were produced by gene replacement. Knockout mutants showed a significant decrease of virulence during apple fruit infection. Virulence was affected by the maturity stage of the fruit (immature, mature and over-mature), and disease severity was notably reduced when the apples were stored at 0 °C. The ΔPeSte12 mutants resulted defective in asexual reproduction, producing less conidia, but this characteristic did not correlate with differences in microscopic morphology. In addition, the ΔPeSte12 mutants produced higher quantity of hydrogen peroxide than the wild type strain. Gene expression analysis revealed that PeSte12 was induced over time during apple infection compared to axenic growth, particularly from 2 dpi, reinforcing its role in virulence. Analysis of transcriptional abundance of several genes in ΔPeSte12 mutants showed that in most of the evaluated genes, PeSte12 seemed to act as a negative regulator during axenic growth, as most of them exhibited an increasing expression pattern along the time period evaluated. The highest expression values corresponded to detoxification, ATPase activity, protein folding and basic metabolism. Gene expression analysis during apple infection showed that 3 out of 9 analysed genes were up regulated; thus, PeSte12 seemed to exert a positive control to particular type of aldolase. These results demonstrate the PeSte12 transcription factor could play an important role in P. expansum's virulence and asexual reproduction.