Deep Genotypic Species Delimitation of Aspergillus Section Flavi Isolated from Brazilian Foodstuffs and the Description of Aspergillus annui sp. nov. and Aspergillus saccharicola sp. nov.

Aspergillus section Flavi is a fungal group that is important in food because it contains spoilage and potentially aflatoxigenic species. Aflatoxins are metabolites that are harmful to human and animal health and have been recognized as the primary natural contaminant in food. Therefore, recognizing the biodiversity of this group in food is necessary to reduce risks to public health. Our study aimed to investigate the diversity of Aspergillus section Flavi isolated from Brazilian foodstuffs such as cassava, sugarcane, black pepper, paprika, Brazil nuts, yerba-mate, peanuts, rice, and corn. A polyphasic approach integrating phenotypic data and multilocus genotypic analyses (CaM, BenA, and RPB2) was performed for 396 strains. Two new species in the Aspergillus subgenus Circumdati section Flavi are proposed using maximum-likelihood analysis, Bayesian inference, and coalescence-based methods: Aspergillus saccharicola sp. nov. and Aspergillus annui sp. nov. A. saccharicola sp. nov. belongs to the series Flavi, is a potentially aflatoxigenic species (B1, B2, G1, and G2), closely related to Aspergillus arachidicola, and was found mostly in sugarcane. A. annui sp. nov. was isolated from samples of sweet paprika. To accommodate A. annui sp. nov., a new series Annuorum was proposed.

Low-cost, specific PCR assays to identify the main aflatoxigenic species of Aspergillus section Flavi.

Aflatoxins are fungal metabolites that are present as contaminants in food globally. Most aflatoxigenic species belong to Aspergillus section Flavi, and the main ones are grouped in the A. flavus clade, where many cryptic species that are difficult to discriminate are found. In this study, we investigated inter- and intraspecific diversity of the A. flavus clade to develop low-cost, species-specific PCR assays for identifying aflatoxigenic species. A total of 269 sequences of the second largest subunit of RNA polymerase II (RPB2) locus were retrieved from GenBank, and primer pairs were designed using data mining to identify A. flavus, A. parasiticus, and A. novoparasiticus. Species-specific amplicons of approximately 620, 350, and 860 bp enabled identification of target species as A. flavus, A. parasiticus, and A. novoparasiticus, respectively.

The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae.

BACKGROUND: Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. RESULTS: A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. CONCLUSIONS: Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.

Molecular characterization of a beta-1,4-endoglucanase from an endophytic Bacillus pumilus strain.

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a beta-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5-8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6-8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50-60 degrees C are required over long incubation periods.

Análise comparativa da estabilidade do DNA de Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) sob diferentes métodos de preservação para uso em RAPD-PCR / Comparative analysis of the stability of DNA of Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) under different methods of preservation for use in RAPD-PCR

The appropriate preservation of the DNA of a certain organism is important for successful application of molecular techniques like RAPD-PCR. This study was designed to compare simple methods of preservation of specimens of Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) with respect to quantity and quality of DNA for use in RAPD-PCR, after different storage periods. Eight methods were tested: freezing (-20°C); ethyl alcohol 70 percent (-20°C and room temperature); absolute ethyl alcohol (-20°C and room temperature); air-dry; and preservation in extraction buffer (whole and homogenized insect). At intervals of 10 to 30 days, insect DNA was extracted, quantified and amplified through RAPD-PCR using primer OPA-04. The quality of extracted DNA was observed on 0.8 percent agarose gel. After 210 days of preservation, freezing (-20°C) showed to be the best method. Satisfactory quantities of DNA were also obtained from insects conserved in absolute ethyl alcohol (-20°C), ethyl alcohol 70 percent (-20°C) and extraction buffer (whole and homogenized insect). Insects conserved in absolute ethyl alcohol (room temperature), ethyl alcohol 70 percent (room temperature) and air-dry conditions were inappropriate for RAPD-PCR studies after 120, 60 and 10 days of storage, respectively.

A preservação adequada do DNA de um determinado organismo é fundamental para o sucesso no uso de técnicas moleculares como RAPD-PCR. O objetivo deste trabalho foi comparar métodos simples de preservação de espécimes de Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) quanto à quantidade e qualidade do DNA para uso em RAPD-PCR, após períodos sucessivos de armazenamento. Avaliaram-se oito métodos: congelamento (-20°C); álcool etílico 70 por cento (-20°C e temperatura ambiente); álcool etílico absoluto (-20°C e temperatura ambiente); secagem ao ar; e preservação em tampão de extração (inseto inteiro e macerado). A intervalos de 10-30 dias, o DNA foi extraído, quantificado e amplificado via RAPD-PCR pelo oligonucleotídeo OPA-04. A qualidade do DNA extraído foi observada em gel de agarose 0,8 por cento. Após 210 dias de armazenamento, o congelamento (-20°C) mostrou ser a melhor técnica. Quantidades satisfatórias de DNA também foram obtidas dos insetos conservados em álcool etílico absoluto (-20°C), álcool etílico 70 por cento (-20°C) e tampão de extração (inseto inteiro e macerado). Insetos conservados em álcool etílico absoluto (temperatura ambiente), em álcool etílico 70 por cento (temperatura ambiente) e secos ao ar mostraram-se impróprios para estudos com RAPD-PCR após 120, 60 e 10 dias de armazenamento, respectivamente.