Daniel L. Mummey

The invasive plant species Centaurea maculosa alters arbuscular mycorrhizal fungal communities in the field

While several recent studies have described changes in microbial communities associated with exotic plant invasion, how arbuscular mycorrhizal fungi (AMF) communities respond to exotic plant invasion is not well known, despite the salient role of this group in plant interactions. Here, we use molecular methods (terminal restriction fragment length polymorphism analyses based on the large subunit of the rRNA gene) to examine AMF community structure in sites dominated by the invasive mycorrhizal forb, Centaurea maculosa Lam. (spotted knapweed), and in adjacent native grassland sites. Our results indicate that significant AMF community alteration occurs following C. maculosa invasion. Moreover, a significant reduction in the number of restriction fragment sizes was found for samples collected in C. maculosa-dominated areas, suggesting reduced AMF diversity. Extraradical hyphal lengths exhibited a significant, on average 24%, reduction in C. maculosa-versus native grass-dominated sites. As both AMF community composition and abundance were altered by C.maculosa invasion, these data are strongly suggestive of potential impacts on AMF-mediated ecosystem processes. Given that the composition of AMF communities has the potential to differentially influence different plant species, our results may have important implications for site restoration after weed invasion.

Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis

Controls on root colonization by arbuscular mycorrhizal fungi (AMF) include host nutrient status, identity of symbionts and soil physico-chemical properties. Here we show, in the field, that the subset of the AMF community colonizing the roots of a common grass species, Dactylis glomerata, was strongly controlled by neighboring roots of a different plant species, Centaurea maculosa, an invasive forb, thus adding a biological spatial component to controls on root colonization. Using an AMF-specific, 18s rDNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis method, significant differences were found between AMF community fingerprints of samples derived from roots of grasses with (GCm) and without (G0) neighboring C. maculosa. There were also significant differences between samples derived from C. maculosa roots (Cmac) and both GCm and G0 roots. Sample ordination indicated three generally distinct groups consisting of Cmac, GCm and G0, with GCm samples being of intermediate distance between G0and Cmac. Our results indicate that, with the presence of C. maculosa, AMF communities of D. glomerata shift to reflect community composition associated with C. maculosa roots. These results highlight the importance of complex spatial distributions of AMF communities at the scale of a root system. An additional dimension to our study is that C. maculosa is an aggressively invasive plant in the intermountain West. Viewed in this light, these results suggest that pervasive influences of this plant on AMF communities, specifically in roots of its competitors, may represent a mechanism contributing to its invasive success. However, further work is clearly required to determine the extent to which AMF genotypic alteration by neighboring plants influences competitive relationships.

Spatial Stratification of Soil Bacterial Populations in Aggregates of Diverse Soils

Most soil microbial community studies to date have focused on homogenized bulk soil samples. However, it is likely that many important microbial processes occur in spatially segregated microenvironments in the soil leading to a microscale biogeography. This study attempts to localize specific microbial populations to different fractions or compartments within the soil matrix. Microbial populations associated with macroaggregates and inner- versus total-microaggregates of three diverse soils were characterized using culture-independent, molecular methods. Despite their relative paucity in most surveys of soil diversity, representatives of Gemmatimonadetes and Actinobacteria subdivision Rubrobacteridae were found to be highly abundant in inner-microaggregates of most soils analyzed. By contrast, clones affiliated with Acidobacteria were found to be relatively enriched in libraries derived from macroaggregate fractions of nearly all soils, but poorly represented in inner-microaggregate fractions. Based upon analysis of 16S rRNA, active community members within microaggregates of a Georgian Ultisol were comprised largely of Gemmatimonadetes and Rubrobacteridae, while within microaggregates of a Nebraska Mollisol, Rubrobacteridae and Alphaproteobacteria were the predominant active bacterial lineages. This work suggests that microaggregates represent a unique microenvironment that selects for specific microbial lineages across disparate soils.

Spatial characterization of arbuscular mycorrhizal fungal molecular diversity at the submetre scale in a temperate grassland

Although arbuscular mycorrhizal fungi (AMF) form spatially complex communities in terrestrial ecosystems, the scales at which this diversity manifests itself is poorly understood. This information is critical to the understanding of the role of AMF in plant community composition. We examined small-scale (submetre) variability of AMF community composition (terminal restriction fragment length polymorphism fingerprinting) and abundance (extraradical hyphal lengths) in two 1 m(2) plots situated in a native grassland ecosystem of western Montana. Extraradical AMF hyphal lengths varied greatly between samples (14-89 m g soil(-1)) and exhibited spatial structure at scales <30 cm. The composition of AMF communities was also found to exhibit significant spatial autocorrelation, with correlogram analyses suggesting patchiness at scales <50 cm. Supportive of overall AMF community composition analyses, individual AMF ribotypes corresponding to specific phylogenetic groups exhibited distinct spatial autocorrelation. Our results demonstrate that AMF diversity and abundance can be spatially structured at scales of <1 m. Such small-scale heterogeneity in the soil suggests that establishing seedlings may be exposed to very different, location dependent AMF communities. Our results also have direct implications for representative sampling of AMF communities in the field.

Culture-Independent Analysis of Midgut Microbiota in the Arbovirus Vector Culicoides sonorensis (Diptera: Ceratopogonidae)

Abstract Differences in midgut microbial communities inhabiting Culicoides spp., insect vectors of virus pathogens, may affect the variation observed in the ability of these biting midges to propagate arthropod-borne viruses. As a first step toward addressing this hypothesis, midgut bacterial communities were compared between Culicoides species expected to be efficient and inefficient vectors of virus pathogens. We used 16S rDNA sequence and restriction fragment information to provisionally identify 36 bacterial genera from guts of wild adult female biting midges, Culicoides sonorensis Wirth and Jones and Culicoides variipennis (Coquillet), from two geographical locations. Bacterial identification was made by sequence analysis of 16S rDNA fragments and by terminal restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S rDNA fragments from adult guts. Of 36 bacterial genera identified, 12 had been previously identified in other insects: Comomonas, Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Stenotrophomonas, Staphylococcus, Chryseobacterium, Moraxella, Acholeplasma, Flavobacterium, and Rickettsia. Significant differences in bacterial community composition were found between all three groups of wild adult females analyzed: live-trapped C. sonorensis, laboratory-emerged C. sonorensis, and laboratory-emerged C. variipennis.

The effects of arbuscular mycorrhizal (AM) fungal and garlic mustard introductions on native AM fungal diversity

Introduced, non-native organisms are of global concern, because biological invasions can negatively affect local communities. Arbuscular mycorrhizal (AM) fungal communities have not been well studied in this context. AM fungi are abundant in most soils, forming symbiotic root-associations with many plant species. Commercial AM fungal inocula are increasingly spread worldwide, because of potentially beneficial effects on plant growth. In contrast, some invasive plant species, such as the non-mycorrhizal Alliaria petiolata, can negatively influence AM fungi. In a greenhouse study we examined changes in the structure of a local Canadian AM fungal community in response to inoculation by foreign AM fungi and the manipulated presence/absence of A. petiolata. We expected A. petiolata to have a stronger effect on the local AM fungal community than the addition of foreign AM fungal isolates. Molecular analyses indicated that inoculated foreign AM fungi successfully established and decreased molecular diversity of the local AM fungal community in host roots. A. petiolata did not affect molecular diversity, but reduced AM fungal growth in the greenhouse study and in a in vitro assay. Our findings suggest that both introduced plants and exotic AM fungi can have negative impacts on local AM fungi.

Suitability of mycorrhiza-defective mutant/wildtype plant pairs (Solanum lycopersicum L. cv Micro-Tom) to address questions in mycorrhizal soil ecology

Despite the importance of arbuscular mycorrhizal fungi (AMF) to ecosystem processes, few experimental tools are available to quantify AMF contributions to process rates. In this study we examine the efficacy of an experimental system consisting of wildtype (WT) and different non-mycorrhizal (Myc−) genotype pairs of tomato (Solanum lycopersicum L.), specifically focusing on cv Micro-Tom. Two conditions necessary to make such a system useful were examined; (1) that the Myc− genotype(s) do not get colonized in a full soil AMF community background, while the WT does, and B) that there are no non-target effects of the Myc− phenotype on soil microbes. We assessed the second condition by growing Myc− genotypes and WT in non-mycorrhizal soil, monitoring plant growth (root, shoot biomass; root length; root diameter size distribution) and soil microbial community structure (PLFA analysis) as indicators of any changes in root tissue quality or rhizodeposition. All tested Myc− genotypes showed a drastically reduced colonization in mycorrhizal soil. However, in non-mycorrhizal soil, M161 had greater root biomass and M20 greater microbial biomass compared to WT. Only one of the Myc− mutants examined fully met the criteria. We conclude that the BC1/WT pair is a powerful experimental system and recommend caution when using Myc− mutants in mycorrhizal ecology.

ACCUMULATION OF ORGANIC CARBON IN RECLAIMED COAL MINE SOILS OF WYOMING

The potential to sequester carbon and increase organic nutrient storage in disturbed soils, such as those reclaimed after surface coal mining, appears to be significant. Quantification of organic carbon accumulation is complicated, however, by the presence of coal and coal dust in these soils. Our preliminary data on organic matter content of reclaimed soils at surface coal mines in Wyoming suggest they are sequestering carbon at a rapid rate. Data from a surface mine reclamation site near Hanna, WY indicate that surface (0-15 cm) soil organic carbon content has increased from a low of 10.9 g C kg soil in 1983 to 18.6 g C kg soil in 1998 and to 20.5 g C kg soil in 2002. Undisturbed soil directly adjacent to the reclaimed site has a mean organic carbon content of 15.1 g kg soil. At a mine near Glenrock, WY, soil organic carbon at a site reclaimed in 1979 increased from an estimated low of 5.8 g C kg soil to a current level of 18.4 g C kg soil. Organic carbon content of undisturbed soils adjacent to the reclaimed area range from 9.9 to 15.7 g C kg soil. In contrast to the elevated organic carbon content, amounts of microbial biomass in reclaimed soils at both mines are lower than in nearby undisturbed soils (ca. 60% or less). We have collected similar data from a number of other surface coal mines in Wyoming. We hypothesize that decomposition rates are slow in reclaimed mine soils due to low microbial activity relative to that in undisturbed soils. Additional

Invasive Plants Rapidly Reshape Soil Properties in a Grassland Ecosystem

In this study, we show how invasive plant species drive rapid shifts in the soil environment from surrounding native communities. Each of the three plant invaders had different but consistent effects on soils. Thus, there does not appear to be a one-size-fits-all strategy for how plant invaders alter grassland soil environments. This work represents a crucial step toward understanding how invaders might be able to prevent or impair native reestablishment by changing soil biotic and abiotic properties. ABSTRACT Plant invasions often reduce native plant diversity and increase net primary productivity. Invaded soils appear to differ from surrounding soils in ways that impede restoration of diverse native plant communities. We hypothesize that invader-mediated shifts in edaphic properties reproducibly alter soil microbial community structure and function. Here, we take a holistic approach, characterizing plant, prokaryotic, and fungal communities and soil physicochemical properties in field sites, invasion gradients, and experimental plots for three invasive plant species that cooccur in the Rocky Mountain West. Each invader had a unique impact on soil physicochemical properties. We found that invasions drove shifts in the abundances of specific microbial taxa, while overall belowground community structure and functional potential were fairly constant. Forb invaders were generally enriched in copiotrophic bacteria with higher 16S rRNA gene copy numbers and showed greater microbial carbohydrate and nitrogen metabolic potential. Older invasions had stronger effects on abiotic soil properties, indicative of multiyear successions. Overall, we show that plant invasions are idiosyncratic in their impact on soils and are directly responsible for driving reproducible shifts in the soil environment over multiyear time scales. IMPORTANCE In this study, we show how invasive plant species drive rapid shifts in the soil environment from surrounding native communities. Each of the three plant invaders had different but consistent effects on soils. Thus, there does not appear to be a one-size-fits-all strategy for how plant invaders alter grassland soil environments. This work represents a crucial step toward understanding how invaders might be able to prevent or impair native reestablishment by changing soil biotic and abiotic properties.

Restoring ecological properties of acidic soils contaminated with elemental sulfur

Elemental sulfur (S0) accumulates in the environment from anthropogenic sources as a byproduct from oil and gas refining and from trap and skeet shooting targets. Bacteria can oxidize S0 to H2SO4, which acidifies soil. We explored whether combinations of soil amendments can be used to remediate acidic soils contaminated with S0 by restoring soil chemistry, plant growth, and bacterial communities in a greenhouse. Results were compared to a contamination gradient in a field that had been limed with CaMg(CO3)2 two years prior. Amendments in the greenhouse included CaCO3 by itself, and in combination with fertilizer, compost, biochar, and chitin. Amended soils were incubated for one week and half of all containers were planted with Poa nevadensis. We sequenced bacterial DNA from a subset of amended soils and along the field gradient. CaCO3 additions in the greenhouse initially raised the pH of contaminated soil to values found in uncontaminated soils. However, pH decreased over time, which was likely caused by the oxidation of S0 to H2SO4. This was also apparent in the field, where CaCO3 additions raised pH to 4 but not to the desired value of 5 or higher. Plants in the greenhouse failed to grow in the unamended contaminated soil, but CaCO3 alone reduced concentrations of toxic cations and resulted in more plant growth than in the uncontaminated soil. CaCO3 also partially restored the bacterial communities in the greenhouse and in the field by increasing richness and diversity to near values found in uncontaminated soil, suggesting that bacteria can be resilient to prolonged acidic conditions. Organic amendments did not provide a significant benefit to restoration. This study demonstrates that acid neutralization alone can restore abiotic and biotic components and productivity of soils contaminated with S0, but multiple CaCO3 applications may be required to avoid future acidification.