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1.
BMC Bioinformatics ; 20(1): 452, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484491

RESUMO

BACKGROUND: Streptomycetes are filamentous microorganisms of high biotechnological relevance, especially for the production of antibiotics. In submerged cultures, the productivity of these microorganisms is closely linked to their growth morphology. Microfluidic lab-on-a-chip cultivation systems, coupled with automated time-lapse imaging, generate spatio-temporal insights into the mycelium development of streptomycetes, therewith extending the biotechnological toolset by spatio-temporal screening under well-controlled and reproducible conditions. However, the analysis of the complex mycelial structure formation is limited by the extent of manual interventions required during processing of the acquired high-volume image data. These interventions typically lead to high evaluation times and, therewith, limit the analytic throughput and exploitation of microfluidic-based screenings. RESULTS: We present the tool mycelyso (MYCElium anaLYsis SOftware), an image analysis system tailored to fully automated hyphae-level processing of image stacks generated by time-lapse microscopy. With mycelyso, the developing hyphal streptomycete network is automatically segmented and tracked over the cultivation period. Versatile key growth parameters such as mycelium network structure, its development over time, and tip growth rates are extracted. Results are presented in the web-based exploration tool mycelyso Inspector, allowing for user friendly quality control and downstream evaluation of the extracted information. In addition, 2D and 3D visualizations show temporal tracking for detailed inspection of morphological growth behaviors. For ease of getting started with mycelyso, bundled Windows packages as well as Docker images along with tutorial videos are available. CONCLUSION: mycelyso is a well-documented, platform-independent open source toolkit for the automated end-to-end analysis of Streptomyces image stacks. The batch-analysis mode facilitates the rapid and reproducible processing of large microfluidic screenings, and easy extraction of morphological parameters. The objective evaluation of image stacks is possible by reproducible evaluation workflows, useful to unravel correlations between morphological, molecular and process parameters at the hyphae- and mycelium-levels with statistical power.


Assuntos
Imageamento Tridimensional , Micélio/citologia , Software , Streptomyces/citologia , Microscopia
2.
Med Mycol ; 57(8): 969-975, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649411

RESUMO

Talaromyces marneffei is a thermally dimorphic fungal pathogen that causes serious infections particularly in patients with human immunodeficiency virus (HIV). Although the mould form typically produces a characteristic red-diffusing pigment, and conidia from penicillate heads, several nonpathogenic Talaromyces/Penicillium species are morphologically and phenotypically similar. While those other species do not exhibit thermal dimorphism, conversion of T. marneffei to the distinctive fission yeast form in vitro is arduous and frequently incomplete. Here we show that T. marneffei can be rapidly and unambiguously discriminated from related nonpathogenic Talaromyces/Penicillium spp., either by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry or conversion to fission yeast after introduction into Galleria mellonella. Conversion of T. marneffei conidia to the fission yeast form in G. mellonella larvae occurred as early as 24 h post inoculation at 37oC. Identification by MALDI-TOF was possible after supplementation of the commercial Bruker database with in-house mass spectral profiles created from either the yeast or mycelial phase of T. marneffei. In addition, we show that in-house generated mass spectral profiles could be successfully used to identify T. marneffei with a recently published on-line MALDI-TOF database, circumventing the need to create extensive in-house additional databases for rarely encountered fungal pathogens.


Assuntos
Lepidópteros/microbiologia , Técnicas Microbiológicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Talaromyces/classificação , Animais , Humanos , Larva/microbiologia , Micélio/citologia , Micélio/crescimento & desenvolvimento , Micoses/microbiologia , Pigmentos Biológicos/metabolismo , Talaromyces/química , Talaromyces/citologia , Talaromyces/isolamento & purificação , Fatores de Tempo
3.
J Microbiol Biotechnol ; 28(11): 1865-1875, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30301325

RESUMO

Enhanced application of solid-state fermentation (SSF) in industrial production and the influence of SSF of Rhizopus K1 on glucoamylase productivity were analyzed using the flat band method. A growth model was implemented through SSF of Rhizopus K1 in this experiment, and spectrophotometric method was used to determine glucoamylase activity. Results showed that in bran and potato culture medium with 70% moisture in a loose state, µ of mycelium reached to 0.15 h-1 after 45 h of culture in a thermostatic water bath incubator at 30°C. Under a low-magnification microscope, mycelial cells appeared uniform, bulky with numerous branches, and were not easily ruptured. The generated glucoamylase activity reached to 55 U/g (dry basis). This study has good utilization value for glucoamylase production by Rhizopus in SSF.


Assuntos
Glucana 1,4-alfa-Glucosidase/biossíntese , Microbiologia Industrial/métodos , Micélio/crescimento & desenvolvimento , Rhizopus/metabolismo , Meios de Cultura , Fermentação , Glucana 1,4-alfa-Glucosidase/análise , Modelos Biológicos , Micélio/citologia , Rhizopus/enzimologia , Rhizopus/crescimento & desenvolvimento , Técnicas de Síntese em Fase Sólida , Espectrofotometria
4.
Plant Dis ; 102(11): 2158-2169, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30252624

RESUMO

Moldy core (MC) of apple is an important disease in Chile, with prevalence observed between 4 and 46% in Fuji, Oregon Spur Red Chief, and Scarlet apple in the 2014-15 and 2015-16 growing seasons. However, there is no information on the identity of the causal agents associated with MC in Chile. The analysis of 653 MC fruit revealed the presence of several genera of filamentous fungi. However, species of Alternaria (67.7%) were by far the most frequently fungi isolated. In total, 41 Alternaria isolates were characterized morphologically and molecularly using Alternaria major allergen Alt a1, calmodulin, and plasma membrane ATPase gene regions. Six small-spored Alternaria spp. were identified; namely, in order of importance, Alternaria tenuissima, A. arborescens, A. alternata, and A. dumosa in sect. Alternaria; A. frumenti in sect. Infectoriae; and A. kordkuyana in sect. Pseudoalternaria. MC symptoms were reproducible and consisted of a light gray to dark olive-green mycelium over the carpel and seed of immature and mature fruit, confirming that the isolates of these Alternaria spp. were pathogenic. These isolates caused brown necrotic lesions with concentric rings on wounded detached apple leaves. This study demonstrated that at least six Alternaria spp. are the cause of MC of apple in Chile. These Alternaria spp. were isolated alone, or with two or more species coexisting in the same fruit. This is the first report of A. tenuissima, A. arborescens, A. frumenti, A. dumosa, and A. kordkuyana associated with MC of apple in Chile and the first report of A. frumenti, A. kordkuyana, and A. dumosa causing MC of apple worldwide.


Assuntos
Alternaria/classificação , Malus/microbiologia , Doenças das Plantas/microbiologia , Alternaria/citologia , Alternaria/genética , Alternaria/patogenicidade , Chile , Frutas/microbiologia , Geografia , Micélio/citologia , Filogenia , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Esporos Fúngicos/citologia
5.
Mycologia ; 110(5): 822-834, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30240341

RESUMO

Sooty blotch and flyspeck (SBFS) fungi infect the cuticle of fruit, including apple fruit, and produce pigmented colonies. A new member of this fungal complex in the genus Peltaster is described on the basis of molecular and morphological evidence. The SBFS complex is a diverse group of ectophytic fungi that reside primarily within the order Capnodiales. Sooty blotch and flyspeck isolates from apple orchards in the central United States were subjected to parsimony and Bayesian analyses based on the internal transcribed spacer regions of nuc rDNA, the partial translation elongation factor 1-α gene, and the partial mitochondrial small subunit rRNA gene. Phylogenetic analysis delineated a new species, Peltaster gemmifer, from P. cerophilus and P. fructicola. Peltaster gemmifer conidiophores bear primary conidia that produce secondary conidia either through budding or through microcyclic conidiation; these were not seen in cultures of P. cerophilus and P. fructicola. On cellulose membrane that was placed on water agar amended with apple juice, P. gemmifer produced brown to black pycnothyria in a superficial brownish mycelial mat, similar to the colonies produced on apple fruit. Findings from the present study add to the >80 named and putative SBFS species so far described worldwide.


Assuntos
Ascomicetos/classificação , Ascomicetos/isolamento & purificação , Malus/microbiologia , Filogenia , Ascomicetos/citologia , Ascomicetos/genética , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Microscopia , Microscopia Eletrônica de Varredura , Micélio/citologia , Micélio/crescimento & desenvolvimento , Micélio/ultraestrutura , Fator 1 de Elongação de Peptídeos/genética , Pigmentos Biológicos/análise , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Análise de Sequência de DNA , Esporos Fúngicos/citologia , Esporos Fúngicos/ultraestrutura , Estados Unidos
6.
PLoS One ; 13(8): e0192803, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30086137

RESUMO

An effective method for research of macro-morphological characterization and its kinetics was developed by studying the macro-morphological characteristics of Mortierella alpina, an oleaginous zygomycete widely used to produce lipids rich in PUFA, in function of culture medium composition and to link morphological features of fungus with the level of lipid production. A number of distinct morphological forms including hollow pellets, fluffy pellets and freely dispersed mycelia were obtained by changing the fermentation factors. By fitting a Logistic curve, the maximum specific growth rate (µmax)was obtained, which determined the final mycelia morphology. µmax of 0.6584 in three kind of morphological forms is the more appropriate. According to the Luedeking-Piret equation fitting, α≠0 and ß≠0, lipid production was partially associated with the hyphal growth, fluffy pellets which turn glucose into lipidwas more effective than the other two kinds of morphological forms.


Assuntos
Técnicas de Cultura Celular por Lotes , Mortierella/citologia , Mortierella/crescimento & desenvolvimento , Meios de Cultura , Fermentação/fisiologia , Cinética , Metabolismo dos Lipídeos , Mortierella/metabolismo , Micélio/citologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo
7.
Fungal Biol ; 122(8): 774-784, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30007428

RESUMO

The laccase production by mycelial antagonistic interaction among white-rot fungi is a very important pathway for lignin degradation research. To gain a better understanding of competitive mechanisms under mycelial antagonistic interaction among three lignin-degrading white-rot basidiomycetes of Trametesversicolor (Tv), Pleurotusostreatus (Po) and Dichomitussqualens (Ds), mycelial morphology and proteins in three co-culture combinations TvPo (Tv cocultivated with Po), PoDs (Po cocultivated with Ds), TvDs (Tv cocultivated with Ds) were compared with corresponding each two mono-cultures. In this study, scanning electron microscopy detection of co-cultures indicated a highly close attachment of fungal hyphae with each other and conidiation could be inhibited under fungal interaction. In addition, a label-free proteomic analysis revealed changes on fungal proteomes existed in their counterpart competitors of co-culture. The maximum number of 1020 differentially expressed proteins (DEPs) were identified in PoDs relative to Po while the minimum number of 367 DEPs were identified in PoDs relative to Ds. Notably, we also found a large number of overexpressed proteins were oxidative stress-related proteins, followed by carbohydrate metabolism-related proteins and energy production-related proteins in all three co-culture combinations compared with control. These results were important for the future exploration of molecular mechanisms underlying lignin-degrading fungal interaction.


Assuntos
Basidiomycota/química , Basidiomycota/crescimento & desenvolvimento , Proteínas Fúngicas/análise , Interações Microbianas , Estresse Oxidativo , Proteoma/análise , Estresse Fisiológico , Microscopia Eletrônica de Varredura , Micélio/citologia , Micélio/crescimento & desenvolvimento
8.
FEMS Yeast Res ; 18(8)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982373

RESUMO

Yarrowia lipolytica is an ascomycetous dimorphic yeast with immense potential for industrial applications, including bioremediation of crude oil-contaminated environments. It has been shown that a dimorphic marine isolate of Y. lipolytica (var. indica) has significant capacity to degrade fatty acids and alkanes, when in its yeast morphology. It has also been demonstrated that polyamines play an important role in the yeast-to-mycelium transition of different strains of Y. lipolytica that are unable to utilize those carbon sources. To determine the role of polyamines on their capacity to utilize oils and hydrocarbons, on the dimorphic transition, and also on other characteristics of the var. indica strain of Y. lipolytica, we proceeded to obtain ornithine decarboxylase minus (odc-) mutants. These mutants behaved as yeasts independently of the concentrations of putrescine added. Further, they conserved the oil-degrading capacity of the parent strain. The odc- mutant can thus be used in fatty acid degradation, and oil spill remediation with distinct advantages.


Assuntos
Poluentes Ambientais/metabolismo , Óleos/metabolismo , Poliaminas/metabolismo , Yarrowia/efeitos dos fármacos , Yarrowia/metabolismo , Biotransformação , Mutação , Micélio/citologia , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Ornitina Descarboxilase/deficiência , Yarrowia/citologia , Yarrowia/crescimento & desenvolvimento
9.
Fungal Biol ; 122(7): 701-716, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880205

RESUMO

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.


Assuntos
Agaricales/crescimento & desenvolvimento , Agaricales/citologia , Agaricales/fisiologia , Cacau/microbiologia , Núcleo Celular , Meios de Cultura , Micélio/citologia , Micélio/crescimento & desenvolvimento , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento
10.
Artigo em Inglês | MEDLINE | ID: mdl-29896454

RESUMO

Aspergillus species are the major cause of health concern worldwide in immunocompromised individuals. Opportunistic Aspergilli cause invasive to allergic aspergillosis, whereas non-infectious Aspergilli have contributed to understand the biology of eukaryotic organisms and serve as a model organism. Morphotypes of Aspergilli such as conidia or mycelia/hyphae helped them to survive in favorable or unfavorable environmental conditions. These morphotypes contribute to virulence, pathogenicity and invasion into hosts by excreting proteins, enzymes or toxins. Morphological transition of Aspergillus species has been a critical step to infect host or to colonize on food products. Thus, we reviewed proteins from Aspergilli to understand the biological processes, biochemical, and cellular pathways that are involved in transition and morphogenesis. We majorly analyzed proteomic studies on A. fumigatus, A. flavus, A. terreus, and A. niger to gain insight into mechanisms involved in the transition from conidia to mycelia along with the role of secondary metabolites. Proteome analysis of morphotypes of Aspergilli provided information on key biological pathways required to exit conidial dormancy, consortia of virulent factors and mycotoxins during the transition. The application of proteomic approaches has uncovered the biological processes during development as well as intermediates of secondary metabolite biosynthesis pathway. We listed key proteins/ enzymes or toxins at different morphological types of Aspergillus that could be applicable in discovery of novel therapeutic targets or metabolite based diagnostic markers.


Assuntos
Aspergilose/microbiologia , Aspergillus/citologia , Aspergillus/metabolismo , Aspergillus/patogenicidade , Proteômica , Aspergillus/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Humanos , Hifas/citologia , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/patogenicidade , Micélio/citologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Micélio/patogenicidade , Micotoxinas/biossíntese , Proteoma/análise , Metabolismo Secundário , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Virulência , Fatores de Virulência/metabolismo
11.
Appl Microbiol Biotechnol ; 102(15): 6627-6636, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29846777

RESUMO

Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and ß-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and ß-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.


Assuntos
Temperatura Alta , Pleurotus/citologia , Pleurotus/metabolismo , Parede Celular/metabolismo , Micélio/química , Micélio/citologia , Pleurotus/química
12.
World J Microbiol Biotechnol ; 34(2): 29, 2018 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-29350302

RESUMO

This study investigated the potential anti-fungal mechanisms of sodium dehydroacetate (SD) against Geotrichum citri-aurantii. The results showed that the cell wall integrity of G. citri-aurantii was not affected, whereas the membrane permeability of G. citri-aurantii mycelia was visibly altered by SD. Dramatic morphological changes of the mycelia, such as loss of cytoplasm, plasmolysis, and dissolution of intracellular substances, were observed by scanning electron microscopy and transmission electron microscopy analyses, indicating that the mycelium is severely damaged by the SD treatment. Furthermore, SD apparently induced a decrease in the intracellular ATP content before 30 min of exposure. An increase in the activity of the Na+/K+-ATPase was also observed, indicating that Na+ ions might enter the cell and thus disturb the energy supply. Taken together, this study's findings suggest that the anti-fungal activity of SD against G. citri-aurantii can be attributed to the disruption of cell membrane permeability and energy metabolism.


Assuntos
Antifúngicos/farmacologia , Geotrichum/citologia , Geotrichum/efeitos dos fármacos , Pironas/farmacologia , Nucleotídeos de Adenina/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Citrus/microbiologia , Citoplasma/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Micélio/citologia , Micélio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Doenças das Plantas/microbiologia , Potássio/metabolismo , Sódio/metabolismo
13.
Mol Plant Pathol ; 19(1): 59-76, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27696683

RESUMO

Plant pathogens of the genus Verticillium pose a threat to many important crops worldwide. They are soil-borne fungi which invade the plant systemically, causing wilt symptoms. We functionally characterized the APSES family transcription factor Vst1 in two Verticillium species, V. dahliae and V. nonalfalfae, which produce microsclerotia and melanized hyphae as resistant structures, respectively. We found that, in V. dahliae Δvst1 strains, microsclerotium biogenesis stalled after an initial swelling of hyphal cells and cultures were never pigmented. In V. nonalfalfae Δvst1, melanized hyphae were also absent. These results suggest that Vst1 controls melanin biosynthesis independent of its role in morphogenesis. The absence of vst1 also had a great impact on sporulation in both species, affecting the generation of the characteristic verticillate conidiophore structure and sporulation rates in liquid medium. In contrast with these key roles in development, Vst1 activity was dispensable for virulence. We performed a microarray analysis comparing global transcription patterns of wild-type and Δvst1 in V. dahliae. G-protein/cyclic adenosine monophosphate (G-protein/cAMP) signalling and mitogen-activated protein kinase (MAPK) cascades are known to regulate fungal morphogenesis and virulence. The microarray analysis revealed a negative interaction of Vst1 with G-protein/cAMP signalling and a positive interaction with MAPK signalling. This analysis also identified Rho signalling as a potential regulator of morphogenesis in V. dahliae, positively interacting with Vst1. Furthermore, it exposed the association of secondary metabolism and development in this species, identifying Vst1 as a potential co-regulator of both processes. Characterization of the putative Vst1 targets identified in this study will aid in the dissection of specific aspects of development.


Assuntos
Proteínas Fúngicas/metabolismo , Micélio/metabolismo , Fatores de Transcrição/metabolismo , Verticillium/crescimento & desenvolvimento , Verticillium/metabolismo , Regulação para Baixo/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Melaninas/biossíntese , Morfogênese/genética , Família Multigênica , Micélio/citologia , Oxirredução , Metabolismo Secundário/genética , Transdução de Sinais/genética , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/fisiologia , Transcrição Genética , Verticillium/patogenicidade
14.
Microsc Res Tech ; 81(1): 13-21, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28987021

RESUMO

Vulvovaginal candidiasis (VVC) is a common gynecologic infection and it occurs when there is overgrowth of the yeast called Candida. VVC diagnosis is usually done by observing a Pap smear sample under a microscope and searching for the conidium and mycelium components of Candida. This manual method is time consuming, subjective and tedious. Any diagnosis tools that detect VVC, semi- or full-automatically, can be very helpful to pathologists. This article presents a computer aided diagnosis (CAD) software to improve human diagnosis of VVC from Pap smear samples. The proposed software is designed based on phenotypic and morphology features of the Candida in Pap smear sample images. This software provide a user-friendly interface which consists of a set of image processing tools and analytical results that helps to detect Candida and determine severity of illness. The software was evaluated on 200 Pap smear sample images and obtained specificity of 91.04% and sensitivity of 92.48% to detect VVC. As a result, the use of the proposed software reduces diagnostic time and can be employed as a second objective opinion for pathologists.


Assuntos
Candidíase Vulvovaginal/diagnóstico , Diagnóstico por Computador/métodos , Teste de Papanicolaou/estatística & dados numéricos , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Micélio/citologia , Sensibilidade e Especificidade , Software , Esporos Fúngicos/citologia , Vagina/microbiologia
15.
Sci Rep ; 7(1): 13226, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038577

RESUMO

Growth of Streptomyces in submerged culture is characterized by the formation of complex mycelial particles, known as pellets or clumps, which strongly influence antibiotic production. Also, many bioactive molecules produced by Streptomyces have great potential to modulate soil bacteria morphological development. However, there has been no effort directed at engineering mycelial morphology using these small molecules. Here, thiostrepton was identified, using a combination of qRT-PCR, semi-preparative HPLC, and MALDI-TOF MS, as a pellet-inducing compound produced by S. laurentii ATCC31255. At sub-inhibitory concentration, thiostrepton stimulated Streptomyces coelicolor M145 pellet formation and antibiotics production were altered, with 3-fold and 2-fold decreases in actinorhodin and undecylprodigiosin yields, respectively. It was also shown that mycelial morphology can be influenced by other antibiotic class at sub-inhibitory concentrations. For instance, in the presence of spectinomycin, S. coelicolor M145, which under typical growth conditions forms large diameter pellets with many protruding hyphae, instead formed small diameter pellets with barely visible hyphae at the edge. Importantly, this morphology produced a 4-fold increase in undecylprodigiosin production and 3-fold decrease in actinorhodin production. These results indicated that these small molecules, previously identified as antimicrobials, also have great potential for influencing mycelial morphology.


Assuntos
Antibacterianos/farmacologia , Streptomyces coelicolor/citologia , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces/metabolismo , Tioestreptona/farmacologia , Antibacterianos/biossíntese , Meios de Cultura , Testes de Sensibilidade Microbiana , Micélio/citologia , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptomyces coelicolor/genética , Streptomyces coelicolor/crescimento & desenvolvimento , Tioestreptona/biossíntese , Tioestreptona/isolamento & purificação
16.
J Microbiol ; 55(11): 877-884, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29076072

RESUMO

In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 µg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl3·6H2O. Spore germination was completely inhibited by 50 µl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.


Assuntos
Fusarium/efeitos dos fármacos , Micélio/efeitos dos fármacos , Controle Biológico de Vetores , Pseudomonas syringae/isolamento & purificação , Pseudomonas syringae/metabolismo , Sideróforos/metabolismo , Sideróforos/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Antibiose , Antifúngicos/farmacologia , DNA Ribossômico , Fusarium/citologia , Fusarium/fisiologia , Micélio/citologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/química , Pseudomonas syringae/genética , Análise de Sequência de DNA , Sideróforos/química
17.
mBio ; 8(4)2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28720727

RESUMO

Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis.IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.


Assuntos
Proteínas Fúngicas/metabolismo , Inativação Gênica , Lectinas/metabolismo , Paracoccidioides/patogenicidade , Fatores de Virulência/metabolismo , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Lectinas/genética , Masculino , Camundongos Endogâmicos BALB C , Micélio/citologia , Micélio/crescimento & desenvolvimento , Paracoccidioides/citologia , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Análise de Sobrevida , Virulência , Fatores de Virulência/genética
18.
J Biosci Bioeng ; 124(4): 452-458, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28625612

RESUMO

Natural yellow pigments produced by submerged fermentation of Monascus purpureus have potential economic value and application in the food industry. In the present study, the relationships among fermentation conditions (in terms of pH and shaking/agitation speed), mycelial morphology and the production of Monascus yellow pigments were investigated in both shake-flask and scale-up bioreactor experiments. In the shake-flask fermentation, the highest yield of the Monascus yellow pigments was obtained at pH 5.0 and a shaking speed of 180 rpm. Microscopic images revealed that these results were associated with the formation of freely dispersed small mycelial pellets with shorter, thicker and multi-branched hyphae. Further investigation indicated that the hyphal diameter was highly correlated with the biosynthesis of the Monascus yellow pigments. In a scaled-up fermentation experiment, the yield of yellow pigments (401 U) was obtained in a 200-L bioreactor, which is the highest yield to the best of our knowledge. The present findings can advance our knowledge on the conditions used for enhancing the production of Monascus yellow pigments in submerged fermentation and facilitate large-scale production of these natural pigments.


Assuntos
Fermentação , Monascus/crescimento & desenvolvimento , Monascus/metabolismo , Micélio/citologia , Pigmentos Biológicos/biossíntese , Reatores Biológicos , Cor , Concentração de Íons de Hidrogênio , Hifas/citologia , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Monascus/citologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Pigmentação
19.
Microbiol Spectr ; 5(3)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28524023

RESUMO

The characteristic growth pattern of fungal mycelia as an interconnected network has a major impact on how cellular events operating on a micron scale affect colony behavior at an ecological scale. Network structure is intimately linked to flows of resources across the network that in turn modify the network architecture itself. This complex interplay shapes the incredibly plastic behavior of fungi and allows them to cope with patchy, ephemeral resources, competition, damage, and predation in a manner completely different from multicellular plants or animals. Here, we try to link network structure with impact on resource movement at different scales of organization to understand the benefits and challenges of organisms that grow as connected networks. This inevitably involves an interdisciplinary approach whereby mathematical modeling helps to provide a bridge between information gleaned by traditional cell and molecular techniques or biophysical approaches at a hyphal level, with observations of colony dynamics and behavior at an ecological level.


Assuntos
Fungos/crescimento & desenvolvimento , Fungos/fisiologia , Micélio/crescimento & desenvolvimento , Micélio/fisiologia , Animais , Transporte Biológico/fisiologia , Biomassa , Ecologia , Ecossistema , Alimentos , Hifas/citologia , Hifas/genética , Hifas/metabolismo , Hifas/fisiologia , Modelos Biológicos , Modelos Teóricos , Micélio/citologia , Micélio/metabolismo , Plantas , Microbiologia do Solo , Água
20.
Mycologia ; 109(1): 153-161, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28402784

RESUMO

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.


Assuntos
Endófitos/crescimento & desenvolvimento , Epichloe/crescimento & desenvolvimento , Festuca/microbiologia , Simbiose , Endófitos/citologia , Epichloe/citologia , Microscopia Confocal , Micélio/citologia , Micélio/crescimento & desenvolvimento , Sementes/microbiologia
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