Hidden Tenants: Microbiota of the Rhizosphere and Phyllosphere of Cordia dodecandra Trees in Mayan Forests and Homegardens.

During domestication, the selection of cultivated plants often reduces microbiota diversity compared with their wild ancestors. Microbiota in compartments such as the phyllosphere or rhizosphere can promote fruit tree health, growth, and development. Cordia dodecandra is a deciduous tree used by Maya people for its fruit and wood, growing, to date, in remnant forest fragments and homegardens (traditional agroforestry systems) in Yucatán. In this work, we evaluated the microbiota's alpha and beta diversity per compartment (phyllosphere and rhizosphere) and per population (forest and homegarden) in the Northeast and Southwest Yucatán regions. Eight composite DNA samples (per compartment/population/region combination) were amplified for 16S-RNA (bacteria) and ITS1-2 (fungi) and sequenced by Illumina MiSeq. Bioinformatic analyses were performed with QIIME and phyloseq. For bacteria and fungi, from 107,947 and 128,786 assembled sequences, 618 and 1092 operating taxonomic units (OTUs) were assigned, respectively. The alpha diversity of bacteria and fungi was highly variable among samples and was similar among compartments and populations. A significant species turnover among populations and regions was observed in the rhizosphere. The core microbiota from the phyllosphere was similar among populations and regions. Forests and homegarden populations are reservoirs of the C. dodecandra phyllosphere core microbiome and significant rhizosphere biodiversity.

Living in honey: bacterial and fungal communities in honey of sympatric populations of Apis mellifera and the stingless bee Melipona beecheii, in Yucatan, Mexico.

Bacterial and fungal communities in the honey of sympatric populations of the bee species Apis mellifera and Melipona beecheii were profiled by amplicon sequencing of the 16S gene and the ITS of the ribosomal DNA. Results showed that the structure of the honey microbiota of these two bee species was very different from each other. Both the bacterial and fungal species in A. mellifera honey were more similar to those of A. mellifera honey reported for other parts of the world than to those in M. beecheii honey. Nevertheless, in both, the most abundant bacterial species belonged to the family Lactobacillaeae.

Seasonal shifts of arbuscular mycorrhizal fungi in Cocos nucifera roots in Yucatan, Mexico.

The diversity and community structure of arbuscular mycorrhizal fungi (AMF) associated with coconut (Cocos nucifera) roots was evaluated by next generation sequencing (NGS) using partial sequences of the 18S rDNA gene and by spore isolation and morphological identification from rhizosphere soil. Root samples from six different Green Dwarf coconut plantations and from one organic plantation surrounded by tropical dry forest along the coastal sand dunes in Yucatan, Mexico, were collected during the rainy and dry seasons. In total, 14 root samples were sequenced with the Illumina MiSeq platform. Additionally, soil samples from the dry season were collected to identify AMF glomerospores. Based on a 95-97% similarity, a total of 36 virtual taxa (VT) belonging to nine genera were identified including one new genus-like clade. Glomus was the most abundant genus, both in number of VT and sequences. The comparison of dry and rainy season samples revealed differences in the richness and composition of AMF communities colonizing coconut roots. Our study shows that the main AMF genera associated with coconut tree roots in all samples were Glomus, Sclerocystis, Rhizophagus, Redeckera, and Diversispora. Based on glomerospore morphology, 22 morphospecies were recorded among which 14 were identified to species. Sclerocystis sinuosa, Sclerocystis rubiformis, Glomus microaggregatum, and Acaulospora scrobiculata were dominant in field rhizosphere samples. This is the first assessment of the composition of AMF communities colonizing coconut roots in rainy and dry seasons. It is of importance for selection of AMF species to investigate for their potential application in sustainable agriculture of coconut.

Data mining of metagenomes to find novel enzymes: a non-computationally intensive method.

Currently, there is a need of non-computationally-intensive bioinformatics tools to cope with the increase of large datasets produced by Next Generation Sequencing technologies. We present a simple and robust bioinformatics pipeline to search for novel enzymes in metagenomic sequences. The strategy is based on pattern searching using as reference conserved motifs coded as regular expressions. As a case study, we applied this scheme to search for novel proteases S8A in a publicly available metagenome. Briefly, (1) the metagenome was assembled and translated into amino acids; (2) patterns were matched using regular expressions; (3) retrieved sequences were annotated; and (4) diversity analyses were conducted. Following this pipeline, we were able to identify nine sequences containing an S8 catalytic triad, starting from a metagenome containing 9,921,136 Illumina reads. Identity of these nine sequences was confirmed by BLASTp against databases at NCBI and MEROPS. Identities ranged from 62 to 89% to their respective nearest ortholog, which belonged to phyla Proteobacteria, Actinobacteria, Planctomycetes, Bacterioidetes, and Cyanobacteria, consistent with the most abundant phyla reported for this metagenome. All these results support the idea that they all are novel S8 sequences and strongly suggest that our methodology is robust and suitable to detect novel enzymes.

A computationally simplistic poly-phasic approach to explore microbial communities from the Yucatan aquifer as a potential sources of novel natural products.

The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products.

Identification and in silico characterization of two novel genes encoding peptidases S8 found by functional screening in a metagenomic library of Yucatán underground water.

Metagenomics is a culture-independent technology that allows access to novel and potentially useful genetic resources from a wide range of unknown microorganisms. In this study, a fosmid metagenomic library of tropical underground water was constructed, and clones were functionally screened for extracellular proteolytic activity. One of the positive clones, containing a 41,614-bp insert, had two genes with 60% and 68% identity respectively with a peptidase S8 of Chitinimonas koreensis. When these genes were individually sub-cloned, in both cases their sub-clones showed proteolytic phenotype, confirming that they both encode functional proteases. These genes -named PrAY5 and PrAY6- are next to each other. They are similar in size (1845bp and 1824bp respectively) and share 66.5% identity. An extensive in silico characterization showed that their ORFs encode complex zymogens having a signal peptide at their 5' end, followed by a pro-peptide, a catalytic region, and a PPC domain at their 3' end. Their translated sequences were classified as peptidases S8A by sequence comparisons against the non-redundant database and corroborated by Pfam and MEROPS. Phylogenetic analysis of the catalytic region showed that they encode novel proteases that clustered with the sub-family S8_13, which according to the CDD database at NCBI, is an uncharacterized subfamily. They clustered in a clade different from the other three proteases S8 found so far by functional metagenomics, and also different from proteases S8 found in sequenced environmental samples, thereby expanding the range of potentially useful proteases that have been identified by metagenomics. I-TASSER modeling corroborated that they may be subtilases, thus possibly they participate in the hydrolysis of proteins with broad specificity for peptide bonds, and have a preference for a large uncharged residue in P1.

Yucatán in black and red: Linking edaphic analysis and pyrosequencing-based assessment of bacterial and fungal community structures in the two main kinds of soil of Yucatán State.

Yucatán State is dominated by two kinds of soil, named "Black Leptosol" and "Red Leptosol", which are interwoven across the State. In this work, we analyzed the relation between the edaphic characteristics and the bacterial and fungal community structures in these two kinds of Leptosol. The results revealed that Black Leptosol (BlaS) had a higher content of calcium carbonates, organic matter, nitrogen, and phosphorus than Red Leptosol (RedS). The most outstanding difference in the bacterial community structure between BlaS and RedS was that while in BlaS Actinobacteria was the most abundant phylum (43.7%), followed by Acidobacteria (26.9%) and Proteobacteria (23.6%), in RedS the bacterial community was strongly dominated by Acidobacteria (83%). Two fungal phyla were identified in both kinds of soil; Ascomycota, with 77% in BlaS and 56% in RedS, and Basidiomycota, with 22% in RedS and only 0.67% in BlaS. The most relevant difference between the two fungal communities was that excepting for Fusarium sp., all the species they had were different. Thus, in contrast with bacterial communities, where most of the major OTUs were present in both kinds of soil, fungal communities appeared to be unique to each kind of Leptosol.

Acidobacteria appear to dominate the microbiome of two sympatric Caribbean Sponges and one Zoanthid.

BACKGROUND: Marine invertebrate-associated microbial communities are interesting examples of complex symbiotic systems and are a potential source of biotechnological products. RESULTS: In this work, pyrosequencing-based assessment from bacterial community structures of sediments, two sponges, and one zoanthid collected in the Mexican Caribbean was performed. The results suggest that the bacterial diversity at the species level is higher in the sediments than in the animal samples. Analysis of bacterial communities' structure showed that about two thirds of the bacterial diversity in all the samples belongs to the phyla Acidobacteria and Proteobacteria. The genus Acidobacterium appears to dominate the bacterial community in all the samples, reaching almost 80% in the sponge Hyrtios. CONCLUSIONS: Our evidence suggests that the sympatric location of these benthonic species may lead to common bacterial structure features among their bacterial communities. The results may serve as a first insight to formulate hypotheses that lead to more extensive studies of sessile marine organisms' microbiomes from the Mexican Caribbean.

Acidobacteria appear to dominate the microbiome of two sympatric Caribbean Sponges and one Zoanthid

BACKGROUND: Marine invertebrate-associated microbial communities are interesting examples of complex symbiotic systems and are a potential source of biotechnological products. RESULTS: In this work, pyrosequencing-based assessment from bacterial community structures of sediments, two sponges, and one zoanthid collected in the Mexican Caribbean was performed. The results suggest that the bacterial diversity at the species level is higher in the sediments than in the animal samples. Analysis of bacterial communities' structure showed that about two thirds of the bacterial diversity in all the samples belongs to the phyla Acidobacteria and Proteobacteria. The genus Acidobacteriumappears to dominate the bacterial community in all the samples, reaching almost 80% in the sponge Hyrtios. CONCLUSIONS: Our evidence suggests that the sympatric location of these benthonic species may lead to common bacterial structure features among their bacterial communities. The results may serve as a first insight to formulate hypotheses that lead to more extensive studies of sessile marine organisms' microbiomes from the Mexican Caribbean.

Estudio de diversidad de accesiones de guayabo (psidium guajava L) mediante el marcador molecular ISTR / Studying the diversity of guava (psidium guajava L) accessions using ISTR molecular markers

Los estudios de variabilidad genética resultan útiles para el manejo racional del material, tanto para su conservación como para el mejoramiento. La Repetición de Secuencias Inversas Marcadas (ISTR) es una técnica basada en la PCR que permite el estudio de la diversidad genética de individuos y poblaciones; identificación de cultivares, entre otras aplicaciones. En este sentido, el objetivo del presente trabajo fue estudiar la diversidad de accesiones de guayabo empleando este marcador molecular. Para el análisis de los datos se generó una matriz de ausencia-presencia de las bandas polimórficas, a partir de la cual se desarrolló un análisis de agrupamiento basado en el coeficiente de Jaccard y el método UPGMA para la construcción del dendrograma con el programa NTSYS-pc. La evaluación de los genotipos de guayabo con el marcador ISTR generó un total de 52 bandas polimórficas, las cuales permitieron diferenciar todos los materiales evaluados, corroborando la utilidad de esta técnica para la identificación de accesiones en la especie. El análisis de agrupamiento permitió evidenciar la formación de cuatro grupos de diversidad definidos y la presencia de cuatro accesiones externas. Las diferencias encontradas en el agrupamiento de las accesiones por ISTR respecto a las obtenidas previamente por AFLP y SSR sugieren que el elegir la técnica más apropiada para determinados estudios puede resultar un proceso difícil. Los resultados discutidos en este trabajo indican la necesidad de realizar estudios integrados en el banco de germoplasma de este cultivo para lograr un manejo más racional de la base genética del mismo.

Studies about genetic variability can be useful for material rational management, as much as for conservation and breeding. The Inverse Sequence Tagged Repeats (ISTR) is a technique based on PCR, which permit the study of genetic diversity for individuals and populations; cultivars identification, within other applications. In this sense, the objective of the present work was to study guava accessions diversity using this molecular marker. For data analysis, an absence-presence matrix for polymorphic bands was generated, from which a cluster analysis was developed, based on Jaccard coefficient and UPGMA method for dendrogram construction using NTSYS-pc program. Guava genotypes evaluation with ISTR marker generated a total of 52 polymorphic bands, which permitted to differentiated all the materials evaluated, corroborating the utility of this technique for accessions identification in the specie. Cluster analysis permitted to evidence the formation of four defined diversity groups and the presence of four external accessions. The differences encountered for accessions clustering with ISTR respected to the ones obtained previously for AFLP and SSR suggest that the election of the most appropriated technique for determinate studies can result a difficult process. The results discussed in this work indicate the necessity to made integrated studies on the germplasm bank of this crop to obtain a more adequate management.