Predicting the HMA-LMA status in marine sponges by machine learning

The dichotomy between high microbial abundance (HMA) and low microbial abundance (LMA) sponges has been observed in sponge-microbe symbiosis, although the extent of this pattern remains poorly unknown. We characterized the differences between the microbiomes of HMA (n=19) and LMA (n=17) sponges (575 specimens) present in the Sponge Microbiome Project. HMA sponges were associated with richer and more diverse microbiomes than LMA sponges, as indicated by the comparison of alpha diversity metrics. Microbial community structures differed between HMA and LMA sponges considering Operational Taxonomic Units (OTU) abundances and across microbial taxonomic levels, from phylum to species. The largest proportion of microbiome variation was explained by the host identity. Several phyla, classes, and OTUs were found differentially abundant in either group, which were considered “HMA indicators” and “LMA indicators”. Machine learning algorithms (classifiers) were trained to predict the HMA-LMA status of sponges. Among nine different classifiers, higher performances were achieved by Random Forest trained with phylum and class abundances. Random Forest with optimized parameters predicted the HMA-LMA status of additional 135 sponge species (1,232 specimens) without a priori knowledge. These sponges were grouped in four clusters, from which the largest two were composed of species consistently predicted as HMA (n=44) and LMA (n=74). In summary, our analyses shown distinct features of the microbial communities associated with HMA and LMA sponges. The prediction of the HMA-LMA status based on the microbiome profiles of sponges demonstrates the application of machine learning to explore patterns of host-associated microbial communities

Dynamics of Seed-Borne Rice Endophytes on Early Plant Growth Stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed

Phylogenetically and spatially close marine sponges harbour divergent bacterial communities

Recent studies have unravelled the diversity of sponge-associated bacteria that may play essential roles in sponge health and metabolism. Nevertheless, our understanding of this microbiota remains limited to a few host species found in restricted geographical localities, and the extent to which the sponge host determines the composition of its own microbiome remains a matter of debate. We address bacterial abundance and diversity of two temperate marine sponges belonging to the Irciniidae family - Sarcotragus spinosulus and Ircinia variabilis – in the Northeast Atlantic. Epifluorescence microscopy revealed that S. spinosulus hosted significantly more prokaryotic cells than I. variabilis and that prokaryotic abundance in both species was about 4 orders of magnitude higher than in seawater. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of S. spinosulus and I. variabilis differed markedly from each other – with higher number of ribotypes observed in S. spinosulus – and from those of seawater. Four PCR-DGGE bands, two specific to S. spinosulus, one specific to I. variabilis, and one present in both sponge species, affiliated with an uncultured sponge-specific phylogenetic cluster in the order Acidimicrobiales (Actinobacteria). Two PCR-DGGE bands present exclusively in S. spinosulus fingerprints affiliated with one sponge-specific phylogenetic cluster in the phylum Chloroflexi and with sponge-derived sequences in the order Chromatiales (Gammaproteobacteria), respectively. One Alphaproteobacteria band specific to S. spinosulus was placed in an uncultured sponge-specific phylogenetic cluster with a close relationship to the genus Rhodovulum. Our results confirm the hypothesized host-specific composition of bacterial communities between phylogenetically and spatially close sponge species in the Irciniidae family, with S. spinosulus displaying higher bacterial community diversity and distinctiveness than I. variabilis. These findings suggest a pivotal host-driven effect on the shape of the marine sponge microbiome, bearing implications to our current understanding of the distribution of microbial genetic resources in the marine realm.This work was financed by the Portuguese Foundation for Science and Technology (FCT - http://www.fct.pt) through the research project PTDC/MAR/101431/2008. CCPH has a PhD fellowship granted by FCT (Grant No. SFRH/BD/60873/2009). JRX’s research is funded by a FCT postdoctoral fellowship (grant no. SFRH/BPD/62946/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Identification of isolated seed-borne strains.

a<p>Rice strains isolated from first (R1-R4) and second (R5-R16) generation of seeds.</p><p>*The 16S rRNA gene sequences of strains R6 and R8 were identical to PCR-DGGE products of the bands 12 and 9, respectively.</p>b<p>Source of the closest rice associated bacteria, LE – Leaf Endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; LS – Leaf surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; PF – Paddy Field (Islam et al., unpublished); PS – Paddy Soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Shrestha1" target="_blank">[28]</a>; R - Rhizosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Steindler1" target="_blank">[25]</a>; RE1 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Hardoim2" target="_blank">[20]</a>; RE2 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a> and SE – Seed endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano1" target="_blank">[5]</a>.</p

Biplot ordination diagrams of rice shoot and root bacterial endophytes.

<p>RDA diagrams generated from PCR-DGGE profiles of endophytic bacterial community sampled from shoot (A and B) and root (C and D) tissues of plants cultivated on K (A and C) and V (B and D) soils are shown. Squares and circle represent PCR-DGGE patterns of bacterial communities from plants submitted to, respectively, flooded and unflooded regimes and exposed to low- (empty symbol) and high- (full symbol) BID. Triangles (control treatment) represent PCR-DGGE patterns of bacterial communities from plants submitted to unflooded regime and cultivated in uninoculated soils. Six replicates of each treatment are shown. Stars represent nominal environmental variables. Arrows represent PCR-DGGE bands in which only the most descriptive communities are shown.</p

Heat map composition of selected bacterial communities.

<p>Distribution of select endophytic bacterial communities (rows) from two soil types (K and V) and four different habitats (root-free and rhizosphere soil, root and shoot endosphere) is shown. Cells are coloured in spectrum of grey that correlates with percentage of observed bacterium in a given habitat. Habitat from which the assessed bacterium was most likely to be originated from ‘artificial’ soil community is labelled with “inoculated”. Unlabelled cells are most likely represented by assessed bacterium originated from rice seeds.</p

Identification of excised PCR-DGGE bands.

a<p>Source of the closest rice associated bacteria: PF – Paddy Field <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Cuong1" target="_blank">[65]</a>; PS – Paddy Soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Shrestha1" target="_blank">[28]</a>; R - Rhizosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Steindler1" target="_blank">[25]</a>; RE1 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Hardoim2" target="_blank">[20]</a> and RE2 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Sun1" target="_blank">[64]</a>.</p

Closest match of sequences obtained in this study against public available rice and <i>Zea</i> seed endophyte sequences.

<p>Closest match of sequences obtained in this study against public available rice and <i>Zea</i> seed endophyte sequences.</p

Dynamics of rice endophytes as revealed by PCR-DGGE profiles of seed, three- and five-week-old rice plants.

<p>Rice endophyte PCR-DGGE patterns of surface-sterilized dehulled seeds and five-day-old shoot, root and remainder of the seeds from two consecutive generations are shown (panel A). PCR-DGGE patterns of root and shoot endosphere community of three- B) and five- C) week-old rice plants cultivated in two soil types. Six replicates per treatments are shown. Arrow heads indicate identified communities from excised PCR-DGGE bands (only numbers) and strains with identical motility (preceded by letter R; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone-0030438-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone-0030438-t002" target="_blank">2</a>), M – marker with a selection of 15 endophyte ribotypes (panel A).</p

Biplot ordination diagrams of rice rhizosphere and bulk soil bacterial communities.

<p>RDA diagrams generated from PCR-DGGE profiles of bacterial community sampled from rhizosphere (A and B) and bulk (C and D) soil of plants cultivated in K (A and C) and V (B and D) soils are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone-0030438-g003" target="_blank">Fig. 3</a> for symbol description.</p