Genetic diversity and pectinolytic activity of epiphytic yeasts from grape carposphere.

The genetic diversity of epiphytic yeasts from grape carposphere is susceptible to environmental variations that determine the predominant carposphere microbiota. Understanding the diversity of yeasts that inhabit grape carposphere in different environments and their pectinolytic activity is a way to understand the biotechnological potential that surrounds us and help improve winemaking. Therefore, this study aimed to evaluate the pectinolytic activity and characterize the genetic diversity of isolated epiphytic yeasts from grape carposphere. Grapes of the Bordeaux cultivar were collected from different regions of Paraná and Rio Grande do Sul States, in Brazil, and the yeasts were isolated from these grape carpospheres. Monosporic isolates were morphologically and genetically characterized on potato dextrose agar medium and by PCR-RFLP and rep-PCR (BOX-PCR) in the ITS1-5.8S-ITS2 region of rDNA. The index of pectinolytic activity of isolates was also evaluated estimating the ratio between the halo diameter of enzymatic degradation and the diameter of the colony when the isolates were grown in cultivation medium containing 10 g/L pectin, 5 g/L yeast extract, 15 g/L agar, 0.12% (w/v) Congo red, and pH 6.2. We observed that the grape carposphere is an environment with a great genetic diversity of epiphytic yeasts of the following genera: Cryptococcus (31.25%), Pichia (25.0%), Candida (25.0%), Dekkera (12.5%), and Saccharomyces (6.25%). The PCR-RFLP technique allowed analyzing existing polymorphism among individuals of a population based on a more restrict and evolutionarily preserved region, mostly utilized to differentiate isolates at the genus level. Approximately 33% of yeast isolates presented pectinolytic activity with potential biotechnological for wine and fruit juice production. This great genetic variability found indicated that it is a potential reservoir of genes to be applied in viniculture improvement programs.

Genomic variability of Pantoea ananatis in maize white spot lesions assessed by AFLP markers.

Measures to control maize white spot (MWS) caused by Pantoea ananatis are preferentially based on resistant cultivars. A lack of knowledge on the genetic variability of pathogens could interfere with the development and utilization of controlling strategies in this pathosystem. The main goals of this study were to investigate the genetic variability of 90 P. ananatis isolates from three different eco-geographical regions of Brazil by amplified fragment length polymorphism (AFLP), and to determine the presence of a universal P. ananatis plasmid in isolates from tropical Brazil. Analysis of genetic similarity by AFLP allowed us to categorize the 90 isolates into two groups. However, no correlation between the collecting sites and genetic groupings was observed. The polymorphism percentage found in P. ananatis ranged between 24.64 and 92.46%, and genetic diversity was calculated to be 0.07-0.09. The analysis of molecular variance showed that 99.18% of genetic variability was within the populations, providing evidence that evolutionary forces were acting on these populations. All P. ananatis isolates showed the P. ananatis universal plasmid (280 or 352 kb). This is the first report on the presence of a universal P. ananatis plasmid from MWS lesions in the tropical area.

Characterization of the inaA gene and expression of ice nucleation phenotype in Pantoea ananatis isolates from Maize White Spot disease.

Maize White Spot (MWS), a foliar disease caused by Pantoea ananatis, could cause up to 60% yield loss. Some strains of P. ananatis harboring the ice nucleation gene inaA catalyze the formation of ice nuclei, causing tissue damage at temperatures slightly below freezing. Little is known about the relationship between the presence of the ina gene in this maize pathogen and its expression during the phenomenon of ice nucleus formation. Here, we attempted to verify the presence of the inaA gene and the expression of phenotype in vitro. The identity of the isolates and the presence of the inaA gene were determined by P. ananatis species-specific primers. The expression of the inaA gene was assessed in vitro by the visualization of ice-crystal formation in water at subzero temperatures. A total of ninety P. ananatis isolates from MWS lesions were characterized. The presence of the inaA gene was confirmed by gel electrophoresis of the 350-400-bp PCR products. The inaA primers did not lead to DNA fragment amplification in three isolates. The ice nucleation phenotype was expressed in 83.34% of the isolates carrying the inaA gene. Our study showed that the ice nucleation in P. ananatis isolated from MWS lesions was dependent on the presence of a functional ina gene in the genome. We also found evidence indicating that some P. ananatis strains have a mutated form of the inaA gene, producing a non-functional ice nucleation protein. This is the first report on inaA gene characterization in P. ananatis isolates from Maize White Spot.

Screening of basidiomycetes in submerged cultivation based on antioxidant activity.

Submerged cultivation of medicinal basidiomycetes is a reproducible and efficient method of producing mycelia and metabolites. The antioxidant activity indicates its medicinal properties and is an important tool for basidiomycete screening. In this study, we analyzed the production of mycelial biomass and exopolysaccharides and the antioxidant activity of basidiomycete strains in submerged cultivation. Twenty-five strains were used for submerged cultivation in extract malt medium, and the production of mycelial biomass and exopolysaccharides was evaluated. Antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl method. Among the 25 evaluated strains, Lentinus crinitus produced the highest biomass, reaching 1190 ± 52 mg·L(-1)day(-1); Agaricus subrufescens strains had the highest exopolysaccharide production from 18.96 ± 0.15 to 20.97 ± 2.10 mg L(-1)·day(-1). Additionally, A. subrufescens showed the highest total antioxidant activity, reinforcing the therapeutic potential of this basidiomycete. No significant correlation was found between mycelial biomass or exopolysaccharide production and antioxidant activity; however, the results depended on each species and the strains of the same species. We found large variations in the production of mycelial biomass and exopolysaccharides and in antioxidant activity among different species and among strains of the same species. Thus, evaluating the total antioxidant activity is an important tool for identifying strains with biotechnological potential.

Photoprotective and Antimutagenic Activity of Agaricus subrufescens Basidiocarp Extracts.

The photoprotective and antimutagenic activity of opened and closed basidiocarps of Agaricus subrufescens (=A. blazei; =A. brasiliensis) obtained by different extraction methods were evaluated on Aspergillus nidulans conidia submitted to ultraviolet (UV) light. The aqueous extracts were obtained by three extraction methods: maceration, infusion, and decoction, at two different extraction times. The extracts of A. subrufescens did not present toxicity for A. nidulans conidia. A suspension of A. nidulans conidia was submitted to extracts before and after the exposure to UV light. All basidiocarp extracts, regardless of the extraction method or development stage, protected A. nidulans conidia against the damaging effects of the mutagenic agent. The antimutagenic and photoprotective activity was strengthened with extracts obtained by 168-h maceration, followed by 24-h maceration and 60-min infusion and, at last, by 30-min infusion. Although the extracts presented protector effect as well as recoverer effect to the action of UV light, the preventive effect was more evident. Differences in the biological activity in function of the different development stages were detected with greater antimutagenic and photoprotective activity for the opened basidiocarps. However, the extraction method is the most important factor to be considered when compared to the basidiocarp development stage to obtain better antimutagenic and photoprotective activity of A. subrufescens basidiocarps.

Localization of auxotrophic and benomyl resistance markers through the parasexual cycle in the beauveria bassiana (Bals.) vuill entomopathogen.

Genetic studies to localize auxotrophic markers and resistance to benomyl fungicide and to analyze gene transfer on the Beauveria bassiana deuteromycete were carried out using the parasexual cycle. Parasexual crosses among strains with complementary genetic markers resulted in vigorous heterocaryons. Selection of the segregant products was made using two methodologies: total isolation and parental elimination. Colonies with recombinant traits were recovered directly from the heterocaryon in all crosses, and no diploid colony was isolated. This shows the high instability of the diploid nucleus in this species. Among the parasexual segregant products preferential recovery of markers in one of the parental strains involved in the crosses was detected, probably because of an inhibition of conidiogenesis from one parental strain to another. Genetic markers were localized in four linkage groups by the parasexual crosses. In the first group markers nic4, nic3, thi2, bio3, ade2, ths2, and ben1R were localized; in the second, the marker met1; in the third, pab1; and in the fourth, bio1. The parental strain 196/A11/3 is a carrier of translocation among the linkage groups I and III.