Márcia Cristina Furlaneto

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

Production and regulation of cuticle-degrading proteases from Beauveria bassiana in the presence of Rhammatocerus schistocercoides cuticle.

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.

A Molecular Method for Detection of Aspergillus carbonarius in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7809, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F809 and OPX7R809) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of Candida spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Development of a simple and rapid Agrobacterium tumefaciens‐mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

Aims:  To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co‐cultivation and to develop an Agrobacterium‐mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers.

Molecular identification of Aspergillus spp. isolated from coffee beans

Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA), a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Parana, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.

Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.

In vitro antifungal activity of the flavonoid baicalein against Candida species.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC(50) (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml(-1). For the three species tested, exposure to baicalein at the MIC(50) concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC(50) (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC(50) produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Genetic variation in the cuticle-degrading protease activity of the entomopathogen Metarhizium flavoviride

Extracellular proteases have been shown to be factors of virulence in fungal entomopathogenicity. We examined the production of the cuticle-degrading extracellular proteases chymoelastase (Pr1) and trypsin (Pr2) in isolates of the fungus Metarhizium flavoviride. Fungal growth was in a mineral medium (MM) containing nitrate, and in MM supplemented with either cuticle from Rhammatocerus schistocercoides or with the non-cuticular substrate casein. The substrates used for growth influenced the expression of both analyzed proteases, the highest protease activities of nearly all isolates having been observed in the medium containing insect cuticle, with more Pr1 than Pr2 being produced. There was a natural variability in the production of cuticle-degrading proteases among isolates, although this was less evident for Pr2. Our data support the hypothesis that the production of Pr1 on insect cuticle is a useful characteristic for the analysis of intraspecific variability of M. flavoviride isolates.