Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation.

Cocoa beans used for chocolate production are fermented seeds of Theobroma cacao obtained by a natural fermentation process. The flavors and chemical compounds produced during the fermentation process make this step one of the most important in fine chocolate production. Herein, an integrative analysis of the variation of microbial community structure, using a shotgun metagenomics approach and associated physicochemical features, was performed during fermentation of fine cocoa beans. Samples of Forastero variety (FOR) and a mixture of two hybrids (PS1319 and CCN51) (MIX) from Bahia, Brazil, were analyzed at 7 different times. In the beginning (0 h), the structures of microbial communities were very different between FOR and MIX, reflecting the original plant-associated microbiomes. The highest change in microbial community structures occurred at the first 24 h of fermentation, with a marked increase in temperature and acetic acid concentration, and pH decrease. At 24-48 h both microbial community structures were quite homogenous regarding temperature, acetic acid, succinic acid, pH, soluble proteins and total phenols. During 72-96 h, the community structure resembles an acidic and warmer environment, prevailing few acetic acid bacteria. Taxonomic richness and abundance at 72-144 h exhibited significant correlation with temperature, reducing sugars, succinic, and acetic acids. Finally, we recommend that dominant microbial species of spontaneous fine cocoa fermentations should be considered as inoculum in accordance with the farm/region and GMP to maintain a differential organoleptic feature for production of fine chocolate. In our study, a starter inoculum composed of Acetobacter pausterianus and Hanseniaspora opuntiae strains is indicated.

Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.

BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

Description of Dioszegia patagonica sp. nov., a novel carotenogenic yeast isolated from cold environments.

During a survey of carotenogenic yeasts from cold and oligotrophic environments in Patagonia, several yeasts of the genus Dioszegia (Tremellales, Agaricomycotina) were detected, including three strains that could not be assigned to any known taxa. Analyses of internal transcribed spacer and D1/D2 regions of the large subunit rRNA gene showed these strains are conspecific with several other strains found in the Italian Alps and in Antarctica soil. Phylogenetic analyses showed that 19 of these strains represent a novel yeast species of the genus Dioszegia. The name Dioszegia patagonica sp. nov. is proposed to accommodate these strains and CRUB 1147T (UFMG 195T=CBMAI 1564T=DBVPG 10618T=CBS 14901T; MycoBank MB 819782) was designated as the type strain. This Dioszegia species accumulates biotechnologically valuable compounds such as carotenoid pigments and mycosporines.

Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae).

The diversity and antimicrobial activity of endophytic fungi associated with the Brazilian medicinal plant Solanum cernuum Vell. were studied during summer and winter seasons. A total of 246 fungal isolates were obtained, including 225 filamentous fungi and 21 yeasts. They were identified by morphological, physiological, and molecular methods. Fifty-five different taxa represented by the phyla Ascomycota (33 taxa), Basidiomycota (21 taxa), and Zygomycota (one taxon) were identified. The most abundant taxa were closely related to Arthrobotrys foliicola , Colletotrichum gloeosporioides , Coprinellus radians , Glomerella acutata , Diatrypella frostii , Phoma glomerata , Mucor sp., Phlebia subserialis , Phoma moricola , Phanerochaete sordida , and Colletotrichum sp. A total of 265 fungal extracts were screened and 64 (26.01%) displayed antimicrobial activities. Among these extracts, 18 (28.12%) presented antibacterial and antifungal activities, 42 (65.62%) displayed selective antibacterial activity, and four (6.25%) exhibited only antifungal activity. The best values of minimum inhibitory concentration were obtained from extracts of Cryptococcus rajasthanensis , Glomerella acutata, Leptosphaeria sp., and Phoma glomerata ranging from 7.8 to 15.62 µg/mL. This study is the first survey of the endophytic fungi community associated with S. cernuum, and our results show that they can represent a promising source of bioactive compounds.

Unraveling potential enzymes and their functional role in fine cocoa beans fermentation using temporal shotgun metagenomics.

Cocoa beans fermentation is a spontaneous process, essential for the generation of quality starting material for fine chocolate production. The understanding of this process has been studied by the application of high-throughput sequencing technologies, which grants a better assessment of the different microbial taxa and their genes involved in this microbial succession. The present study used shotgun metagenomics to determine the enzyme-coding genes of the microbiota found in two different groups of cocoa beans varieties during the fermentation process. The statistical evaluation of the most abundant genes in each group and time studied allowed us to identify the potential metabolic pathways involved in the success of the different microorganisms. The results showed that, albeit the distinction between the initial (0 h) microbiota of each varietal group was clear, throughout fermentation (24-144 h) this difference disappeared, indicating the existence of selection pressures. Changes in the microbiota enzyme-coding genes over time pointed to the distinct ordering of fermentation at 24-48 h (T1), 72-96 h (T2), and 120-144 h (T3). At T1, the significantly more abundant enzyme-coding genes were related to threonine metabolism and those genes related to the glycolytic pathway, explained by the abundance of sugars in the medium. At T2, the genes linked to the metabolism of ceramides and hopanoids lipids were clearly dominant, which are associated with the resistance of microbial species to extreme temperatures and pH values. In T3, genes linked to trehalose metabolism, related to the response to heat stress, dominated. The results obtained in this study provided insights into the potential functionality of microbial community succession correlated to gene function, which could improve cocoa processing practices to ensure the production of more stable quality end products.

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica.

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.

Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil.

The purpose of this study was to examine antimicrobial activity of endophytic fungi isolated from the leaves, stems, and roots of 54 species of Orchidaceae collected in a Brazilian tropical ecosystem. In total, 382 filamentous fungi and 13 yeast isolates were obtained and cultured to examine the production of crude extracts. Thirty-three percent of the isolates displayed antimicrobial activity against at least one target microorganism. The multivariate statistical analyses conducted indicate that the extracts of endophytic fungi isolated from leaves of terrestrial orchids in semideciduous forest were more active against Escherichia coli, whereas extracts of endophytic fungi from roots of rupicolous orchids collected in rock fields were more active against Candida krusei and Candida albicans. Among the fungi that were screened in the study, 22 isolates held their antimicrobial activities after replication and were therefore selected for assessment of the minimum inhibitory concentration (MIC), which ranged from 62.5 to 250 microg/mL and 7.8 to 250 microg/mL against bacteria and fungi, respectively. One isolate of Alternaria sp. and one isolate of Fusarium oxysporum presented the strongest antibacterial activity. Three Fusarium isolates, Epicoccum nigrum, and Sclerostagonospora opuntiae showed the greatest MIC values against the pathogenic yeasts. This study is the first survey investigating the bioactive potential of endophytic fungi associated with tropical Orchidaceae species present in Brazilian ecosystems.

16S rRNA Gene Amplicon Based Metagenomic Signatures of Rhizobiome Community in Rice Field During Various Growth Stages.

Rice is a major staple food across the globe. Its growth and productivity is highly dependent on the rhizobiome where crosstalk takes place between plant and the microbial community. Such interactions lead to selective enrichment of plant beneficial microbes which ultimately defines the crop health and productivity. In this study, rhizobiome modulation is documented throughout the development of rice plant. Based on 16S rRNA gene affiliation at genus level, abundance, and diversity of plant growth promoting bacteria increased during the growth stages. The observed α diversity and rhizobiome complexity increased significantly (p < 0.05) during plantation. PCoA indicates that different geographical locations shared similar rhizobiome diversity but exerted differential enrichment (p < 0.001). Diversity of enriched genera represented a sigmoid curve and subsequently declined after harvest. A major proportion of dominant enriched genera (p < 0.05, abundance > 0.1%), based on 16S rRNA gene, were plant growth promoting bacteria that produces siderophore, indole-3-acetic acid, aminocyclopropane-1-carboxylic acid, and antimicrobials. Hydrogenotrophic methanogens dominated throughout cultivation. Type I methanotrophs (n = 12) had higher diversity than type II methanotrophs (n = 6). However, the later had significantly higher abundance (p = 0.003). Strong enrichment pattern was also observed in type I methanotrophs being enriched during water logged stages. Ammonia oxidizing Archaea were several folds more abundant than ammonia oxidizing bacteria. K-strategists Nitrosospira and Nitrospira dominated ammonia and nitrite oxidizing bacteria, respectively. The study clarifies the modulation of rhizobiome according to the rice developmental stages, thereby opening up the possibilities of bio-fertilizer treatment based on each cultivation stages.

A multiscale study of fungal endophyte communities of the foliar endosphere of native rubber trees in Eastern Amazon.

Hevea brasiliensis is a native hyperdiverse tree species in the Amazon basin with great economic importance since it produces the highest quality natural rubber. H. brasiliensis, in its natural habitat, may harbor fungal endophytes that help defend against phytopathogenic fungi. In this work, we investigated the fungal endophytic communities in two pristine areas in Eastern Amazon (Anavilhanas National Park - ANP and Caxiuanã National Forest - CNF) at different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). Using a culture-based approach, 210 fungal endophytes were isolated from 240 sampling units and assigned to 46 distinct MOTUs based on sequencing of the nrITS DNA. The community compositions of the endophytomes are different at both regional and local scales, dominated by very few taxa and highly skewed toward rare taxa, with many endophytes infrequently isolated across hosts in sampled space. Colletotrichum sp. 1, a probably latent pathogen, was the most abundant endophytic putative species and was obtained from all individual host trees in both study areas. Although the second most abundant putative species differed between the two collection sites, Clonostachys sp. 1 and Trichoderma sp. 1, they are phylogenetically related (Hypocreales) mycoparasites. Thus, they probably exhibit the same ecological function in the foliar endosphere of rubber tree as antagonists of its fungal pathogens.

Fungal endophytes associated with three South American Myrtae (Myrtaceae) exhibit preferences in the colonization at leaf level.

Fungal endophytes associated with Myrtaceae from Brazil and Argentina were isolated at three levels of nesting: leaf, individual host trees, and site collection. The alternating logistic regression (ALR) was used to model the data because it offers a computationally convenient method for fitting regression structures involving large clusters. The objectives of this study were to determine: (i) whether the colonization pattern is influenced by environmental variables, (ii) if there is some leaf part they prefer to colonize; (iii) if there is some fungal endophyte aggregation between hierarchical levels; (iv) what the distance effect is on the fungal association. The environmental variables were statistically significant only for Xylaria, i.e., when the elevation and water precipitation increase and the temperature decreases, the odds ratio of finding another fungal endophyte of that genus previously found increases. Sordariomycetes, Xylariales, and Xylaria exhibited leaf fragment preference to petiole and tip. Fungal endophytes showed association within leaf. The horizontal transmission mode and the dispersal limitation may explain this association at the leaf level. Moreover, our results suggest that when a fungal endophyte infects a leaf or host tree individual, the odds ratio of dispersal inside them is greater.

Diversity and distribution of fungal communities in lakes of Antarctica.

This study assessed the diversity and distribution of filamentous fungi obtained from water sampled from six lakes in the Antarctic Peninsula. One hundred and twenty-eight fungal isolates were purified and identified by analysis of nuclear rDNA ITS region sequences as belonging to 31 fungal different operational taxonomic units (OTUs). The most frequently isolated fungi were Geomyces pannorum and Mortierella sp.; these species occurred in six and three of the lakes sampled, respectively, and displayed the highest total colony-forming unit per L. Different species that have not been found to these lakes and/or had adapted to cold conditions were found. In general, the fungal community displayed low richness and high dominance indices. The species Cadophora cf. luteo-olivacea, Cadophora malorum, Davidiella tassiana, G. pannorum, Mortierella cf. alpina and Thelebolus cf. microsporus that were found in the lakes in question were also previously found in other cold ecosystems, such as Arctic, temperate and Alpine regions. The results of this study suggest the presence of an interesting aquatic fungal web, including symbionts, weak and strong saprophytes and parasite/pathogen fungal species. This aquatic web fungal may be a useful community model for further ecological and evolutionary studies of extreme habitats.

Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes.

Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with high transparency, surrounded by well-developed forests and located at San Carlos de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we characterized yeast distribution and diversity along a south-to-north transect and established a relationship between the ability to produce photoprotective compounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of yeast at different collection points. Subsurface water samples were filtered for yeast isolation. Total yeast counts ranged between 22 and 141 CFU L(-1) , and the highest values corresponded to the most impacted sites. Littoral sites had a low proportion of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably a result of the high transparency of the water and the increased UV exposure. The yeast community from NH Lake showed a high species richness and a uniform distribution of taxa between pelagic and border collection points. Yeasts were identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and Cryptococcus victoriae were the most frequently found species, representing 14.4% and 13.6% of the total yeast isolates, respectively. Most of the yeast isolates demonstrated at least one extracellular enzymatic activity (mainly cellulase and lipase activities), which suggested that these microorganisms are metabolically active in the lake.

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors.

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7 percent produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 æM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60 percent at 20 æg/ml (59 and 90 æM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 æg/ml (0.6-1.5 mM).