Composition and Diversity of Fungal Decomposers of Submerged Wood in Two Lakes in the Brazilian Amazon State of Para´

Hindawi International Journal of Microbiology Volume 2020, Article ID 6582514, 9 pages https://doi.org/10.1155/2020/6582514 Research Article Composition and Diversity of Fungal Decomposers of Submerged Wood in Two Lakes in the Brazilian Amazon State of Para´ Eveleise SamiraMartins Canto ,1,2 Ana Clau´ dia AlvesCortez,3 JosianeSantana Monteiro,4 Flavia Rodrigues Barbosa,5 Steven Zelski ,6 and João Vicente Braga de Souza3 1Programa de Po´s-Graduação da Rede de Biodiversidade e Biotecnologia da Amazoˆnia Legal-Bionorte, Manaus, Amazonas, Brazil 2Universidade Federal do Oeste do Para´, UFOPA, Santare´m, Para´, Brazil 3Instituto Nacional de Pesquisas da Amazoˆnia, INPA, Laborato´rio de Micologia, Manaus, Amazonas, Brazil 4Museu Paraense Emilio Goeldi-MPEG, Bele´m, Para´, Brazil 5Universidade Federal de Mato Grosso, UFMT, Sinop, Mato Grosso, Brazil 6Miami University, Department of Biological Sciences, Middletown, OH, USA Correspondence should be addressed to Eveleise Samira Martins Canto; [email protected] and Steven Zelski; [email protected] Received 25 August 2019; Revised 20 February 2020; Accepted 4 March 2020; Published 9 April 2020 Academic Editor: Giuseppe Comi Copyright © 2020 Eveleise Samira Martins Canto et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aquatic ecosystems in tropical forests have a high diversity of microorganisms, including fungi, which are important decomposers of submerged wood. Despite the importance of their role in decomposition, research concerning the diversity of freshwater fungi from Brazilian Amazonian environments is scarce. *e aim of this work was to describe the composition and diversity of fungi present on submerged wood in two lakes of the Brazilian Amazon (State of Pará).Fragments of decaying wood (30 samples per lake) were collected from the Lakes Jua´ and Maica.´ *e wood samples were inspected for 6 months in the presence of fungal reproductive structures. Fungi observed in the wood were identified morphologically. Twenty-three taxa were identified in the Lake Jua´ (10 sexual and 13 asexual) and 26 taxa in the Lake Maica´ (17 sexual, 9 asexual). ITS sequences were obtained for 14 taxa to aid in identification. In the Lakes Juáand Maicá,the diversity indices were H’: 2.6514 and H’: 2.8174, respectively. *e Sørensen index of the fungal communities in the studied lakes was 0.3673. *is study is the first to describe the fungal biodiversity of two important aquatic environments in Pará,Brazil. 1. Introduction woody debris [5, 6]. *is group is polyphyletic, with the majority of species belonging to the subphylum Pezizo- Freshwater fungi include species that inhabit water for all or mycotina in the classes Leotiomycetes, Sordariomycetes, and part of their life cycle or any species adapted to colonize Dothideomycetes [2, 7]. Members of the Eurotiomycetes [8] predominantly aquatic or semiaquatic substrates in nature and Orbiliomycetes [9] are less common. Shearer and Raja [1]. *ese organisms serve as key agents in the decompo- [10] reported that freshwater ascomycetes are distributed in sition of submerged dead plant material. Fungal biomass is a relatively few orders: Helotiales, Pleosporales, Sordariales, source of nutrition for other organisms in aquatic ecosys- Savoryellales, Microascales, Eurotiales, and Jahnulales. *e tems [2, 3]. Fungal enzymatic activity also modifies plant number of known taxa has increased in recent years due to substrates to make them more palatable to invertebrate broader sampling and the use of molecular methods for shredders and scrapers [4]. identification [11, 12]. Freshwater ascomycetes are a group of fungi known to *e Amazon basin is arguably the most complex net- actively participate in the decomposition of submerged work of aquatic habitats on the planet [13]. *is vast region 2 International Journal of Microbiology contains a variety of aquatic environments that include large structures on the collected wood samples. *e structures rivers, lakes, swamps, and seasonally inundated floodplains found were transferred, with dissection needles, to micro- [13, 14]. Aquatic ecosystems can be roughly classified into scope slides containing distilled water. *e identification of three main types: clear waters [15], white waters [16], and sexual ascomycetes was performed based on the micro- black waters [14, 17]. Water characteristics such as tem- morphology of ascomata, hamathecium, asci, and asco- perature, dissolved oxygen, and amount of organic matter spores using an Axioskop 40 microscope (Zeiss) as described may influence the diversity of freshwater fungal species [18]. previously [22, 29, 30]. Aqueous lactophenol cotton blue *e first published studies of freshwater fungi in Amazonian solution was used to determine the staining reactions of the waters (Peru) were conducted by Matsushima in white water apical ascus apparatus. Asexual ascomycetes were identified rivers [19]. More recent studies have described new species by investigating the types of conidiogenesis, conidiophore, of ascomycetes in the Peruvian Amazon [20–24] and in the and conidia, using an Axioskop 40 microscope (Zeiss). Brazilian states of Para´ [25, 26] and Amazonas [27, 28]. In Isolation of fungal was carried out by transferring the fungal general, the microbial community of the Amazon region is structures to the surface of Antibiotic Water Agar (20 g/L still underexplored, and studies describing the diversity and agar and chloramphenicol 250 mg/L), Potato Dextrose Agar ecology of fungi in the Amazon region are urgently needed. (PDA) (Kasvi), and Malt Extract Agar (MEA) (malt extract *erefore, we sought to investigate new environments such 30 g/L, mycological peptone 5g/L and 15g/L agar, pH as the Tapajos´ River, one of the largest clear water tributaries 5.4 ± 0.2). *e developed colonies were transferred to PYG of the Amazon River [17]. *e present work aimed to de- agar (1.25 g/L peptone, 1.25 g/L yeast extract, 5 g/L glucose, scribe the composition and diversity of fungi present on 250 mg/L chloramphenicol, and 18 g/L agar) [22] submerged decomposing wood in two lakes in the Tapajos´ (Figures 2(d)–2(g)). River basin near Santarem,´ in the Brazilian state of Para.´ 2. Materials and Methods 2.5. DNA Extraction, Amplification, and Sequencing. DNA extraction was performed according to the protocol of 2.1. Study Sites. Samples of submerged wood were collected Ferrer et al. [31]. Fungi were grown in test tubes containing from Lake Jua´ (2°25′57″S, 54° 46′39″W) and Lake Maica´ 10 mL of potato dextrose broth (120 g potato, 10 g dextrose, (2°27′29.0″S, 54° 40′10.8″W) and, essentially, the embay- and 1000 mL distilled water) for 10 days and transferred to ment of the lower Tapajos´ and Amazon Rivers, respectively. Eppendorf tubes (2 mL) containing 500 μL of buffer (2.5 mg Lake Jua´ receives water that drains from a stream and the SDS, 7.0 mg NaCl, 3.65 mg EDTA, 20 mL of Tris-HCl and Tapajo´s. Lake Maica´ is a mix of surface drainages and the white 100 mL of DI water, and 5 μL of ß-mercaptoethanol). *e water of the Amazon River [14] (Figures 1, 2(a), and 2(b)). microtubes were subjected to freezing (−20 °C) and heating (65°C for 1 h) in order to rupture the cellular structures. Next, 500 μL of a phenol/chloroform/isoamyl alcohol so- 2.2. Water Characterization. Physicochemical variables lution (v/v/v: 25/24/1) was added to the microtubes and were measured using a ProfiLine 197i WTW multiparameter vortexed until a homogeneous suspension was obtained. *e ° probe (Wellheim, Germany). Temperature ( C), pH, elec- suspension was then centrifuged (14,000 rpm, 15 min) and trical conductivity (μS), dissolved oxygen concentration the supernatant was transferred to a new microtube (1.5 mL). (mg/L), and turbidity (NTU) were recorded for each sample Isopropanol was added to the supernatant in equal volume, collection. and the mixture was homogenized and incubated at −20°C overnight to precipitate the DNA. *e precipitated DNA was 2.3. Sample Collection. A single collection was made at each centrifuged (14,000 rpm for 15 min), washed twice with 70% location in Lake Jua´ in October 2017 and in Lake Maica´ in ethanol, and resuspended in 100 μL of sterile water distilled. November 2017. At each site, 30 submerged wood fragments Molecular data were gathered by sequencing the ITS that showed signs of active decomposition were collected. region of rDNA according to the procedure of White et al. *e lengths of the samples ranged from 6 to 22 cm and [32]. *e reaction yielded a final volume of 50 μL, consisting diameters ranged from 6 to 15 cm (Figure 2(c)). *ese of PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), substrates were transported in sealable plastic bags 1.5 mM MgCl 2, 0.5 μM of ITS-1 (5’-TCCGTAGGT- (20 cm × 10 cm), with a small amount of local water, to the GAACCTGCGG-3’) and ITS-4 (5’-TCCTCCGCTTATT- Laboratory of Multidisciplinary Teaching in Applied Biol- GATATGC-3’), 200 μM dNTPs, 1.5 U Taq DNA polymerase, ogy-Labio, Universidade Federal do Oeste do Para´ and 100 ng of fungal DNA. *ermocycling was performed (UFOPA). In the laboratory, the samples were gently rinsed on an Applied Biosystems ProFlex PCR System with initial ° with running water, placed in moist chambers (plastic boxes denaturation at 94 C for 5 min, followed by 35 cycles of DNA ° ° with moist paper towels), and incubated at room temper- denaturation at 94 C for 1 min, annealing at 55 C for 1 min, ° ° ature with 12/12 h light/dark conditions. extension at 72 C for 2 min, and a final extension at 72 C for 10 min. Two positive controls and one negative control were also tested.

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