Deirdre Gleeson

Low Pore Connectivity Increases Bacterial Diversity in Soil

ABSTRACT One of soil microbiologys most intriguing puzzles is how so many different bacterial species can coexist in small volumes of soil when competition theory predicts that less competitive species should decline and eventually disappear. We provide evidence supporting the theory that low pore connectivity caused by low water potential (and therefore low water content) increases the diversity of a complex bacterial community in soil. We altered the pore connectivity of a soil by decreasing water potential and increasing the content of silt- and clay-sized particles. Two textures were created, without altering the chemical properties or mineral composition of the soil, by adding silt- and clay-sized particles of quartz to a quartz-based sandy soil at rates of 0% (sand) or 10% (silt+clay). Both textures were incubated at several water potentials, and the effect on the active bacterial communities was measured using terminal restriction fragment length polymorphism (TRFLP) of bacterial 16S rRNA. Bacterial richness and diversity increased as water potential decreased and soil became drier (P < 0.012), but they were not affected by texture (P > 0.553). Bacterial diversity increased at water potentials of ≤2.5 kPa in sand and ≤4.0 kPa in silt+clay, equivalent to ≤56% water-filled pore space (WFPS) in both textures. The bacterial community structure in soil was affected by both water potential and texture (P < 0.001) and was correlated with WFPS (sum of squared correlations [δ2] = 0.88, P < 0.001). These findings suggest that low pore connectivity is commonly experienced by soil bacteria under field conditions and that the theory of pore connectivity may provide a fundamental principle to explain the high diversity of bacteria in soil.

Characterization of Fungal Community Structure on a Weathered Pegmatitic Granite

This study exploited the contrasting major element chemistry of adjacent, physically separable crystals of framework and sheet silicates in a pegmatitic granite to investigate the mineralogical influences of fungal community structure on mineral surfaces. Large intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were individually extracted, together with whole-rock granite. Environmental scanning electron microscopy (ESEM) revealed a diversity of fungal structures, with microcolonial fungi and fungal hyphae clearly visible on surfaces of all mineral types. Fungal automated ribosomal intergenic spacer analysis (FARISA) was used to generate a ribotype profile for each mineral sample and a randomization test revealed that ribotype profiles, or community fingerprints, differed between different mineral types. Canonical correspondence analysis (CCA) revealed that mineral chemistry affected individual fungal ribotypes, and strong relationships were found between certain ribotypes and particular chemical elements. This finding was further supported by analysis of variance (ANOVA) of the 16 most abundant ribotypes within the community. Significantly, individual ribotypes were largely restricted to single mineral types and ribotypes clustered strongly on the basis of mineral type. CCA also revealed that Al, Si, and Ca had a significant impact on fungal community structure within this system. These results show that fungal community structure was driven by the chemical composition of mineral substrates, indicating selective pressure by individual chemical elements on fungal populations in situ.

Minerals in soil select distinct bacterial communities in their microhabitats.

We tested the hypothesis that different minerals in soil select distinct bacterial communities in their microhabitats. Mica (M), basalt (B) and rock phosphate (RP) were incubated separately in soil planted with Trifolium subterraneum, Lolium rigidum or left unplanted. After 70 days, the mineral and soil fractions were separated by sieving. Automated ribosomal intergenic spacer analysis was used to determine whether the bacterial community structure was affected by the mineral, fraction and plant treatments. Principal coordinate plots showed clustering of bacterial communities from different fraction and mineral treatments, but not from different plant treatments. Permutational multivariate anova (permanova) showed that the microhabitats of M, B and RP selected bacterial communities different from each other in unplanted and L. rigidum, and in T. subterraneum, bacterial communities from M and B differed (P<0.046). permanova also showed that each mineral fraction selected bacterial communities different from the surrounding soil fraction (P<0.05). This study shows that the structure of bacterial communities in soil is influenced by the mineral substrates in their microhabitat and that minerals in soil play a greater role in bacterial ecology than simply providing an inert matrix for bacterial growth. This study suggests that mineral heterogeneity in soil contributes to the spatial variation in bacterial communities.

Soil Microbial Community Successional Patterns during Forest Ecosystem Restoration

ABSTRACT Soil microbial community characterization is increasingly being used to determine the responses of soils to stress and disturbances and to assess ecosystem sustainability. However, there is little experimental evidence to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration. This study utilized a chronosequence of developing jarrah (Eucalyptus marginata) forest ecosystems, rehabilitated after bauxite mining (up to 18 years old), to examine changes in soil bacterial and fungal community structures (by automated ribosomal intergenic spacer analysis [ARISA]) and changes in specific soil bacterial phyla by 16S rRNA gene microarray analysis. This study demonstrated that mining in these ecosystems significantly altered soil bacterial and fungal community structures. The hypothesis that the soil microbial community structures would become more similar to those of the surrounding nonmined forest with rehabilitation age was broadly supported by shifts in the bacterial but not the fungal community. Microarray analysis enabled the identification of clear successional trends in the bacterial community at the phylum level and supported the finding of an increase in similarity to nonmined forest soil with rehabilitation age. Changes in soil microbial community structure were significantly related to the size of the microbial biomass as well as numerous edaphic variables (including pH and C, N, and P nutrient concentrations). These findings suggest that soil bacterial community dynamics follow a pattern in developing ecosystems that may be predictable and can be conceptualized as providing an integrated assessment of numerous edaphic variables.

Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost

Cold, salt and frost are important environmental stresses in forest trees and may significantly reduce productivity. Elevated levels of proline are associated with these stresses and may help alleviate their effects. Transgenic hybrid larch Larix leptoeuropaea has been produced expressing a Vigna aconitifolia gene for pyrroline 5-carboxylate synthase, the rate-limiting step in proline synthesis. Embryogenic masses of hybrid larch were co-cultivated with Agrobacterium tumefaciens harbouring a binary vector expressing the gene. The integration of the gene into the plant genome was confirmed by Southern blot and by proline content analysis. There was an approximately 30-fold increase in proline level in transgenic tissue compared to non-transformed controls. The transgenic tissue lines were significantly more resistant to cold, salt, and freezing stresses and grew under conditions (200mM NaCl or 4 °C) that completely inhibited the growth of control cell lines. Our results indicated that introduction of proline over-accumulation into forest trees might be an effective strategy for ameliorating the effects of environmental stresses.

Meter-Scale Diversity of Microbial Communities on a Weathered Pegmatite Granite Outcrop in the Wicklow Mountains, Ireland; Evidence for Mineral Induced Selection?

In this study we examined the microbial community composition developed on three different silicate minerals (muscovite, feldspar and quartz) sampled from three spatially separate domains along a sub-vertical 10 metre long pegmatitic granite outcrop. We investigated the influence of mineral type on bacterial and fungal community structure. A DNA-based community fingerprinting approach (ARISA—automated ribosomal intergenic spacer analysis) was used to assess the nature and extent of microbial diversity. The molecular biology approach was combined with multivariate statistics: canonical analysis of principal coordinates (CAP) and permutational multivariate analysis of variance (PERMANOVA) to identify the main geochemical factors that influence microbial community structure in situ. We found that bacterial and fungal community structure was strongly influenced by the chemistry of the silicate mineral, with many bacterial and fungal ribotypes limited to a single mineral type, and to a much lesser extent by the sampling location along the outcrop. The novelty of our findings is that mineral type exerted a very strong selection on bacterial and fungal community structure on the length scales ranging from a few centimetres to several metres. A number of 49 bacterial and 45 fungal strains were isolated from the studied mineral surfaces. Several archaea clones belonging to Crenarchaeota phylum were retrieved from the mineral samples and soil.

Microbialite taphonomy and biogenicity: new insights from NanoSIMS

We here show that nano-scale mapping of elements commonly utilized in biological cycles provides a promising new additional line of evidence when evaluating the extent of the contribution of biology to microbialites. Our case study comes from Lake Clifton in Western Australia, a unique environment where living domical and conical microbialites occur in close proximity to ≤ 4000-year-old fossilized equivalents. The outer margins of a partially lithified, actively growing Lake Clifton microbialite are characterized by abundant filamentous cyanobacteria within a loosely cemented aragonite matrix. Nano-scale chemical maps have been successfully matched to specific morphological features such as trichomes, sheaths and putative extracellular polymeric substances (EPS). A suite of elements (C, O, Mg, N, Si, S) is concentrated within cyanobacterial sheaths, with carbon, magnesium, nitrogen and sulfur also enriched within trichomes and putative EPS. Calcium distribution highlights the sites of aragonite mineralization. In contrast, the fossilized Lake Clifton microbialite contains only rare, extensively degraded cyanobacterial filaments, the mean diameter of which is <50% of the living equivalents. Nevertheless, nano-scale chemical maps can again be matched with morphological features. Here, poorly preserved filamentous microfossils are highlighted by enrichments in nitrogen and sulfur. Magnesium is no longer concentrated within the filaments, instead it co-occurs with calcium and oxygen in the calcite cement. Extension of this study to a ~2720-million-year-old stromatolitic microbialite from the Tumbiana Formation of Western Australia shows that similar nano-scale signals, in particular nitrogen and sulfur enrichments, are characteristic of stromatolite laminations, even when morphological microfossils are absent. The close similarities of nano-scale elemental distributions in organic material from modern and ancient microbialites show that this technique provides a valuable addition to the morphological investigation of such structures, particularly in non-fossiliferous ancient examples.

Effect of Sheep Urine Deposition on the Bacterial Community Structure in an Acidic Upland Grassland Soil

ABSTRACT The effect of the addition of synthetic sheep urine (SSU) and plant species on the bacterial community composition of upland acidic grasslands was studied using a microcosm approach. Low, medium, and high concentrations of SSU were applied to pots containing plant species typical of both unimproved (Agrostis capillaris) and agriculturally improved (Lolium perenne) grasslands, and harvests were carried out 10 days and 50 days after the addition of SSU. SSU application significantly increased both soil pH (P < 0.005), with pH values ranging from pH 5.4 (zero SSU) to pH 6.4 (high SSU), and microbial activity (P < 0.005), with treatment with medium and high levels of SSU displaying significantly higher microbial activity (triphenylformazan dehydrogenase activity) than treatment of soil with zero or low concentrations of SSU. Microbial biomass, however, was not significantly altered by any of the SSU applications. Plant species alone had no effect on microbial biomass or activity. Bacterial community structure was profiled using bacterial automated ribosomal intergenic spacer analysis. Multidimensional scaling plots indicated that applications of high concentrations of SSU significantly altered the bacterial community composition in the presence of plant species but at different times: 10 days after application of high concentrations of SSU, the bacterial community composition of L. perenne-planted soils differed significantly from those of any other soils, whereas in the case of A. capillaris-planted soils, the bacterial community composition was different 50 days after treatment with high concentrations of SSU. Canonical correspondence analysis also highlighted the importance of interactions between SSU addition, plant species, and time in the bacterial community structure. This study has shown that the response of plants and bacterial communities to sheep urine deposition in grasslands is dependent on both the grass species present and the concentration of SSU applied, which may have important ecological consequences for agricultural grasslands.

Fine endophytes (Glomus tenue) are related to Mucoromycotina, not Glomeromycota

Fine endophytes (FE),Glomus tenue, are traditionally considered to be arbuscular mycorrhizal fungi (AMF) with distinctive microscopic morphology when stained. FE have fine hyphae (c. 1.5 lm diameter) which branch intra-cellularly in a distinctive fan-like pattern (Gianinazzi-Pearson et al., 1981; Abbott, 1982). The hyphae contain small swellings along their length, sometimes referred to as vesicle-like swellings (Hall, 1977). FE form arbuscules (or arbuscule-like structures) with fine elements in a tapered, conical shape (Greenall, 1963; Merryweather & Fitter, 1998). Spores of FE are very small (< 20 lm) compared to the majority of Glomeromycota, and colourless (Hall, 1977). Morphological variations indicate that FE may consist of multiple species (Thippayarugs et al., 1999), hence we use the term FE to indicate a species group. Within the kingdom Fungi, both morphological and genetic characteristics are used to determine taxonomic classification (St€ urmer, 2012). In 2001, all AMFwere placed within the phylum Glomeromycota (Sch€ ußler et al., 2001). In the listing of glomeromycotan species by Sch€ ußler &Walker (2010), some members of the genusGlomuswere not revised due to insufficient taxonomic knowledge, and this included FE. A key reason for classifying FE within the Glomeromycota was the presence of arbuscules, considered apomorphic for the phylum (Morton, 1990).However, the morphological features of root colonization by FE are distinct from other, coarse, AMF so their placement within the genus Glomus and the Glomeromycota was questioned (Hall, 1977; Sch€ ußler &Walker, 2010), and their status as mycorrhizal fungi is ambivalent. Accurate determination of FE usually requires magnification ≥9100, hence, where assessments of AMF colonization use lower magnifications theymay not be identified. Furthermore, FEmaybe undetected if samples are not processed within 2 d of harvesting (Orchard et al., 2016a). Nevertheless, FE are globally distributed and prolific within many ecosystems, examples include: pastures and native bushland of New Zealand (Crush, 1973) and Australia (Abbott&Robson, 1982;McGee, 1989),Venezuelan cloud forests (Rabatin et al., 1993), riverine and alpine regions of Europe (Read &Haselwandter, 1981; Turnau et al., 1999; Binet et al., 2011) and an old-field in the United States (Hilbig &Allen, 2015). However, the difficulty of isolating and, hence, genetically characterizing FE has hindered the determination of their phylogenetic placement.

Afforestation alters community structure of soil fungi.

Relatively little is known about the effect of afforestation on soil fungal communities. This study demonstrated that afforestation altered fungal community structure and that changes were correlated to pools of soil C. Pasture at three locations on the same soil type was afforested with Eucalyptus globulus or Pinus pinaster. The structure of fungal communities under the three land uses was measured after 13y using automated ribosomal intergenic spacer analysis (ARISA). Afforestation significantly altered the structure of fungal communities. The effect of location on the structure of fungal communities was limited to pasture soils; although these contained the same plant species, the relative composition of each species varied between locations. Differences in the structure of fungal communities between pasture, E. globulus and P. pinaster were significantly correlated with changes in the amount of total organic C and microbial biomass-C in soil. Afforestation of patches of agricultural land may contribute to conserving soil fungi in agricultural landscapes by supporting fungal communities with different composition to agricultural soils.