Metabarcoding of Soil Fungal Communities in Rupestrian Grassland Areas Preserved and Degraded by Mining: Implications for Restoration.

Rupestrian grasslands are vegetation complexes of the Cerrado biome (Brazilian savanna), exhibiting simultaneously great biodiversity and important open-pit mining areas. There is a strong demand for the conservation of remaining areas and restoration of degraded. This study evaluated, using next-generation sequencing, the diversity and ecological aspects of soil fungal communities in ferruginous rupestrian grassland areas preserved and degraded by bauxite mining in Brazil. In the preserved and degraded area, respectively, 565 and 478 amplicon sequence variants (ASVs) were detected. Basidiomycota and Ascomycota comprised nearly 72% of the DNA, but Ascomycota showed greater abundance than Basidiomycota in the degraded area (64% and 10%, respectively). In the preserved area, taxa of different hierarchical levels (Agaromycetes, Agaricales, Mortierelaceae, and Mortierella) associated with symbiosis and decomposition were predominant. However, taxa that colonize environments under extreme conditions and pathogens (Dothideomycetes, Pleoporales, Pleosporaceae, and Curvularia) prevailed in the degraded area. The degradation reduced the diversity, and modified the composition of taxa and predominant ecological functions in the community. The lack of fungi that facilitate plant establishment and development in the degraded area suggests the importance of seeking the restoration of this community to ensure the success of the ecological restoration of the environment. The topsoil of preserved area can be a source of inocula of several groups of fungi important for the restoration process but which occur in low abundance or are absent in the degraded area.

Cryptic fungal diversity revealed by DNA metabarcoding in historic wooden structures at Whalers Bay, Deception Island, maritime Antarctic.

We provide the first assessment of fungal diversity associated with historic wooden structures at Whalers Bay (Heritage Monument 71), Deception Island, maritime Antarctic, using DNA metabarcoding. We detected a total of 177 fungal amplicon sequence variants (ASVs) dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Monoblepharomycota, Rozellomycota, and Zoopagomycota. The assemblages were dominated by Helotiales sp. 1 and Herpotrichiellaceae sp. 1. Functional assignments indicated that the taxa detected were dominated by saprotrophic, plant and animal pathogenic, and symbiotic taxa. Metabarcoding revealed the presence of a rich and complex fungal community, which may be due to the wooden structures acting as baits attracting taxa to niches sheltered against extreme conditions, generating a hotspot for fungi in Antarctica. The sequences assigned included both cosmopolitan and endemic taxa, as well as potentially unreported diversity. The detection of DNA assigned to taxa of human and animal opportunistic pathogens raises a potential concern as Whalers Bay is one of the most popular visitor sites in Antarctica. The use of metabarcoding to detect DNA present in environmental samples does not confirm the presence of viable or metabolically active fungi and further studies using different culturing conditions and media, different growth temperatures and incubation periods, in combination with further molecular approaches such as shotgun sequencing are now required to clarify the functional ecology of these fungi.

DNA Metabarcoding Reveals Cryptic Diversity in Forest Soils on the Isolated Brazilian Trindade Island, South Atlantic.

Located 1140 km from the South American coastline in the South Atlantic Ocean and with an age of 4 million years, Trindade Island is the most recent volcanic component of Brazilian territory. Its original native vegetation has been severely damaged by human influence, in particular through the introduction of exotic grazing animals such as goats. However, since the complete eradication of goats and other feral animals in the late 1990s, the island's vegetation has been recovering, and even some endemic species that had been considered extinct have been rediscovered. In this study, we set out to characterize the contemporary cryptic diversity in soils of the recovering native forest of Trindade Island using metabarcoding by high throughput sequencing (HTS). The sequence diversity obtained was dominated by microorganisms, including three domains (Bacteria, Archaea, and Eukarya) and five kingdoms (Fungi, Metazoa, Protozoa, Chromista, and Viridiplantae). Bacteria were represented by 20 phyla and 116 taxa, with Archaea by only one taxon. Fungi were represented by seven phyla and 250 taxa, Viridiplantae by five phyla and six taxa, Protozoa by five phyla and six taxa, Metazoa by three phyla and four taxa and Chromista by two phyla and two taxa. Even after the considerable anthropogenic impacts and devastation of the island's natural forest, our sequence data reveal the presence of a rich and complex diversity of microorganisms, invertebrates, and plants and provide important baseline biodiversity information that will contribute to ecological restoration efforts on the island.

Diversity and ecology of fungal assemblages present in lake sediments at Clearwater Mesa, James Ross Island, Antarctica, assessed using metabarcoding of environmental DNA.

We detected the fungal assemblages present in lake sediments on James Ross Island, Antarctica, using DNA metabarcoding. A total of 132 amplicon sequence variants (ASVs) were assigned, dominated by taxa of the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. The less common phyla Chytridiomycota, Rozellomycota, Monoblepharomycota, Basidiobolomycota, Aphelidiomycota and the fungus-like Straminopila were also detected. Fungal sp. 1, Fungal sp. 2, Spizellomycetales sp. 1, Rozellomycotina sp. 1, Talaromyces rubicundus and Betamyces sp. dominated the assemblages. In general, the assemblages displayed high diversity and richness, and moderate dominance. Saprophytic, pathogenic and symbiotic fungi were detected. The metabarcoding data indicated that Antarctic lakes may represent a hotspot of fungal diversity in Antarctica. The sediments of these lakes may accumulate different fungal fragments and active fungal mycelia and their propagules, deposited over long periods of time. Lakes in the Antarctic Peninsula region are sensitive environments threatened by the effects of regional climatic changes. The abundance of sequences of little-known Rozellomycota and Chytridiomycota (Spizellomycetales) taxa in these ecosystems highlights the need for further studies to identify if they are metabolically active in the sediments and whether they have potentially pathogenic capabilities.

Diversity, distribution and ecology of fungal communities present in Antarctic lake sediments uncovered by DNA metabarcoding.

We assessed fungal diversity in sediments obtained from four lakes in the South Shetland Islands and James Ross Island, Antarctica, using DNA metabarcoding. We detected 218 amplicon sequence variants (ASVs) dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota, Mucoromycota and Chytridiomycota. In addition, the rare phyla Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Monoblepharomycota, Rozellomycota and Zoopagomycota as well as fungal-like Straminopila belonging to the phyla Bacillariophyta and Oomycota were detected. The fungal assemblages were dominated by unknown fungal taxa (Fungal sp. 1 and Fungal sp. 2), followed by Talaromyces rubicundus and Dactylonectria anthuriicola. In general, they displayed high diversity, richness and moderate dominance. Sequences representing saprophytic, pathogenic and symbiotic fungi were detected, including the phytopathogenic fungus D. anthuriicola that was abundant, in the relatively young Soto Lake on Deception Island. The lake sediments studied contained the DNA of rich, diverse and complex fungal communities, including both fungi commonly reported in Antarctica and other taxa considered to be rare. However, as the study was based on the use of environmental DNA, which does not unequivocally confirm the presence of active or viable organisms, further studies using other approaches such as shotgun sequencing are required to elucidate the ecology of fungi in these Antarctic lake sediments.

Large-scale Degradation of the Tocantins-Araguaia River Basin.

The Tocantins-Araguaia Basin is one of the largest river systems in South America, located entirely within Brazilian territory. In the last decades, capital-concentrating activities such as agribusiness, mining, and hydropower promoted extensive changes in land cover, hydrology, and environmental conditions. These changes are jeopardizing the basin's biodiversity and ecosystem services. Threats are escalating as poor environmental policies continue to be formulated, such as environmentally unsustainable hydropower plants, large-scale agriculture for commodity production, and aquaculture with non-native fish. If the current model persists, it will deepen the environmental crisis in the basin, compromising broad conservation goals and social development in the long term. Better policies will require thought and planning to minimize growing threats and ensure the basin's sustainability for future generations.

Functional and structural characterization of a novel GH3 ß-glucosidase from the gut metagenome of the Brazilian Cerrado termite Syntermes wheeleri.

In this study, a GH3 family ß-glucosidase (Bgl7226) from metagenomic sequences of the Syntermes wheeleri gut, a Brazilian Cerrado termite, was expressed, purified and characterized. The enzyme showed two optimum pHs (pH 7 and pH 10), and a maximum optimum temperature of about 40 °C using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate. Bgl7226 showed higher enzymatic activity at basic pH, but higher affinity (Km) at neutral pH. However, at neutral pH the Bgl7226 enzyme showed higher catalytic efficiency (kcat/Km) for pNPG substrate. Predictive analysis about the enzyme structure-function relationship by sequence alignment suggested the presence of multi-domains and conserved catalytic sites. Circular dichroism results showed that the secondary structure composition of the enzyme is pH-dependent. Small conformational changes occurred close to the optimum temperature of 40 o C, and seem important for the highest activity of Bgl7226 observed at pH 7 and 10. In addition, the small transition in the unfolding curves close to 40 o C is typical of intermediates associated with proteins structured in several domains. Bgl7226 has significant ß-glucosidase activity which could be attractive for biotechnological applications, such as plant roots detoxification; specifically, our group is interested in cassava roots (Manihot esculenta) detoxification.

Latitude and chlorophyll a density drive the distribution of carbohydrate-active enzymes in the planktonic microbial fraction of the epipelagic zone.

Microbes drive the majority of the global carbon cycle. The effect of environmental conditions on selecting microbial functional diversity is well established, and recent studies have revealed the effects of geographic distances on selecting the functional components of marine microbial communities. Our study is the first attempt at establishing the effects of environmental factors on driving the marine carbohydrate-active enzyme (CAZyme) distribution. We characterized the diversity of CAZyme genes and investigated the correlations between their distributions and biogeographic parameters (latitude, longitude, distance from the equator, site depth, water depth, chlorophyll density, salinity and temperature). Therefore, we accessed a subset of surface water samples (38 metagenomes) from the Global Ocean Sampling project. Only chlorophyll and latitude altered the distribution patterns of CAZymes, revealing the existence of two latitudinal gradients (positive and negative) of marine CAZyme abundance. Considering the importance of carbohydrates in microbial life, characterization of the spatial patterns of the genetic repertoire involved in carbohydrate metabolism represents an important step in improving our understanding of the metabolic strategies associated with the microbial marine carbon cycle and their effects on the productivity of marine ecosystems.

Cyanobacterial biodiversity of semiarid public drinking water supply reservoirs assessed via next-generation DNA sequencing technology.

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

Structural and functional characterization of a novel lipolytic enzyme from a Brazilian Cerrado soil metagenomic library.

OBJECTIVE: To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features. RESULTS: Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67-29,411.98 Da and 2.58-2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0-9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% ß-sheet, consistent with the 3D structure predicted by homology. CONCLUSION: Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.

Diversity and Pathogenicity of Rhizoctonia Species from the Brazilian Cerrado.

Eighty-one Rhizoctonia-like isolates were identified based on morphology and nuclei-staining methods from natural and agricultural soils of the Cerrado (Brazilian savanna). The nucleotide similarity analysis of ITS1-5.8S-ITS2 regions identified 14 different taxa, with 39.5% of isolates assigned to Waitea circinata (zeae, oryzae, and circinata varieties), while 37.0% belonged to Thanatephorus cucumeris anastomosis groups (AGs) AG1-IB, AG1-ID, AG1-IE, AG4-HGI, and AG4-HGIII. Ceratobasidium spp. AG-A, AG-F, AG-Fa, AG-P, and AG-R comprised 23.5%. Rhizoctonia zeae (19.8%), R. solani AG1-IE (18.6%), and binucleate Rhizoctonia AG-A (8.6%) were the most frequent anamorphic states found. Root rot severity caused by the different taxa varied from low to high on common beans, and tended to be low to average in maize. Twenty-two isolates were pathogenic to both hosts, suggesting difficulties in managing Rhizoctonia root rots with crop rotation. These results suggest that cropping history affects the geographical arrangement of AGs, with a prevalence of AG1 in the tropical zone from central to north Brazil while the AG4 group was most prevalent from central to subtropical south. W. circinata var. zeae was predominant in soils under maize production. To our knowledge, this is the first report on the occurrence of W. circinata var. circinata in Brazil.

Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and associated biotechnological applications.

Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes.

Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes).

Microbial Community Profile and Water Quality in a Protected Area of the Caatinga Biome.

The Caatinga is a semi-arid biome in northeast Brazil. The Paraguaçú River is located in the Caatinga biome, and part of its course is protected by the National Park of Chapada Diamantina (PNCD). In this study we evaluated the effect of PNCD protection on the water quality and microbial community diversity of this river by analyzing water samples obtained from points located inside and outside the PNCD in both wet and dry seasons. Results of water quality analysis showed higher levels of silicate, ammonia, particulate organic carbon, and nitrite in samples from the unprotected area compared with those from protected areas. Pyrosequencing of the 16S rRNA genes revealed that Burkholderiales was abundant in samples from all three sites during both seasons and was represented primarily by the genus Polynucleobacter and members of the Comamonadaceae family (e.g., genus Limnohabitans). During the dry season, the unprotected area showed a higher abundance of Flavobacterium sp. and Arthrobacter sp., which are frequently associated with the presence and/or degradation of arsenic and pesticide compounds. In addition, genes that appear to be related to agricultural impacts on the environment, as well as those involved in arsenic and cadmium resistance, copper homeostasis, and propanediol utilization, were detected in the unprotected areas by metagenomic sequencing. Although PNCD protection improves water quality, agricultural activities around the park may affect water quality within the park and may account for the presence of bacteria capable of pesticide degradation and assimilation, evidencing possible anthropogenic impacts on the Caatinga.

The Gut Microbiota of Workers of the Litter-Feeding Termite Syntermes wheeleri (Termitidae: Syntermitinae): Archaeal, Bacterial, and Fungal Communities.

The gut microbiota of termites allows them to thrive on a variety of different materials such as wood, litter, and soil. For that reason, they play important roles in the decomposition of biomass in diverse biomes. This function is essential in the savanna, where litter-feeding termites are one of the few invertebrates active during the dry season. In this study, we describe the gut microbiota of workers (third and fourth instars) of the species Syntermes wheeleri, a litter-feeding termite from the Brazilian savanna. Results of 16S and 18S ribosomal RNA (rRNA) gene-targeted pyrosequencing using primers sets specific to each domain have revealed its bacterial, archaeal, and fungal diversities. Firmicutes accounted for more than half of the operational taxonomic units of the Bacteria domain. The most abundant fungal species were from the class Dothideomycetes of the phylum Ascomycota. The methanogenic orders Methanobacteriales, Methanosarcinales, and Methanomicrobiales of the phylum Euryarchaeota accounted for the greatest part of the Archaea detected in this termite. A comparison of the gut microbiota of the two instars revealed a difference in operational taxonomic unit (OTU) abundance but not in species richness. This description of the whole gut microbiota represents the first study to evaluate relationships among bacteria, archaea, fungi, and host in S. wheeleri.

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production.

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted ß-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum.

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.

Optimized microplate beta-1,3-glucanase assay system for Trichoderma spp. screening.

Beta-1,3-glucanase is an important cell wall-degrading enzyme involved in mycoparasitism by Trichoderma spp. during antagonism against phytopathogenic fungi. A simple microplate-based method to assay beta-1,3-glucanase activity is described here as an alternative to an expensive tube-assay method. The reaction volume of the micro-assay was reduced to 130 microl from the 1150 microl used in the standard beta-1,3-glucanase macro-assay. Statistical analyses showed significant difference in sensitivity between the micro- and the macro-assay. The micro-method was optimized using the Response Surface Quadratic Model. The sensitivity of the optimized micro-method was shown to be four-fold greater than the macro-assay and two-fold higher than the micro-assay. The optimized micro-assay was significantly more sensitive in all of the twenty examined isolates during Trichoderma spp. beta-1,3-glucanase screening. We conclude that this modified and optimized method is more convenient, faster, cheaper and more reproducible than the traditional tube-assay.