Cunninghamella echinulata DSM1905 biofilm-based L-asparaginase production in pneumatically-driven bioreactors.

We evaluated by comparing the performance of three pneumatically-driven bioreactors in the production of L-asparaginase (L-ASNase), an enzyme used to treat leukaemia and lymphoma. A two-step screening process was conducted to detect Cunninghamella spp. strains producing L-ASNase. Cunninghamella echinulata DSM1905 produced the highest levels of L-ASNase during screening assays. Subsequently, fermentations were performed in bubble column (BCR), airlift (ALR), and hybrid fixed-bed airlift (FB-ALR) bioreactors to determine the best upstream bioprocess. Mycelial biomass production was higher in BCR than in ALR and FB-ALR (p ≤ 0.0322). The activity of L-ASNase produced in FB-ALR, in which the fungus grew as a consistent biofilm, was significantly higher (p ≤ 0.022) than that from ALR, which was higher than that of BCR (p = 0.036). The specific activity of ALR and FB-ALR presented no differences (p = 0.073), but it was higher than that of BCR (p ≤ 0.032). In conclusion, C. echinulata DSM1905, grown under the biofilm phenotype, produced the highest levels of L-ASNase, and FB-ALR was the best upstream system for enzyme production.

Metabolomics of the interaction between a consortium of entomopathogenic fungi and their target insect: Mechanisms of attack and survival.

One of the most concerning pests that attack strawberries in Brazil is Duponchelia fovealis (Zeller), a non-native moth with no registered control methods to date. Our group recently observed that a fungal consortium formed by two strains of Beauveria bassiana (Balsamo) increased the mortality of D. fovealis more than inoculation with each strain on its own. However, the molecular interaction between the fungal consortium and the caterpillars is unknown. Thus, in this work, we sought to pioneer the evaluation of the molecular interaction between a fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to understand the biocontrol process involved in this interaction and the defense system of the caterpillar. Seven days after D. fovealis were inoculated with the consortium, the dead and surviving caterpillars were analyzed using GC-MS and LC-MS. Some of the metabolites identified in dead caterpillars have primarily antioxidant action. Other metabolites may have insecticidal potential, such as diltiazem-like and tamsulosin-like compounds, as well as 2,5-dimethoxymandelic acid. In surviving caterpillars, the main mechanisms are pro-inflammatory from 2-Palmitoylglycerol metabolite and the antifungal action of the metabolite Aegle marmelos Alkaloid-C. The metabolites identified in dead caterpillars may explain the increased mortality caused by the consortium due to its antioxidant mechanism, which can suppress the caterpillars' immune system, and insecticide action. In surviving caterpillars, the main resistance mechanisms may involve the stimulus to the immunity and antifungal action.

Antifungal activity of essential oils and their combinations against storage fungi.

We aimed to evaluate the fungicidal activity of essential oils (EOs) from Baccharis dracunculifolia (Asteraceae), Baccharis uncinella (Asteraceae), Mentha arvensis (Lamiaceae), Salvia officinalis (Lamiaceae), Melaleuca alternifolia (Myrtaceae), and Cymbopogon nardus (Poaceae) in the in vitro control of mycotoxin-producing strains of Aspergillus niger, Aspergillus nomius, Aspergillus flavus, and Fusarium graminearum. EOs' chemical composition was analyzed by gas chromatography-mass spectrometry, and a total of 19, 21, 18, 20, 17, and 15 compounds were identified in B. dracunculifolia, B. uncinella, S. officinalis, M. arvensis, M. alternifolia, and C. nardus EOs, respectively. Contact and volatilization bioassays were performed, for which M. alternifolia and C. nardus EOs had the greatest fungicidal effect (> 90%). Therefore, these EOs were evaluated for minimum inhibitory concentration, medium inhibitory concentration, and sporulation. Effects from the combined use of EOs were also evaluated. EOs interacted in combination, displaying an additive effect against F. graminearum and A. flavus and an antagonistic effect against the remaining isolates. We conclude that C. nardus EO was effective in the control of storage pathogens and that combined EOs can improve their antifungal effects.

Fungal consortium of two Beauveria bassiana strains increases their virulence, growth, and resistance to stress: A metabolomic approach.

The use of two or more microorganisms in a microbial consortium has been increasingly applied in the biological control of diseases and pests. Beauveria bassiana is one of the most widely studied fungal species in biological control, yet little is known about its role in fungal consortiums. In a previous study, our group found that a consortium formed by two strains of B. bassiana had significantly greater biocontrol potential against the polyphagous caterpillars Duponchelia fovealis (Lepidoptera: Crambidae) than either strain on its own. In this study, we use GC-MS and LC-MS/MS to evaluate and discuss the metabolomics of the consortium. A total of 21 consortium biomarkers were identified, corresponding to 14 detected by LC-MS/MS and seven by GC-MS. Antioxidant and anti-inflammatory mechanisms are the main properties of the metabolites produced by the consortium. These metabolites can depress the insect's immune system, increasing its vulnerability and, hence, the fungal virulence of the consortium. In light of these results, we propose an action model of insect mortality due to the metabolites secreted by the consortium. The model includes the inhibition of defense mechanisms such as pro-inflammatory interleukin secretion, cell migration, cell aggregation, Dif, Dorsal and Relish gene transcription, and JAK/STAT and JNK signaling pathways. It also promotes the cleaning of oxidative molecules, like ROS, NOS, and H2O2, and the induction of virulence factors.

Streptomyces spp. Isolated from Marine and Caatinga Biomes in Brazil for the Biological Control of Duponchelia fovealis.

Actinobacteria have been drawing attention due to their potential for the development of new pest control products. We hereby assess the effects of Streptomyces isolated from marine and caatinga biomes against Duponchelia fovealis Zeller (Lepidoptera: Crambidae), a pest associated with the strawberry culture at a global scale. To this end, eggs deposited by adults were immersed for 5 s in a bacterial suspension, and the larvae were fed on leaflets placed in glass tubes containing bacterial suspensions. In both treatments, the control was a saline solution. The bioassays demonstrated that the Streptomyces strains were able to cause the death of D. fovealis eggs (≈ 40%) and larvae (≈ 65%) compared to untreated eggs (1.4%) and larvae (2.0%). The crude extract of strain T49 and the chitinase extract of strain T26 affected larval growth when applied directly to the thorax of first-instar larvae (larval-adult lifespan of 65.3 ± 0.5 days and 67.5 ± 0.7 days, respectively; survival of 61.2 ± 1.2%) in relation to the control treatment (larval-adult lifespan of 41.75 ± 0.2 days and survival of 83.7 ± 2.6%). The Streptomyces spp. strains T41, T49, and T50 caused antifeeding activity. Apart from larval mortality, the adults that emerged from the larvae exposed to the extracts presented morphological abnormalities, and the moths' chitin spectra showed clear alterations to the pupa and wings. Our studies show, for the very first time, that Streptomyces isolated from the marine environment and the Caatinga biome are effective at provoking the mortality of D. fovealis and are promising agents for developing new products with biological control properties.

Isolation of Beauveria Strains and Their Potential as Control Agents for Lema bilineata Germar (Coleoptera: Chrysomelidae).

Lema bilineata Germar (Coleoptera: Chrysomelidae) was recently reported to damage Physalis peruviana crops in Brazil. Given the potential for inflicting damage on other Solanaceae species and the lack of alternatives for controlling this pest, we assessed the pathogenicity of 15 Beauveria isolates against L. bilineata adults in vitro. In addition, three of these isolates were tested for their ovicidal effect against L. bilineata eggs. Fungal strains were isolated from mummified corpses of L. bilineata collected in a non-commercial field in Paraná, Brazil. The isolates were identified as Beauveria bassiana using molecular markers. Lema bilineata adults were susceptible to conidial suspensions of all these isolates at a concentration of 108 conidia mL-1. Deaths caused by fungal extrusion were confirmed. Three strains were found to be more virulent against L. bilineata adults and showed ovicidal effects. This is the first study on entomopathogenic fungi isolated from dead insects collected from P. peruviana crops and tested against L. bilineata carried out in Brazil. The results obtained in the laboratory indicate the high potential of the use of three B. bassiana strains against L. bilineata as a biocontrol agent.

Unveiling Xylanolytic Enzymes Production of Talaromyces wortmannii DR49 on Industrial Agro Wastes

Abstract Xylan degradation is an important step in different industries, such as in biorefinery for biomass hydrolysis. Talaromyces wortmannii is a known fungus due to second metabolite production but only few works showed the xylanolytic potential of this fungus. In this way, the aim of this study was to evaluate the production of xylanolytic enzymes from T. wortmannii DR49 on industrial agro wastes. Cultivation in shake flask showed highest xylanase titration (10.3 U/mL; 9.5 U/mL) for wheat bran (WB) and hydrothermal pretreated sugar cane bagasse (HB); in β-xylosidase production WB and xylose were the best carbon sources (0.57 U/mL; 0.34 U/mL) respectively. STR cultivation revealed that 29°C and pH 6.0 were the best conditions for xylanase (14.5 U/mL) and β-xylosidase (1.7 U/mL) production. T. wortmannii DR49 showed to be a potential candidate for xylanolytic enzymes production using agro wastes in bioreactors, which has never been previously reported in this fungus.

Compatibility between Entomopathogenic Fungi and Egg Parasitoids (Trichogrammatidae): A Laboratory Study for Their Combined Use to Control Duponchelia fovealis.

The European pepper moth, Duponchelia fovealis (Lepidoptera: Crambidae), is a key pest in strawberry production. Entomopathogenic fungi (EF) and parasitoids of the Trichogrammatidae family are effective biological control agents of this pest with the potential to be used jointly for improved efficacy. This study aims to evaluate the susceptibility of Trichogramma atopovirilia and Trichogramma pretiosum to two Beauveria bassiana strains (B2 and B3) and two commercial bioinsecticides (Bovemax® and Methamax®) by applying them to D. fovealis eggs in pre- and post-parasitism periods. Pre-parasitism application of B2 and B3 did not affect the percentage of D. fovealis eggs parasitized by either Trichogramma species, except in the case of T. atopovirilia when eggs were sprayed with B3 at 1.5 × 105 conidia mL-1 (16.7% less than the control). In contrast, eggs sprayed with 1.5 × 108 conidia mL-1 of the commercial bioinsecticides were not parasitized by any Trichogramma species. Overall, the EF tested reduced the parasitism rate, adult emergence, and longevity of Trichogramma adults by less than 30% in all cases. The adverse effects of the B. bassiana strains and commercial products on the biological traits of both Trichogramma species were minimal, meaning that these agents can be used jointly in D. fovealis control strategies.

Inhibition of growth and ochratoxin A production in Aspergillus species by fungi isolated from coffee beans.

Ochratoxin A (OTA) is a mycotoxin found in several agricultural commodities. Produced by Aspergillus spp., it is nephrotoxic and hepatotoxic and can be carcinogenic. Preventive measures are preventing fungal growth and OTA production. In this study, fungal strains (Rhizopus oryzae, Lichtheimia ramosa, Aspergillus westerdijkiae, Aspergillus niger, Aspergillus tamarii, Aspergillus sp., and Aspergillus fumigatus) isolated from coffee beans were identified for their abilities to inhibit the growth of Aspergillus ochraceus, Aspergillus westerdijkiae, Aspergillus carbonarius, and Aspergillus niger, and OTA production. All fungi strains tested were able to inhibit growth of the four Aspergillus species and OTA production, where A. niger showed the best results in both tests. L. ramosa showed the lowest growth-reducing potential, while the other fungal strains had a growth-reducing potential higher than 70% against all Aspergillus species tested. Regarding OTA production, L. ramosa and Aspergillus sp. completely inhibited the mycotoxin production by A. ochraceus and non-toxigenic strain A. niger completely inhibited OTA production by A. niger. Our findings indicate that the strains tested can be used as an alternative means to control growth of OTA-producing fungi and production of the mycotoxin in coffee beans.

Microbiological quality of Brazilian artisanal cheese and fermented sausages / Qualidade microbiológica de queijos e salames artesanais brasileiros

Cheeses and sausages are ready-to-eat foods, and the products prepared by artisanal process are susceptible to microbial contamination. Few studies on the quality of foods from different Brazilian regions have been done. In this context, this study aimed at evaluating the microbiological quality of 32 cheese and 13 sausages samples purchased from artisanal food stores or producers markets located in the metropolitan areas of 10 capital cities of Brazilian states. Microbiological analyses were performed to determine the counts of microbial contamination indicators, including Escherichia coli and coagulase-positive staphylococci, as well as for evaluating the presence of Listeria monocytogenes and Salmonella spp. using bacteriological methods. E. coli was detected in 50.0 % of samples, coagulase-positive staphylococci in 34.4 %, and Salmonella spp. in 6.3 % of cheese samples. In fermented sausage samples, coagulase-positive staphylococci were isolated from 23.1 % samples and Salmonella spp. from 7.7 %. According to the Brazilian Sanitary Legislation, 63.0 % of cheese samples and 23.0 % of artisanal fermented sausage samples from the metropolitan areas of 10 Brazilian capital cities were unsuitable for consumption, indicating the importance of conducting the close monitoring of these foods and the application of the effective measures for preventing food-borne outbreaks.

Queijos e salames são alimentos prontos para consumo e quando artesanalmente produzidos são suscetíveis à contaminação microbiana. A importância deste estudo em dez capitais brasileiras deve-se à carência de dados obtidos simultaneamente abrangendo-se diferentes regiões do Brasil. O objetivo do presente trabalho foi de avaliar a qualidade microbiológica de 32 amostras de queijos e 13 de salames artesanais, adquiridos em casas de produtos artesanais ou feiras de produtores nas regiões metropolitanas de dez capitais brasileiras. Análises microbiológicas com as respectivas contagens de indicadores de contaminação microbiana, Escherichia coli e Staphylococcus coagulase positiva, e a pesquisa de Listeria monocytogenes e Salmonella spp. foram realizadas por meio de métodos bacteriológicos. Nas amostras de queijos foram observadas E. coli em 50,0 %, Staphylococcus coagulase positiva em 34,4 % e Salmonella spp. em 6,3 %. Nas amostras de salames foram detectadas Staphylococcus coagulase positiva em 23,1 % e Salmonella spp. em 7,7 %. De acordo com a legislação sanitária brasileira, 63,0 % das amostras de queijos e 23,0 % das amostras de salames artesanais coletadas da Região Metropolitana de dez capitais brasileiras estavam impróprias para o consumo, o que demonstra a importância de realizar monitoramento próximo e efetivo para prevenir surtos de origem alimentar.

Microbiological quality of Brazilian artisanal cheese and fermented sausages / Qualidade microbiológica de queijos e salames artesanais brasileiros

Cheeses and sausages are ready-to-eat foods, and the products prepared by artisanal processare susceptible to microbial contamination. Few studies on the quality of foods from different Brazilian regions have been done. In this context, this study aimed at evaluating the microbiological quality of 32 cheese and 13 sausages samples purchased from artisanal food stores or producers markets located in the metropolitan areas of 10 capital cities of Brazilian states. Microbiological analyses were performed to determine the counts of microbial contamination indicators, including Escherichia coli and coagulase-positive staphylococci, as well as for evaluating the presence of Listeria monocytogenes and Salmonella spp. using bacteriological methods.E. coli was detected in 50.0 % of samples, coagulase-positive staphylococci in 34.4 %, and Salmonella spp. in 6.3 % of cheese samples. In fermented sausage samples, coagulase-positive staphylococci were isolated from 23.1 % samples and Salmonella spp. from 7.7 %. According tothe Brazilian Sanitary Legislation, 63.0 % of cheese samples and 23.0 % of artisanal fermentedsausage samples from the metropolitan areas of 10 Brazilian capital cities were unsuitable for consumption, indicating the importance of conducting the close monitoring of these foodsand the application of the effective measures for preventing food-borne outbreaks.

Queijos e salames são alimentos prontos para consumo e quando artesanalmente produzidos são suscetíveis à contaminação microbiana. A importância deste estudo em dez capitais brasileiras deve-se à carência de dados obtidos simultaneamente abrangendo-se diferentes regiões do Brasil. O objetivo do presente trabalho foi de avaliar a qualidade microbiológica de 32 amostras de queijos e 13 de salames artesanais, adquiridos em casas de produtos artesanais ou feiras de produtores nas regiões metropolitanas de dez capitais brasileiras. Análises microbiológicas com as respectivas contagens de indicadores de contaminação microbiana, Escherichia coli e Staphylococcus coagulase positiva, e a pesquisa de Listeria monocytogenes e Salmonella spp. foram realizadas por meio de métodos bacteriológicos. Nas amostras de queijos foram observadas E. coli em 50,0 %, Staphylococcus coagulase positiva em 34,4 % e Salmonella spp. em 6,3 %. Nas amostras de salames foram detectadas Staphylococcus coagulase positiva em 23,1 % e Salmonella spp. em 7,7 %. De acordo com a legislação sanitária brasileira, 63,0 % das amostras de queijos e 23,0 % das amostras de salames artesanais coletadas da Região Metropolitana de dez capitais brasileiras estavam impróprias para o consumo, o que demonstra a importância de realizar monitoramento próximo e efetivo para prevenir surtos de origem alimentar.

Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 µg, streptomycin 10 µg, and ceftazidime 30 µg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

Enhancing of sugar cane bagasse hydrolysis by Annulohypoxylon stygium glycohydrolases.

The aim of this study was to develop a bioprocess for the production of ß-glucosidase and pectinase from the fungus Annulohypoxylon stygium DR47. Media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase at pH 4.0 and 10.13 U/mL of ß-glucosidase at pH 5.0. In addition, the enzymes extracts were able to replace partially Celluclast 1.5L in sugar cane bagasse hydrolysis. Proteomic analysis from A. stygium cultures revealed accessory enzymes, mainly belong to the families GH3 and GH54, that would support enhancement of commercial cocktail saccharification yields. This is the first report describing bioreactor optimization for enzyme production from A. stygium with a view for more efficient degradation of sugar cane bagasse.

Qualidade microbiológica da água e dialisato em clínicas de hemodiálise do Sul do Brasil / Microbiological quality of water and dialysate from haemodialysis units in Southern Brazil

Infectious diseases in renal patients may be associated with the dialysis water quality, which may be contaminated with microorganisms. In Brazil, the water quality is evaluated by analyzing total coliforms, heterotrophic bacteria, and bacterial endotoxin, but not Pseudomonas sp. and fungi. Water samples from haemodialysis units in Curitiba/PR were investigated on their conformity with the standard established by the Brazilian Health Ministry. Total coliforms, heterotrophic bacteria,P. aeruginosa and fungi counts were performed according to APHA, and LAL methodology for detecting bacterial endotoxin. All of the samples showed the total coliforms counts ≤1.1 MPN/100 mL,and ≥ 95 % of analyzed samples complied with the standards for heterotrophic bacteria counting.P. aeruginosa was recovered from 4 % of samples. In 15 % of samples, bacterial endotoxin was detected in values above the limit established by legislation. Yeasts were isolated from 26 % samples and filamentous fungi from 58 %, being 46 % characterized as melanized fungi. The fungi genera were Cladosporium spp., Penicillium spp., Beauveria spp., Exophiala spp., Fusarium spp., Aspergillus spp., Trichoderma spp, Acremonium spp. and Rinocladiella spp.. The study highlights the significance ofP. aeruginosa and fungi detection in those systems, as these microorganisms are potentially pathogenic to immunocompromised patients...

The capability of endophytic fungi for production of hemicellulases and related enzymes.

BACKGROUND: There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G. RESULTS: The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, ß-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial ß-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. CONCLUSIONS: The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

Occurrence of sulphate reducing bacteria (SRB) associated with biocorrosion on metallic surfaces in a hydroelectric power station in Ibirama (SC) - Brazil

The aim of this study was evaluate, two methods for the detection and identification of sulphate reducing bacteria (SRB): ML medium and PCR with specific primers for SRB groups. SRB were detected through the selective medium only on carbon steel, which showed corrosion. Employing specific PCR primer, SBR were detected from all the metallic components assayed, even those that did not present visible corrosion spots, such stainless steel and copper alloys. Despite the presence or absence of corrosion at the later stages effectively by using the selective medium,, the initial stages of the corrosion could only be detected by the amplification of total DNA with SRB specific primers. The early detection of SRB could be employed for preventing the damages on metal surfaces before the installation of corrosion processes. Strategies for reducing the time spent on SRB isolation and identification could be auxiliary tools for controlling the corrosion of materials.

Potential of Fourier transform infrared spectroscopy (FT-IR) to differentiate environmental Aspergillus fungi species A. niger, A. ochraceus, and A. westerdijkiae using two different methodologies.

We assessed the ability of Fourier transform infrared spectroscopy (FT-IR) to differentiate three important and morphologically similar Aspergillus species: A. ochraceus and A. westerdijkiae, and A. niger. Fungi were processed by two methods, powdered mycelia and conidiospore-saline solution, and then recorded in a spectrometer. Second derivatives with nine points of smoothing were applied as spectra data pretreatment. Partial least squares regression was used for the species comparison models and a prediction test was used to evaluate the models. The powdered-mycelia methodology correctly identified 100% of the prediction test set to discriminate A. niger from A. ochraceus and A. westerdijkiae; in addition, it had a 86.6% success rate in discriminating A. ochraceus and A. westerdijkiae. This is the first time a study assessed the ability of FT-IR to differentiate A. niger, A. ochraceus, and A. westerdijkiae, and we believe this technique is very promising for classifying and distinguish fungi isolates.

Black yeasts-like fungi isolated from dialysis water in hemodialysis units.

Hemodialysis in patients with chronic renal failure promotes the removal of toxic substances, water, and minerals from the body and often takes place in specialized clinics. Microbial contamination of dialysis fluid is a serious problem in therapy. One of the sources of contamination is the water used to prepare the dialysate. In Brazil, legislation regulating the microbiological quality of water for dialysis does not cover waterborne microbes such as Pseudomonas, mycobacteria, and fungi. The aim of the present study was to quantify, isolate, and identify fungi present in water systems in six hemodialysis units in Curitiba, Paraná state, Brazil. Fungi were analyzed by surface plating and membrane filtration. Isolates were identified by morphology, while the dematiaceous fungi were identified by sequencing the rDNA ITS region. It was found that 66 % of the samples presented fungi, while black fungi were present in 46 % of all samples. Twenty-eight isolates from treated water for dialysis and dialysate were identified by sequencing and were found to be Exophiala pisciphila, E. cancerae, E. equina, and Rhinocladiella similis. The presence of dematiaceous fungi may pose a risk for debilitated hospitalized patients.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species.

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia ("espinheira-santa") in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Col1 and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.