Ione Parra Barbosa-Tessmann

Decolorization of Industrial Dyes by a Brazilian Strain of Pleurotus pulmonarius Producing Laccase As the Sole Phenol-Oxidizing Enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

The objective of this work was to compare the polymerase chain reaction (PCR) using lesion scrapping with other conventional techniques for the diagnosis of the American tegumentary leishmaniasis (ATL). For this, patients with cutaneous lesions suspected to be ATL were studied. The DNA was amplified with the MP1L/MP3H primers. From the 156 studied patients, 79 (50.6%) presented positive parasite direct search (PD), 81 (51.9%) had positive Montenegro skin test (MST), and 90 (57.7%) presented PD and/or MST positive. The PCR was positive in all of the positive-PD patients (100% sensitivity), in 91.1% of the positive PD and/or MST patients, and in 27.3% of the patients that presented negative PD and positive MST. The PCR positivity was similar to the PD (P = 0.2482) and inferior to the MST (P = 0.0455), and to the PD/MST association (P = 0.0133). The high PCR sensitivity, and positivity in those cases where the PD was negative, highlights the importance of this technique as an auxiliary tool for the diagnosis of ATL.

A new PCR approach for the identification of Fusarium graminearum

The main objective of this work was to develop a PCR protocol for the identification of Fusarium graminearum, based on a pair of primers targeted to a segment of the 3´coding region of the gaoA gene that codes for the enzyme galactose oxidase (GO). This region has low homology with the same region of GO genes from other fungi. Genomic DNA from 17 strains of Fusarium spp. isolated from diseased cereals, from several other Fusarium species, and from other fungi genera was analyzed in a PCR assay using this primer set. The 17 strains of Fusarium spp. were also analyzed for the GO enzyme production in submerse fermentation in a new formulated liquid medium. All strains that were morphologically and molecularly identified as F. graminearum were able to secrete the enzyme and had a positive result in the used PCR protocol. No DNA fragment was amplified using genomic DNA from other Fusarium species and species of other fungi genera. The results suggest that the proposed PCR protocol is specific and can be considered as a new molecular tool for the identification of F. graminearum. In addition, the new formulated medium is a cheap alternative for screening for GO screening production by F. graminearum.

Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536bp was detected from 50ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.

Occurrence of zearalenone in wheat- and corn-based products commercialized in the State of Paraná, Brazil

The productivity of wheat and corn crops depends on climatic conditions and resistance against phytopathogenic fungi such as those of the genus Fusarium. Some species of this genus produce zearalenone (ZEA), a mycotoxin with hyperestrogenic effects. The objective of this study was to investigate the presence of ZEA in samples of cracked wheat (n = 109), popcorn (n = 51) and corn grits (n = 50) commercialized in the State of Paraná, Brazil. Commercial samples of each crop were collected between September 2007 and June 2008 and analyzed by thin-layer chromatography. The method used for detection of the mycotoxin in wheat and corn derivatives presented a recovery rate of 94.5% and 99.5%, respectively, detection limit of 40 μg.kg−1 and quantification limit of 55 μg.kg−1. No contamination with ZEA was detected in cracked wheat samples. Among the corn derivatives, only one cracked corn sample was contaminated with ZEA (64 μg.kg−1). Despite the low contamination observed, monitoring the occurrence of mycotoxins in foods is important to ensure safety.

A new species of Fusarium producer of galactose oxidase

Fifty‐two isolates of Fusarium species and one of Gibberella fujikuroi were tested for galactose oxidase (GO) production. Five Fusarium isolates contained GO activity in the culture filtrate: three F. graminearum and one each F. moniliforme f. sp. subglutinans and F. acuminatum. This is the first time F. acuminatum is reported to be a producer of GO enzyme. GO enzyme activity produced by isolates was assayed through a time course. Moreover, GO protein was partially purified from the most productive four isolates to show that the activity measured in the culture filtrates was due to the presence of GO protein.

Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities

Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.

New PCR Assays for the Identification of Fusarium verticillioides, Fusarium subglutinans, and Other Species of the Gibberella fujikuroi Complex

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.

Identification of new galactose oxidase genes in Fusarium spp.

Galactose oxidase (GO) converts galactose to an aldehyde and has several biotechnological applications, including cancer diagnosis. It is mainly produced by Fusarium austroamericanum but is also produced by Fusarium acuminatum and by isolates of the Fusarium graminearum and Gibberella fujikuroi complexes. The F. austroamericanum GO gaoA gene has been cloned, but the GO genes from other secreting species have not been characterized. Problems associated with the F. austroamericanum GO such as high pI and low catalytic efficiency and thermostability, and the difficult purification process makes the search for homologous genes attractive. In this work, the GO genes from Fusarium verticillioides and Fusarium subglutinans, two species of the G. fujikuroi complex, were cloned, sequenced, and analyzed. New GO genes were found in databases and were used to construct a phylogenetic tree, which revealed the existence of three orthologous lineages of GO genes in Fusarium spp. In addition, RT‐PCR analyses revealed that the new GO cloned gene may be endogenously expressed in F. subglutinans but not in F. verticillioides, in the used culture conditions. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

A deteccao de fungos micotoxigenicos em alimentos e importante porque sua presenca pode indicar uma possivel contaminacao com as micotoxinas associadas. Fusarium graminearum e um patogeno de trigo e um produtor de micotoxinas. A reacao da polimerase em cadeia (PCR) e empregada na identificacao especifica de F. graminearum. No entanto, essa metodologia nao tem sido comumente empregada na deteccao de F. graminearum em alimentos. Assim, o presente trabalho teve como objetivo desenvolver uma metodologia molecular para detectar F. graminearum em amostras comerciais de trigo para quibe. Dois metodos foram testados. No primeiro, uma amostra desse cereal foi contaminada com micelio de F. graminearum. O DNA genomico foi extraido dessa mistura e utilizado em uma reacao de PCR especifica para F. graminearum. A especie F. graminearum foi detectada somente em amostras altamente contaminadas. No segundo metodo, amostras de trigo para quibe foram inoculadas em um meio de cultura solido e isolados com caracteristicas culturais de F. graminearum foram obtidos. O DNA extraido desses isolados foi utilizado em reacoes de PCR especificas para F. graminearum. Um isolado obtido teve o genotipo de producao de tricotecenos determinado por PCR. A metodologia estabelecida poderia ser utilizada em levantamentos de contaminacao de alimentos com F. graminearum.