Vol. 9(5), pp. 301-306, 4 February, 2015 DOI: 10.5897/AJMR2014.7130 Article Number: FBC3F6550465 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyrighht of this article http://www.academicjournals.org/AJMR Full Length Research Paper Characterization ofo Aspergilluus section Nigri isolates from leaf litter and soil in the Atlantic Forest of Brazil Tatiana Faria Maia1, Jean Luiz Simões-Araújo2, Carlos Antonio Inácio3 and Marcelo Elias Fraga4* 1Programa de Pós-graduação em Fitossanidade e Biotecnologia Aplicada, Universidade Federal Rural do Rio de Janeiro, Brazil. 2Embrapa Agrobiologia, Brazil. 3Departamento de Entomologia e Fitopatologia, Universidade Federal Rural do Rio de Janeiro, Brazil. 4Departamento de Microbiologia e Imunologia Veterinária, Universidade Fedeeral Rural do Rio de Janeiro, Brazil. Received 17 September, 2014; Accepted 1 December, 2014 Section Nigri is among the most important from the genus Aspergillus, since it possesses economical and biotechnological properties. Its identification is considered quite hard and complex. Due to this fact, Aspergillus taxonomy has been carried out by morphological, biochemical and molecular data. Morphological characterization besides molecular analyses was performed to properly identify Aspergillus section Nigri. A set of 42 Aspergiillus section Nigri isolates were obtained from Brazilian Atlantic Rainforest. From these isolates, 16.7% originated from leaf litter layer and 83.3%, from soil. The isolates were divided into three different groups according to annalyzed characteristics. 26 were identified as Aspergillus aculeatus, 12 as Aspergillus aculeatinus and 4 belonged to a group called “Aspergillus niger Aggregate”, considered as biseriate. Key words: Fungi, taxonomy, morphological identification, polymerase chain reaction (PCR), ITS. INTRODUCTION Fungi from the genus Aspergillus have a significant are considered the most important (Gams et al., 1985), impact on modern society (Pitt and Hocking, 2009) as thhey are used in industrial fermentation to hydrolyze because they cause food bio-deterioration and are enzymes and are source of extracellular enzymes pottential pathogens to men and animals in general (Abarca et al., 2004). The use of these fungi as enzymes (Geiser et al., 2007). However, such microorganisms are producers is recognized by Food and Drug Administration of big concern because they have bio-technologic (FDA); some species of the Aspergillus genus possess pottential to produce chemical compound, mycotoxins, Generally Regardeed as Safe (GRAS) status, due to theiir enzymes, organic acids, phenolic compounds, amongst low toxicity and historical use in industrial food production other substances (Varga et al., 2003). and drink. A good example is the Aspergillus niger Of all the genera, Aspergillus species from section Nigri lineage (Abarca et al., 20044). The Nigri section is *Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0International License 302 Afr. J. Microbiol. Res. considered by many taxonomists as one of the most (Potato dextrose agar) medium at 28°C for 5 days and about 1 cm2 difficult to identify, due to high genetic diversity, which of mycelium from cultures was collected and used for DNA makes identification based only on phenotypic charac- extraction. The best result was observed with the protocol indicated by Vicente (2000). The DNA was quantified in Qubit 2.0 teristics a hard work (Samson et al., 2007). Therefore, Fluorometer (Invitrogen). many authors have been highlighting the importance of polyphasic taxonomy, by combining different identification tools, using morphological, physiological and biochemical Amplification of ITS region from rDNA by PCR characteristics, as well as metabolites production and The oligonucleotides primers ITS1 (5’-TTC CGT AGG TGA ACC molecular data. Their main goal is to clarify the complex TGC GG- 3’) and ITS4 (5’ TCC TCC GCT TAT TGA TAT GC 3’) taxonomy of section Nigri species (Varga et al., 2011; (White et al., 1990) were used for molecular characterization of Silva et al., 2011; Simões et al., 2013). New molecular Aspergillus section Nigri isolates. The polymerase chain reaction approaches have contributed to such studies and have (PCR) was carried out with 2 μL of DNA (between 100 and 200 ng); allowed significant break-throughs in fungi taxonomical 10 μL of Taq buffer to PCR (10X); 6 μL of MgCl2 (25 mM); 1 μL of organization, acknow-ledging the existence of an dNTP (10 mM); 5 μ/μL GoTaq DNA polymerase; 1.2 μL of DMSO; 1.5 μL of each primer; ITS1 (10 pmol/μL) and DNase free water to a elevated biodiversity on section Nigri (Krijgsheld et al., final volume of 50 μL. The PCRs were performed on a thermo 2013). cycler (Mastercycle 22331 Hamburg Eppendorf AG). The In this context, the present work aims to use amplification conditions were: a denaturation stage at 95°C for 4 morphological, biochemical and molecular data to identify min, followed by 30 cycles of denaturation at 95°C for 45 s, Aspergillus species from section Nigri isolates from leaf annealing at 55°C for 30 s and extension at 72°C for 10 min. The electrophoresis was performed using 1% agarose gel stained with litter and soil in the Atlantic Rainforest of Brazil. -1 ethidium bromide solution (0.5 μg.mL ) and visualized in a transiluminator (KODAK Scientific Imaging Systems). MATERIALS AND METHODS Amplified fragments sequencing Leaf litter and soil samples and isolation The PCR purified product was used for sequencing reactions on an The collection areas of leaf litter and soil samples are located in automatic DNA sequencer ABI PRISM 3100 Genetic Analyzer Mid-West Minas Gerais, at Sete Lagoas city, belonging to the (Applied Biosystems). The contiguous DNA sequences were Atlantic Rainforest’s biome of Brazil. Both samples, leaf litter and assembled with ITS1 and ITS2 independent sequence reactions soil, were collected on two different periods, the first being humid, using Phred/Phrad programs (Ewing et al., 1998). at the end of January and the second dry, at the beginning of September in 2012. Nine forest fragments were sampled on three different depths (leaf litter, 0 to 5 and 5 to 10 cm) with three Similarity and phylogeny analyses repetitions in each depth, totaling 162 samples. After collection, the packaging containing the samples was stored at 4°C until analysis. The similarity research was performed in the Genbank, using Basic The isolations were performed on five grams of soil and leaf litter Local Alignment Search Tool (BLAST) program re-suspended in an Erlenmeyer flask with 45 mL of 0.1% peptone (http://www.ncbi.nlm.nih.gov). The ITS1-ITS4 similarly sequences and Tween 80; and later agitated for 30 min on a vertical were imported from BioEdit Sequence Alignment Editor program homogenizer. The cultural media used was Dichloran Rose Bengal (Hall, 1999) to alignment, using CLUSTALW program (Thompson et Chloramphenicol Agar (DRBC), accomplished with aliquots of 0.1 al., 1994) and manual edition. The phylogenetic tree was built -2 -6 mL from respective dilutions (10 to 10 ). The Petri dishes were through comparison of 550 pb using the Neighbor-Joining method. incubated on greenhouses at 25°C for five days (Fraga and Pereira, The tree’s topology toughness was evaluated through Kimura 2- 2012). parameter method and phylogenetic analysis was conducted on Molecular Analyses Evolutive Genetics (MEGA.5) program (Tamura et al., 2011). Morphological analyses For morphological identification, the inoculations were accom- plished through conidial suspension of isolates from Aspergillus RESULTS AND DISCUSSION section Nigri on solution containing Agar and Tween 80. From such suspension 0.2 mL was used for inoculation on three equidistant Morphological characterization points in the Petri dishes containing Czapek yeast agar (CYA) and malt extract agar (MEA). It was done in triplicate in each medium. The 42 isolates were divided into 3 groups according to The incubation was performed at 25 and 37°C on CYA and 25°C on morphological characteristics. Group I: 26 isolates MEA for seven days; afterwards, macroscopic and microscopic characteristics were registered (Samson et al., 2007). (62%); group II: 12 isolates (28.5%), and Group III: 4 isolates (9.5%). Table 1 shows the macroscopic and microscopic characteristics of Aspergillus section Nigri of Molecular analyses isolates species. Groups I and II present all uniseriate strains; in other words, present only phialide, conidia DNA extraction varying from dark and light brown, echinulated on both The DNA extraction of 42 isolates from section Nigri was carried out globular and sub-globular shape and hyaline hyphae. In using four different protocols. The fungi were cultivated on PDA group III all strains were biseriate, presenting Maia et al. 303 Table 1. Phenotypic characters of Aspergillus section Nigri isolates obtained from leaf litter and soil in the Atlantic Rainforest of Brazil. Colony color Colony diameter (mm) Isolates CYA (25°C) verse / MEA CYA Sclerotia (groups) MEA (25 °C) verse / reverse CYA (25°C) reverse (25°C) (37°C) (color) Macroscopic Dark brown/ Black with yellowish halo / I >80 55-75 15-28 Yellowish Light Brown colorless with yellowish tones Dark II Black/colorless >80 75-85 20-35 Orange brown/yellowed Dark brown to black/ colorless to Dark Brown to III 50-85 54-79 47-70 - yellowish with gray Black/colorless tones Microscopic