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Several materials have been characterized using proximal sensors, but still incipient efforts have been driven to plant tissues. Eucalyptus spp. cultivation in Brazil covers approximately 7.47 million hectares, requiring faster methods to assess plant nutritional status. This study applies portable X-ray fluorescence (pXRF) spectrometry to (i) distinguish Eucalyptus clones using pre-processed pXRF data; and (ii) predict the contents of eleven nutrients in the leaves of Eucalyptus (B, Ca, Cu, Fe, K, Mg, Mn, N, P, S, and Zn) aiming to accelerate the diagnosis of nutrient deficiency. Nine hundred and twenty samples of Eucalyptus leaves were collected, oven-dried, ground, and analyzed using acid-digestion (conventional method) and using pXRF. Six machine learning algorithms were trained with 70% of pXRF data to model conventional results and the remaining 30% were used to validate the models using root mean square error (RMSE) and coefficient of determination (R 2 ). The principal component analysis clearly distinguished developmental stages based on pXRF data. Nine nutrients were accurately predicted, including N (not detected using pXRF spectrometry). Results for B and Mg were less satisfactory. This method can substantially accelerate decision-making and reduce costs for Eucalyptus foliar analysis, constituting an ecofriendly approach which should be tested for other crops.

Anthracnose, caused by Colletotrichum lindemuthianum, is a disease affecting the common bean plant, Phaseolus vulgaris. To establish infection, the phytopathogen must survive the toxic compounds (phytoanticipins and phytoalexins) that are produced by the plant as a defense mechanism. To study the detoxification and efflux mechanisms in C. lindemuthianum, the abcCl1 gene, which encodes an ABC transporter, was analyzed. The abcCl1 gene (4558 pb) was predicted to encode a 1450-amino acid protein. Structural analysis of 11 genome sequences from Colletotrichum spp. showed that the number of ABC transporters varied from 34 to 64. AbcCl1 was classified in the ABC-G family of transporters, and it appears to be orthologs to ABC1 from Magnaporthe grisea and FcABC1 from Fusarium culmorum, which are involved in pleiotropic drug resistance. A abcT3 (ΔabcCl1) strain showed reduction on aggressivity when inoculated on bean leaves that presented diminishing anthracnose symptoms, which suggests the important role of AbcCl1 as a virulence factor and in fungal resistance to host compounds. The expression of abcCl1 increased in response to different toxic compounds, such as eugenol, hygromycin, and pisatin phytoalexin. Together, these results suggest that AbcCl1 is involved in fungal resistance to the toxic compounds produced by plants or antagonistic microorganisms., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

In agreement with other fungal infections, immunoprotection in pulmonary paracoccidioidomycosis (PCM) is mediated by Th1/Th17 cells whereas disease progression by prevalent Th2/Th9 immunity. Treg cells play a dual role, suppressing immunity but also controlling excessive tissue inflammation. Our recent studies have demonstrated that the enzyme indoleamine 2,3 dioxygenase (IDO) and the transcription factor aryl hydrocarbon receptor (AhR) play an important role in the immunoregulation of PCM. To further evaluate the immunomodulatory activity of AhR in this fungal infection, Paracoccidioides brasiliensis infected mice were treated with two different AhR agonists, L-Kynurenin (L-Kyn) or 6-formylindole [3,2-b] carbazole (FICZ), and one AhR specific antagonist (CH223191). The disease severity and immune response of treated and untreated mice were assessed 96 hours and 2 weeks after infection. Some similar effects on host response were shared by FICZ and L-Kyn, such as the reduced fungal loads, decreased numbers of CD11c+ lung myeloid cells expressing activation markers (IA, CD40, CD80, CD86), and early increased expression of IDO and AhR. In contrast, the AhR antagonist CH223191 induced increased fungal loads, increased number of pulmonary CD11c+ leukocytes expressing activation markers, and a reduction in AhR and IDO production. While FICZ treatment promoted large increases in ILC3, L-Kyn and CH223191 significantly reduced this cell population. Each of these AhR ligands induced a characteristic adaptive immunity. The large expansion of FICZ-induced myeloid, lymphoid, and plasmacytoid dendritic cells (DCs) led to the increased expansion of all CD4+ T cell subpopulations (Th1, Th2, Th17, Th22, and Treg), but with a clear predominance of Th17 and Th22 subsets. On the other hand, L-Kyn, that preferentially activated plasmacytoid DCs, reduced Th1/Th22 development but caused a robust expansion of Treg cells. The AhR antagonist CH223191 induced a preferential expansion of myeloid DCs, reduced the number of Th1, Th22, and Treg cells, but increased Th17 differentiation. In conclusion, the present study showed that the pathogen loads and the immune response in pulmonary PCM can be modulated by AhR ligands. However, further studies are needed to define the possible use of these compounds as adjuvant therapy for this fungal infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Araújo, Loures, Preite, Feriotti, Galdino, Costa and Calich.)

Phytic acid stores 60-90% of the inorganic phosphorus in legumes, oil seeds, and cereals, making it inaccessible for metabolic processes in living systems. In addition, given its negative charge, phytic acid complexes with divalent cations, starch, and proteins. Inorganic phosphorous can be released from phytic acid upon the action of phytases. Phytases are phosphatases produced by animals, plants, and microorganisms, notably Aspergillus niger, and are employed as animal feed additive, in chemical industry and for ethanol production. Given the industrial relevance of phytases produced by filamentous fungi, this work discusses the functional characterization of fungal phytase-coding genes/proteins, highlighting the physicochemical parameters that govern the enzymatic activity, the development of phytase super-producing strains, and key features for industrial applications.

Objective this study aimed to determine the thermal favorability for the oidium (Oidium caricae) and early blight (Asperisporium caricae) in areas of edaphoclimatic aptitude for the papaya (Carica papaya) in the Espírito Santo state, Brazil. The edaphoclimatic zoning was based on the overlapping of maps that characterize the average annual air temperature obtained by multiple linear regression, annual water deficiency calculated by the Thornthwaite and Matter method (1955) and favorable soil classes to the development of papaya. The results indicated that as regards the edaphoclimatic zoning for the papaya crop it was observed that 71.70% of the area is classified as apt for its development. In relation to agrometeorological favorability for the occurrence of fungal diseases, there was a favorability of 7.64% for the development without restrictions of the oidium and a predominance of 64,56% of favorability with thermal restriction. For the early blight fungus, it was observed that the zones of favorability without restriction correspond to 11.66% of the area apt for the papaya cultivation and that 55.13% of the area has favorability with restriction of humidity. The edaphoclimatic zoning for the papaya crop showed compatibility with the most productive areas of this crop in the state of Espírito Santo. The zoning of thermal favorability for the occurrence of papaya fungal diseases proved to be a valuable tool for studies of plant diseases, allowing the establishment of plans for the allocation of resistant varieties, in order to minimize the risks of loss of crop productivity due to the disease. This methodology presents potential to be used in other areas, cultures and phytopathological diseases., (Copyright © 2020. Published by Elsevier Ltd.)

AhR is a ligand-activated transcription factor that plays an important role in the innate and adaptive immune responses. In infection models, it has been associated with host responses that promote or inhibit disease progression. In pulmonary paracoccidioidomycosis, a primary fungal infection endemic in Latin America, immune protection is mediated by Th1/Th17 cells and disease severity with predominant Th2/Th9/Treg responses. Because of its important role at epithelial barriers, we evaluate the role of AhR in the outcome of a pulmonary model of paracoccidioidomycosis. AhR -/- mice show increased fungal burdens, enhanced tissue pathology and mortality. During the infection, AhR -/- mice have more pulmonary myeloid cells with activated phenotype and reduced numbers expressing indoleamine 2,3 dioxygenase 1. AhR-deficient lungs have altered production of cytokines and reduced numbers of innate lymphoid cells (NK, ILC3 and NCR IL-22). The lungs of AhR -/- mice showed increased presence Th17 cells concomitant with reduced numbers of Th1, Th22 and Foxp3 + Treg cells. Furthermore, treatment of infected WT mice with an AhR-specific antagonist (CH223191) reproduced the main findings obtained in AhR -/- mice. Collectively our data demonstrate that in pulmonary paracoccidioidomycosis AhR controls fungal burden and excessive tissue inflammation and is a possible target for antifungal therapy.

The Brazilian Savannah, known as Cerrado, has the richest flora in the world among the savannas, with a high degree of endemic species. Despite the global ecological importance of the Cerrado, there are few studies focused on the modeling of the volume and biomass of this forest formation. Volume and biomass estimation can be performed using allometric models, artificial intelligence (AI) techniques and mixed regression models. Thus, the aim of this work was to evaluate the use of AI techniques and mixed models to estimate the volume and biomass of individual trees in vegetation of Brazilian central savanna. Numerical variables (diameter at height of 1.30 m of ground, total height, volume and biomass) and categorical variables (species) were used for the training and fitting of AI techniques and mixed models, respectively. The statistical indicators used to evaluate the training and the adjustment were the correlation coefficient, bias and Root mean square error relative. In addition, graphs were elaborated as complementary analysis. The results obtained by the statistical indicators and the graphical analysis show the great potential of AI techniques and mixed models in the estimation of volume and biomass of individual trees in Brazilian savanna vegetation. In addition, the proposed methodologies can be adapted to other biomes, forest typologies and variables of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

In human paracoccidioidomycosis (PCM), a primary fungal infection typically diagnosed when the disease is already established, regulatory T cells (Treg) cells are associated with disease severity. Experimental studies in pulmonary PCM confirmed the detrimental role of these cells, but in most studies, Tregs were depleted prior to or early during infection. These facts led us to study the effects of Treg cell depletion using a model of ongoing PCM. Therefore, Treg cell depletion was achieved by treatment of transgenic C57BL/6 DTR/eGFP (DEREG) mice with diphtheria toxin (DT) after 3 weeks of intratracheal infection with 1 × 10 6 Paracoccidioides brasiliensis yeasts. At weeks 6 and 10 post-infection, DT-treated DEREG mice showed a reduced number of Treg cells associated with decreased fungal burdens in the lungs, liver and spleen, reduced tissue pathology and mortality. Additionally, an increased influx of activated CD4 + and CD8 + T cells into the lungs and elevated production of Th1/Th17 cytokines was observed in DT-treated mice. Altogether, our data demonstrate for the first time that Treg cell depletion in ongoing PCM rescues infected hosts from progressive and potentially fatal PCM; furthermore, our data indicate that controlling Treg cells could be explored as a novel immunotherapeutic procedure.

Immunotherapy has become an important ally in the fight against distinct types of cancer. However, the metabolic plasticity of the tumor environment frequently influences the efficacy of therapeutic procedures, including those based on immunological tools. In this scenario, immunometabolic adjuvants arise as an alternative toward the development of more efficient cancer therapies. Here we demonstrated that the combination of melatonin, a neuroimmunomodulator molecule, and an indoleamine 2,3-dioxygenase (IDO) inhibitor (1-methyl-DL-tryptophan, DL-1MT) improves the efficacy of an immunotherapy (gDE7) targeting human papillomavirus (HPV)-associated tumors. Melatonin or IDO inhibitors (D-1MT and DL-1MT) directly reduced proliferation, migration, adhesion and viability of a tumor cell line (TC-1), capable to express the HPV-16 E6 and E7 oncoproteins, but could not confer in vivo antitumor protection effects. Nonetheless, combination of gDE7 with melatonin or D-1MT or DL-1MT enhanced the antitumor protective immunity of gDE7-based vaccine in mice. Notably, expression of IDO1 in stromal cells and/or immune cells, but not in tumor cells, inhibited the antitumor effects of the gDE7, as demonstrated in IDO1-deficient mice. Finally, co-administration of gDE7, melatonin and DL-1MT further improved the protective antitumor effects and the numbers of circulating E7-specific CD8 + T cells in mice previously transplanted with TC-1 cells. The unprecedented combination of melatonin and IDO inhibitors, as immunometabolic adjuvants, thus, represents a new and promising alternative for improving the efficacy of immunotherapeutic treatments of HPV-associated tumors.

Resistance to primary fungal pathogens is usually attributed to the proinflammatory mechanisms of immunity conferred by interferon-γ activation of phagocytes that control microbial growth, whereas susceptibility is attributed to anti-inflammatory responses that deactivate immunity. This study challenges this paradigm by demonstrating that resistance to a primary fungal pathogen such as Paracoccidiodes brasiliensis can be mediated by disease tolerance, a mechanism that preserves host fitness instead of pathogen clearance. Among the mechanisms of disease tolerance described, a crucial role has been ascribed to the enzyme indoleamine-2,3 dioxygenase (IDO) that concomitantly controls pathogen growth by limiting tryptophan availability and reduces tissue damage by decreasing the inflammatory process. Here, we demonstrated in a pulmonary model of paracoccidioidomycosis that IDO exerts a dual function depending on the resistant pattern of hosts. IDO activity is predominantly enzymatic and induced by IFN-γ signaling in the pulmonary dendritic cells (DCs) from infected susceptible (B10.A) mice, whereas phosphorylated IDO (pIDO) triggered by TGF-β activation of DCs functions as a signaling molecule in resistant mice. IFN-γ signaling activates the canonical pathway of NF-κB that promotes a proinflammatory phenotype in B10.A DCs that control fungal growth but ultimately suppress T cell responses. In contrast, in A/J DCs IDO promotes a tolerogenic phenotype that conditions a sustained synthesis of TGF-β and expansion of regulatory T cells that avoid excessive inflammation and tissue damage contributing to host fitness. Therefore, susceptibility is unexpectedly mediated by mechanisms of proinflammatory immunity that are usually associated with resistance, whereas genetic resistance is based on mechanisms of disease tolerance mediated by pIDO, a phenomenon never described in the protective immunity against primary fungal pathogens.

In infectious diseases, the enzyme indoleamine 2,3 dioxygenase-1 (IDO1) that catalyzes the tryptophan (Trp) degradation along the kynurenines (Kyn) pathway has two main functions, the control of pathogen growth by reducing available Trp and immune regulation mediated by the Kyn-mediated expansion of regulatory T (Treg) cells via aryl hydrocarbon receptor (AhR). In pulmonary paracoccidioidomycosis (PCM) caused by the dimorphic fungus Paracoccidioides brasiliensis , IDO1 was shown to control the disease severity of both resistant and susceptible mice to the infection; however, only in resistant mice, IDO1 is induced by TGF-β signaling that confers a stable tolerogenic phenotype to dendritic cells (DCs). In addition, in pulmonary PCM, the tolerogenic function of plasmacytoid dendritic cells was linked to the IDO1 activity. To further evaluate the function of IDO1 in pulmonary PCM, IDO1-deficient (IDO1 -/- ) C57BL/6 mice were intratracheally infected with P. brasiliensis yeasts and the infection analyzed at three postinfection periods regarding several parameters of disease severity and immune response. The fungal loads and tissue pathology of IDO1 -/- mice were higher than their wild-type controls resulting in increased mortality rates. The evaluation of innate lymphoid cells showed an upregulated differentiation of the innate lymphoid cell 3 phenotype accompanied by a decreased expansion of ILC1 and NK cells in the lungs of infected IDO1 -/- mice. DCs from these mice expressed elevated levels of costimulatory molecules and cytokine IL-6 associated with reduced production of IL-12, TNF-α, IL-1β, TGF-β, and IL-10. This response was concomitant with a marked reduction in AhR production. The absence of IDO1 expression caused an increased influx of activated Th17 cells to the lungs with a simultaneous reduction in Th1 and Treg cells. Accordingly, the suppressive cytokines IL-10, TGF-β, IL-27, and IL-35 appeared in reduced levels in the lungs of IDO1 -/- mice. In conclusion, the immunological balance mediated by the axis IDO/AhR is fundamental to determine the balance between Th17/Treg cells and control the severity of pulmonary PCM.

The NOD-like receptor P3 (NLRP3) inflammasome is an intracellular multimeric complex that triggers the activation of inflammatory caspases and the maturation of IL-1β and IL-18, important cytokines for the innate immune response against pathogens. The functional NLRP3 inflammasome complex consists of NLRP3, the adaptor protein apoptosis-associated speck-like protein, and caspase-1. Various molecular mechanisms were associated with NLRP3 activation including the presence of extracellular ATP, recognized by the cell surface P2X7 receptor (P2X7R). Several pattern recognition receptors on innate immune cells recognize Paracoccidioides brasiliensis components resulting in diverse responses that influence adaptive immunity and disease outcome. However, the role of NLRP3 inflammasome was scantily investigated in pulmonary paracoccidioidomycosis (PCM), leading us to use an intratracheal (i.t.) model of infection to study the influence of this receptor in anti-fungal immunity and severity of infection. For in vivo studies, C57BL/6 mice deficient for several NLRP3 inflammasome components ( Nlrp3 -/- , Casp1/11 -/- , Asc -/- ) as well as deficient for ATP receptor ( P2x7r -/- ) were infected via i.t. with P. brasiliensis and several parameters of immunity and disease severity analyzed at the acute and chronic periods of infection. Pulmonary PCM was more severe in Nlrp3 -/- , Casp1/11 -/- , Asc -/- , and P2x7r -/- mice as demonstrated by the increased fungal burdens, mortality rates and tissue pathology developed. The more severe disease developed by NLRP3, ASC, and Caspase-1/11 deficient mice was associated with decreased production of IL-1β and IL-18 and reduced inflammatory reactions mediated by PMN leukocytes and activated CD4 + and CD8 + T cells. The decreased T cell immunity was concomitant with increased expansion of CD4 + CD25 + Foxp3 regulatory T (Treg) cells. Characterization of intracellular cytokines showed a persistent reduction of CD4 + and CD8 + T cells expressing IFN-γ and IL-17 whereas those producing IL-4 and TGF-β appeared in increased frequencies. Histopathological studies showed that all deficient mouse strains developed more severe lesions containing elevated numbers of budding yeast cells resulting in increased mortality rates. Altogether, these findings led us to conclude that the activation of the NLRP3 inflammasome has a crucial role in the immunoprotection against pulmonary PCM by promoting the expansion of Th1/Th17 immunity and reducing the suppressive control mediated by Treg cells.

Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Plasmacytoid dendritic cells (pDCs), considered critical for immunity against viruses, were recently associated with defense mechanisms against fungal infections. However, the immunomodulatory function of pDCs in pulmonary paracoccidiodomycosis (PCM), an endemic fungal infection of Latin America, has been poorly defined. Here, we investigated the role of pDCs in the pathogenesis of PCM caused by the infection of 129Sv mice with 1 x 106 P. brasiliensis-yeasts. In vitro experiments showed that P. brasiliensis infection induces the maturation of pDCs and elevated synthesis of TNF-α and IFN-β. The in vivo infection caused a significant influx of pDCs to the lungs and increased levels of pulmonary type I IFN. Depletion of pDCs by a specific monoclonal antibody resulted in a less severe infection, reduced tissue pathology and increased survival time of infected mice. An increased influx of macrophages and neutrophils and elevated presence of CD4+ and CD8+ T lymphocytes expressing IFN-γ and IL-17 in the lungs of pDC-depleted mice were also observed. These findings were concomitant with decreased frequency of Treg cells and reduced levels of immunoregulatory cytokines such as IL-10, TGF-β, IL-27 and IL-35. Importantly, P. brasilienis infection increased the numbers of pulmonary pDCs expressing indoleamine 2,3-dioxygenase-1 (IDO), an enzyme with immunoregulatory properties, that were reduced following pDC depletion. In agreement, an increased immunogenic activity of infected pDCs was observed when IDO-deficient or IDO-inhibited pDCs were employed in co-cultures with lymphocytes Altogether, our results suggest that in pulmonary PCM pDCs exert a tolerogenic function by an IDO-mediated mechanism that increases Treg activity., Competing Interests: The authors have declared that no competing interests exist.

Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI-1, acyl homoserine lactone (AHL) common to gram-negative bacteria; however, it has a receptor for AI-1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs-1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12-HSL, increased biofilm formation and promoted expression of biofilm formation genes (lpfA, fimF, fliF, glgC) and virulence genes (hilA, invA, invF). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: The goals of the present study were to identify, to analyse the phylogenetic relations and to evaluate the genetic variability in Diaporthe endophytic isolates from common bean., Methods and Results: Diaporthe sp., D. infecunda and D. phaseolorum strains were identified using multilocus phylogeny (rDNA ITS region; EF1-α, β-tubulin, and calmodulin genes). IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) molecular markers reveal the existence of high genetic variability, especially among D. infecunda isolates., Conclusions: It was concluded that the multilocus phylogenetic approach was more effective than individual analysis of ITS sequences, in identifying the isolates to species level, and that IRAP and REMAP markers can be used for studying the genetic variability in the genus Diaporthe particularly at the intraspecific level., Significance and Impact of the Study: The combined use of molecular tools such as multilocus phylogenetic approach and molecular markers, as performed in this study, is the best way to distinguish endophytic strains of Diaporthe isolated from common bean (Phaseolus vulgaris L.)., (© 2015 The Society for Applied Microbiology.)

Paracoccidioidomycosis (PCM), is a pulmonary fungal disease whose severity depends on the adequate development of T cell immunity. Although regulatory T (Treg) cells were shown to control immunity against PCM, deleterious or protective effects were described in different experimental settings. To clarify the function of Treg cells in pulmonary PCM, loss-and gain-of-function approaches were performed with Foxp3GFP knock-in mice and immunodeficient Rag1-/- mice, respectively, which were intratracheally infected with 106 yeast cells. The activity of Foxp3-expressing Treg cells in pulmonary PCM was determined in Foxp3GFP transgenic mice. First, it was verified that natural Treg cells migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, adoptive transfer of diverse T cell subsets to Rag1-/- mice subsequently infected by the pulmonary route demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone reduced fungal growth and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Treg cells combined with CD4+Foxp3- T cells generated more efficient and balanced immune Th1/Th2/Th17 responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and increased survival rates. Altogether, these loss- and gain-of-function approaches allow us to clearly demonstrate the dual role of Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation.

Endophytic bacteria play a key role in the biocontrol of phytopathogenic microorganisms. In this study, genotypic diversity was analyzed via repetitive element PCR (rep-PCR) of endophytic isolates of the phylum Actinobacteria that were previously collected from leaves of cultivars of common bean (Phaseolus vulgaris). Considerable variability was observed, which has not been reported previously for this phylum of endophytic bacteria of the common bean. Furthermore, the ethanol extracts from cultures of various isolates inhibited the growth of pathogenic bacteria in vitro, especially Gram-positive pathogens. Extracts from cultures of Microbacterium testaceum BAC1065 and BAC1093, which were both isolated from the 'Talismã' cultivar, strongly inhibited most of the pathogenic bacteria tested. Bean endophytic bacteria were also demonstrated to have the potential to inhibit the quorum sensing of Gram-negative bacteria. This mechanism may regulate the production of virulence factors in pathogens. The ability to inhibit quorum sensing has also not been reported previously for endophytic microorganisms of P. vulgaris. Furthermore, M. testaceum with capacity to inhibit quorum sensing appears to be widespread in common bean. The genomic profiles of M. testaceum were also analyzed via pulsed-field gel electrophoresis, and greater differentiation was observed using this method than rep-PCR; in general, no groups were formed based on the cultivar of origin. This study showed for the first time that endophytic bacteria from common bean plants exhibit high variability and may be useful for the development of strategies for the biological control of diseases in this important legume plant.

Dectin-1 is a pattern recognition receptor (PRR) that recognizes β-glucans and plays a major role in the immunity against fungal pathogens. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. To investigate the role of dectin-1 in the innate immunity of resistant (A/J) and susceptible (B10.A) mice to P. brasiliensis infection, we evaluated the role of curdlan (a dectin-1 agonist) and laminarin (a dectin-1 antagonist) in the activation of macrophages from both mouse strains. We verified that curdlan has a negligible role in the activation of B10.A macrophages but enhances the phagocytic and fungicidal abilities of A/J macrophages. Curdlan up-regulated the expression of costimulatory molecules and PRRs in A/J macrophages that express elevated levels of dectin-1, but not in B10.A cells. In addition, curdlan treatment inhibited arginase-1 and enhanced NO-synthase mRNA expression in infected A/J macrophages but had not effect in B10.A cells. In contrast, laminarin reinforced the respective M2/M1 profiles of infected A/J and B10.A macrophages. Following curdlan treatment, A/J macrophages showed significantly higher Syk kinase phosphorylation and expression of intracellular pro-IL-1β than B10.A cells. These findings led us to investigate if the NRLP3 inflammasome was differently activated in A/J and B10.A cells. Indeed, compared with B10.A cells A/J macrophages showed an increased expression of NALP3, ASC, and IL-1β mRNA. They also showed elevated caspase-1 activity and secreted high levels of mature IL-β and IL-18 after curdlan treatment and P. brasiliensis infection. Our data demonstrate that soluble and particulate β-glucans exert opposed modulatory activities on macrophages of diverse genetic patterns. Moreover, the synergistic action of dectin-1 and NALP3 inflammasome were for the first time associated with the innate response of resistant hosts to P. brasiliensis infection.

Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15 kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag gene is constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.

Salmonella spp are among the main causative agents of foodborne diseases. Some phenotypes associated with increased drug resistance and virulence are regulated by quorum sensing (QS). In the present study, the autoinducer (AI)-1- and -2-mediated QS mechanisms were characterized in Salmonella enterica serovar Enteritidis PT4 for the first time. Salmonella Enteritidis did not produce AI-1. Phylogenetic analysis of nucleotides encoding the SdiA protein, the response regulator of AI-1-mediated QS, and comparative alignment of its amino acids showed that the gene and protein are conserved within the same bacterial genus. Thus, bacteria of the same genus respond to the same AIs. However, this finding did not preclude the possibility that Salmonella Enteritidis might respond to AIs released from bacteria of a different genus, which might confer a competitive advantage to this pathogen. We found that the regulation of AI-2-mediated QS in Salmonella Enteritidis is similar to that in serovar Typhimurium. The elucidation of the AI-1- and AI-2-mediated QS mechanisms in Salmonella Enteritidis will contribute to the development of new control strategies for this pathogen by indicating new targets for antimicrobial drugs.

The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the mannose receptor (MR) are C-type lectin receptors (CLRs) previously reported to cooperate with toll-like receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4(+) and CD8(+) T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1, and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, wild type (WT), Dectin-1(-/-), TLR-2(-/-), and TLR-4(-/-) DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4(+) and CD8(+) T cells. In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR-4, and MR.

Aims: To evaluate the diversity of endophytic fungi from the leaves of the common bean and the genetic diversity of endophytic fungi from the genus Colletotrichum using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) analyses., Methods and Results: The fungi were isolated by tissue fragmentation and identified by analysing the morphological features and sequencing the internal transcribed spacer (ITS) regions and the rDNA large subunit (LSU). Twenty-seven different taxa were identified. Colletotrichum was the most commonly isolated genera from the common bean (32.69% and 24.29% of the total isolates from the Ouro Negro and Talismã varieties, respectively). The IRAP and REMAP analyses revealed a high genetic diversity in the Colletotrichum endophytic isolates and were able to discriminate these isolates from the phytopathogen Colletotrichum lindemuthianum., Conclusions: Fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris endophytic community, and the IRAP and REMAP markers can be used to rapidly distinguish between C. lindemuthianum and other Colletotrichum members that are frequently found as endophytes., Significance and Impact of the Study: This is the first report of the diversity of endophytic fungi present in the common bean and the use of IRAP and REMAP markers to assess the genetic diversity of endophytic fungi from the genus Colletotrichum., (© 2014 The Society for Applied Microbiology.)

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages.

The phy gene, which encodes a phytase in Penicillium chrysogenum CCT 1273, was cloned into the vector pAN-52-1-phy and the resulting plasmid was used for the cotransformation of Penicillium griseoroseum PG63 protoplasts. Among the 91 transformants obtained, 23 were cotransformants. From there, the phytase activity of these 23 transformants was evaluated and P. griseoroseum T73 showed the highest. The recombinant strain P. griseoroseum T73 contained the phy gene integrated in at least three sites of the genome and showed a 5.1-fold increase in phytase activity in comparison to the host strain (from 0.56 ± 0.2 to 2.86 ± 0.4 U μg protein(-1)). The deduced PHY protein has 483 amino acids; an isoelectric point (pI) higher than that reported for phytases from filamentous fungi (7.6); higher activity at pH 2.0 (73%), pH 5.0 (100%) and 50 °C; and is stable at pH values 3.0-8.0 and temperatures 70-80 °C. PHY produced by the recombinant strain P. griseoroseum T73 was stable after four weeks of storage at -20, 8 and 25 °C and was effective in releasing Pi, especially from soybeans. The data presented here show that P. griseoroseum is a successful host for expression of heterologous protein and suggest the potential use of PHY in the animal nutrition industry., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre-symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre-symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4-fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23-fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT-PCR 30 days after fungus-plant contact, suggesting that RAS-mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation., Methodology/principal Findings: In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology., Conclusions/significance: Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation.

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Background: Sclerotinia sclerotiorum is a necrotrophic fungus that is pathogenic to many plants. Genomic analysis of its revealed transposable element expansion that has strongly influenced the evolutionary trajectory of several species. Transposons from the Tc1-Mariner superfamily are thought to be ubiquitous components of fungal genomes and are generally found in low copy numbers with large numbers of deleterious mutations in their transposase coding sequence., Results: This study shows that the genome of S. sclerotiorum has a large number of copies of Tc1-Mariner transposons, and in silico analysis shows evidence that they were recently active. This finding was confirmed by expressed sequence tag (EST) analysis. Fourteen new Tc1-Mariner transposon families that were distributed throughout the genome were identified, and in some cases, due to the excision/retention of introns, different transcripts were observed for the same family, which might be the result of an efficient strategy to circumvent mutations that generate premature stop codons in the RNA sequence. In addition, the presence of these introns shows that the transposase protein has a flexible coding sequence and, consequently, conformation. No evidence for RIP-like gene silencing mechanisms, which are commonly found in fungi, was found in the identified Tc1-Mariner elements, and analysis of the genomic insertion sites of these elements showed that they were widely distributed throughout the genome with some copies located near the 3' regions of genes. In particular, EST analysis demonstrated that one of these copies was co-expressed with a gene, which showed the potential for these elements to undergo exaptation., Conclusions: Fourteen novel Tc1-Mariner families were characterized. Some families had evidence of introns, which might or might not be excised depending on the family or element in question, and this finding demonstrates a possible strategy for overcoming possible mutations that generate premature stop codons in a RNA sequence. Tc1-Mariner elements likely play an important role in the structure and evolution of the S. sclerotiorum genome.

The fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8-31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes.

Dectin-1, the innate immune receptor that recognizes β-glucan, plays an important role in immunity against fungal pathogens. Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. This fact motivated us to use dectin-1-sufficient and -deficient mice to investigate the role of β-glucan recognition in the immunity against pulmonary paracoccidioidomycosis. Initially, we verified that P. brasiliensis infection reinforced the tendency of dectin-1-deficient macrophages to express an M2 phenotype. This prevalent antiinflammatory activity of dectin-1(-/-) macrophages resulted in impaired fungicidal ability, low nitric oxide production, and elevated synthesis of interleukin 10 (IL-10). Compared with dectin-1-sufficient mice, the fungal infection of dectin-1(-/-) mice was more severe and resulted in enhanced tissue pathology and mortality rates. The absence of dectin-1 has also impaired the production of T-helper type 1 (Th1), Th2, and Th17 cytokines and the activation and migration of T cells to the site of infection. Remarkably, dectin-1 deficiency increased the expansion of regulatory T cells and reduced the differentiation of T cells to the IL-17(+) phenotype, impairing the migration of IL-17(+)CD8(+) T cells and polymorphonuclear cells to infected tissues. In conclusion, dectin-1 exerts an important protective role in pulmonary paracoccidioidomycosis by controlling the innate and adaptive phases of antifungal immunity., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen., (© 2014 by The Mycological Society of America.)

Background: Cochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ~ 1.2-Mb of DNA encoding genes responsible for the production of T-toxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species., Results: A detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions., Conclusions: New families of transposons were identified. In C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus.

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.

A number of parameters, including culture medium pH, affect growth and enzyme production by microorganisms. In the present study, the production and secretion of pectin lyase (PL) and polygalacturonase (PG) by recombinant strains of Penicillium griseoroseum cultured in mineral-buffered media (MBM; initial pH 6.8) and mineral-unbuffered medium (MUM; initial pH 6.3) were evaluated. Under these culture conditions, no change in the transcriptional levels of plg1 and pgg2 was observed. However, the levels of secreted total protein ranged from 7.80 ± 1.1 to 3.25 ± 1.50 µg ml(-1) in MBM and MUM, respectively, and were evaluated by SDS-PAGE. PL and PG enzymatic activities decreased 6.4 and 3.6 times, respectively, when P. griseoroseum was cultivated under acidic pH conditions (MUM). Furthermore, differences were observed in the hypha and mycelium morphology. These findings suggest that acidic growing conditions affect PL and PG secretion, even though the transcription and translation processes are successful. The data obtained in this study will help to establish optimal culture conditions that increase production and secretion of recombinant proteins by filamentous fungi., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Chagas disease is a major cause of cardiomyopathy and sudden death in our country. It has a high mortality when their patients develop New York Heart Association (NYHA) class IV., Objective: The objective of this study is to analyze the clinical outcome of patients with Chagas' cardiomyopathy with congestive heart failure with optimized pharmacological therapy, undergoing cardiac resynchronization therapy., Methods: Between January 2004 and February 2009, 72 patients with Chagas' cardiomyopathy in NYHA class III and IV underwent cardiac resynchronization therapy and were monitored to assess their clinical evolution. We used the t test or the Wilcoxon test to compare the same variable in two different times. A P value < 0.05 was established as statistically significant., Results: The average clinical follow-up was 46.6 months (range 4-79 months). At the end of the evaluation, 87.4% of patients were in NYHA class I or II (P<0.001). There was response to therapy in 65.3% of patients (P<0.001), with an overall mortality of 34.7%., Conclusion: In patients with chronic Chagas cardiomyopathy undergoing cardiac resynchronization therapy, we found the following statistically significant changes: improvement in NYHA class and increase of left ventricle ejection fraction, a decrease of the systolic final diameter and systolic final left ventricle volume and improvement of patient survival.

Background: Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM., Methodology/principal Findings: Wild type (WT) and IL-10(-/-) C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10(-/-) mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10(-/-) and WT mice were i.t. infected with 1×10(6) Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10(-/-) mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10(-/-) mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4(+) and CD8(+) T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10(-/-) mice., Conclusions/significance: Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.

In this study, we demonstrate that ClIRAP primers designed using the transposable element RetroCl1 sequence from Colletotrichum lindemuthianum can be used to generate an efficient IRAP (inter-retrotransposon amplified polymorphism) molecular marker to study intra- and inter-species diversity in fungi. It has been previously demonstrated that primers generated from this TRIM-like element can be used in the Colletotrichum species. We now prove that the RetroCl1 sequence can also be used to analyze diversity in different fungi. IRAP profiles were successfully generated for 27 fungi species from 11 different orders, and intra-species genetic variability was detected in six species. The ClIRAP primers facilitate the use of the IRAP technique for a variety of fungi without prior knowledge of the genome., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: In this study, a gene that encodes a carboxylesterase (carb) in Penicillium expansum GF was cloned, sequenced and overexpressed by Penicillium griseoroseum PG63, and the enzyme was characterized., Methods and Results: The recombinant strain, P. griseoroseum T55, obtained upon transformation using the plasmid pAN-52-1-carb, showed integration of the carb gene into at least two heterologous sites of the genome by Southern blotting. Furthermore, the recombinant strain T55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG63 strain when both were cultured without inducers. Based on the secondary structure and multiple sequence alignments with carboxylesterases, cholinesterase and lipase, a three-dimensional model was obtained. The α/β barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with Ser(213)-Glu(341)-His(456) as the putative catalytic triad. CARB preferentially hydrolysed acyl chains with eight carbon atoms, and its activity was optimal at a pH of 7·0 and a temperature of 25°C. CARB exhibited stability in alkaline pH, high activity under mesophilic conditions and stability in organic solvents., Conclusion: The CARB protein is potentially useful in bioremediation, food and chemical/pharmaceutical industries., Significance and Impact of the Study: This study is the first to report the development of a recombinant strain superproducing a Penicillium sp. carboxylesterase., (Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.)

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.

Colletotrichum lindemuthianum is the causal agent of plant bean anthracnose, one of the most important diseases affecting the common bean. We investigated the structure and expression of the nit1 gene (nitrate reductase) of C. lindemuthianum. The nit1 gene open reading frame contains 2787 bp, interrupted by a single 69-bp intron. The predicted protein has 905 amino acids; it shows high identity with the nitrate reductase of C. higginsianum (79%) and C. graminicola (73%). Expression of nit1 in C. lindemuthianum was evaluated in mycelia grown on different nitrogen sources under conditions of activation and repression. The gene was expressed after 15 min of induction with nitrate, reaching maximum expression at 360 min. The transcription was repressed in mycelia grown in media enriched with ammonia, urea or glutamine. Twenty nit1⁻ mutants were obtained in a medium treated with chlorate. Ten of these mutants were characterized by DNA hybridization, which identified point mutations, a deletion and an insertion. These rearrangements in the nit1 gene in the different mutants may have occurred through activity of transposable elements.

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques - fragment plating and the innovative dilution-to-extinction culturing - to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill's index, N1. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.

Fungi of the Colletotrichum genus are among the most prominent phytopathogens that cause diseases with a considerable economic impact, such as anthracnose. The hemibiotrophic fungus Colletotrichum lindemuthianum (teleomorph Glomerella cingulata f. sp. phaseoli) is the causal agent of the anthracnose of the common bean; and similarly to other phytopathogens, it uses multiple strategies to gain access to different carbon sources from its host. In this study, we examine mfs1, a newly identified C. lindemuthianum hexose transporter. The mfs1 gene is expressed only during the necrotrophic phase of the fungus' interaction within the plant and allows it to utilize the available sugars during this phase. The deletion of mfs1 gene resulted in differential growth of the fungus in a medium that contained glucose, mannose or fructose as the only carbon source. This study is the first to describe a hexose transporter in the hemibiotrophic pathogen C. lindemuthianum and to demonstrate the central role of this protein in capturing carbon sources during the necrotrophic development of the plant/pathogen interaction.

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.

Background: Mycosphaerella fijiensis is a ascomycete that causes Black Sigatoka in bananas. Recently, the M. fijiensis genome was sequenced. Repetitive sequences are ubiquitous components of fungal genomes. In most genomic analyses, repetitive sequences are associated with transposable elements (TEs). TEs are dispersed repetitive DNA sequences found in a host genome. These elements have the ability to move from one location to another within the genome, and their insertion can cause a wide spectrum of mutations in their hosts. Some of the deleterious effects of TEs may be due to ectopic recombination among TEs of the same family. In addition, some transposons are physically linked to genes and can control their expression. To prevent possible damage caused by the presence of TEs in the genome, some fungi possess TE-silencing mechanisms, such as RIP (Repeat Induced Point mutation). In this study, the abundance, distribution and potential impact of TEs in the genome of M. fijiensis were investigated., Results: A total of 613 LTR-Gypsy and 27 LTR-Copia complete elements of the class I were detected. Among the class II elements, a total of 28 Mariner, five Mutator and one Harbinger complete elements were identified. The results of this study indicate that transposons were and are important ectopic recombination sites. A distribution analysis of a transposable element from each class of the M. fijiensis isolates revealed variable hybridization profiles, indicating the activity of these elements. Several genes encoding proteins involved in important metabolic pathways and with potential correlation to pathogenicity systems were identified upstream and downstream of transposable elements. A comparison of the sequences from different transposon groups suggested the action of the RIP silencing mechanism in the genome of this microorganism., Conclusions: The analysis of TEs in M. fijiensis suggests that TEs play an important role in the evolution of this organism because the activity of these elements, as well as the rearrangements caused by ectopic recombination, can result in deletion, duplication, inversion and translocation. Some of these changes can potentially modify gene structure or expression and, thus, facilitate the emergence of new strains of this pathogen.

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris) leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10(2) to 2.8 x 10(3) CFU g(-1) of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

Planting resistant varieties is the most effective control measure against the angular leaf spot of dry beans, a fungal disease caused by Pseudocercospora griseola. However, dry bean varieties with durable resistance are not easily obtained. Knowledge about the genetic variability of the pathogen population is key for the success of dry bean breeding programs aimed at developing resistant materials, but finding suitable operationally simple and genetically accurate markers is not an easy task. The aim of this study was to assess the suitability of the ISSR-PCR technique to quantify the genetic variability of P. griseola isolates. Total DNA of 27 P. griseola isolates from Goiás, Minas Gerais, Espírito Santo, and Paraná States was extracted and amplified using specific primers for ISSR. Using cluster analysis, 27 genotypes were identified. The ISSR-PCR technique was suitable for assessing intraspecific variability of P. griseola. The ISSR-PCR marker was found to be highly sensitive to genetic variation and can aid in elucidating the genetic structure of the population of this plant pathogen as a support tool for the dry bean breeding programs.