Impact of Endochitinase-Transformed White Spruce on Soil Fungal Biomass and Ectendomycorrhizal Symbiosis▿

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp

Culture-Dependant and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods

Contrasting the Community Structure of Arbuscular Mycorrhizal Fungi from Hydrocarbon-Contaminated and Uncontaminated Soils following Willow (<i>Salix</i> spp. L.) Planting

<div><p>Phytoremediation is a potentially inexpensive alternative to chemical treatment of hydrocarbon-contaminated soils, but its success depends heavily on identifying factors that govern the success of root-associated microorganisms involved in hydrocarbon degradation and plant growth stimulation. Arbuscular mycorrhizal fungi (AMF) form symbioses with many terrestrial plants, and are known to stimulate plant growth, although both species identity and the environment influence this relationship. Although AMF are suspected to play a role in plant adaptation to hydrocarbon contamination, their distribution in hydrocarbon-contaminated soils is not well known. In this study, we examined how AMF communities were structured within the rhizosphere of 11 introduced willow cultivars as well as unplanted controls across uncontaminated and hydrocarbon-contaminated soils at the site of a former petrochemical plant. We obtained 69 282 AMF-specific 18S rDNA sequences using 454-pyrosequencing, representing 27 OTUs. Contaminant concentration was the major influence on AMF community structure, with different AMF families dominating at each contaminant level. The most abundant operational taxonomic unit in each sample represented a large proportion of the total community, and this proportion was positively associated with increasing contamination, and seemingly, by planting as well. The most contaminated soils were dominated by three phylotypes closely related to <i>Rhizophagus irregularis</i>, while these OTUs represented only a small proportion of sequences in uncontaminated and moderately contaminated soils. These results suggest that <i>in situ</i> inoculation of AMF strains could be an important component of phytoremediation treatments, but that strains should be selected from the narrow group that is both adapted to contaminant toxicity and able to compete with indigenous AMF species.</p></div

Mean relative abundance of AMF families from all samples in the uncontaminated blocks (N1, N3, N5; n = 12 per block), the moderately contaminated block (C3; n = 12), and the highly contaminated blocks (C4, C5; n = 3 per block).

<p>Labels underneath block numbers indicate no hydrocarbon contamination (NC), low hydrocarbon contamination (LC), and high hydrocarbon contamination (HC).</p

Measures of the AMF phylogenetic structure recorded across soils with no hydrocarbon contamination (NC), low hydrocarbon contamination (LC), and high hydrocarbon contamination (HC).

1<p>NC: not contaminated; LC: low contamination; HC: high contamination.</p>2<p>Standard effect size of the mean pairwise distance.</p

Redundancy analysis (RDA) showing the relationship between contaminant level, willow cultivar identity, and the abundance of AMF OTUs.

<p>The adjusted R² value indicates the amount of variance in AMF community composition accounted for by the constraining variables (contaminant level and willow identity). The location of contaminant level and cultivar labels represent factor centroids. Red numbers indicate AMF OTUs, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102838#pone-0102838-g001" target="_blank">Figure 1</a>. Black open triangles represent individual samples.</p

Bayesian phylogenetic tree based on nuclear small subunit (SSU) rDNA consensus sequences showing the distribution of the 27 OTUs recorded in Varennes (red labels) among the <i>Glomeromycota</i> tree.

<p>Sequence data were analysed with the SSU sequences (black labels) from Krüger et al. (2012) and the closest match recovered from MaarjAM database. Circles on nodes indicate Bayesian posterior probabilities ranging from 0.9 (white) to 1 (black). Node size also decreases with decreasing probabilities. The scale represents the branch length corresponding to expected substitutions per site.</p

Mean relative abundance of most dominant OTU across blocks.

<p>Error bars indicate standard error. Labels underneath block numbers indicate no hydrocarbon contamination (NC), low hydrocarbon contamination (LC), and high hydrocarbon contamination (HC).</p

Composition of the standard and selective media (based on one liter of medium) used to isolate soil fungi.

<p>Each medium contained 200 mg / L of streptomycin, and 100 mg / L of ampicillin.</p><p>Composition of the standard and selective media (based on one liter of medium) used to isolate soil fungi.</p

Proportional Venn diagram showing the distribution of bacterial (A) and fungal (B) OTUs between culture-independent (CI, light grey) and culture-dependent (CD, dark grey) methods.

<p>Heatmap distribution of the relative abundance of bacterial (C) and fungal (D) OTUs recorded with CI (light grey) and CD (dark grey) methods. OTUs are in rows and colour intensity indicates relative abundance, with black indicating the highest relative abundances observed for slightly contaminated (SC), contaminated (C) and highly contaminated (HC) composite soil sediments recovered with CI and CD methods. OTUs are presented by descending number of reads from top to bottom, based on proportion of total reads recorded in CI and CD datasets. The top dendrogram shows the hierarchical clustering of bacterial and fungal communities recovered for each PAH contamination level based on Bray-Curtis dissimilarity.</p