Contrasting Water Quality Treatments Result in Structural and Functional Changes to Wetland Plant-Associated Microbial Communities in Lab-Scale Mesocosms.

The impact of contrasting water quality treatments on wetland plant-associated microbial communities was investigated in this study using 12 lab-scale wetland mesocosms (subsurface flow design) planted with reed canary grass (Phalaris arundinacea) or water speedwell (Veronica anagallis-aquatica) over a 13-week period. Mesocosms received water collected from two sites along the Grand River (Ontario, Canada) designated as having either high or poor water quality according to Grand River Conservation Authority classifications. All mesocosms were established using sediment collected from the high water quality site and received water from this source pre-treatment. Resulting changes to microbial community structure were assessed using PCR-denaturing gel gradient electrophoresis (DGGE) on microbial 16S rDNA sequences extracted from rhizoplane, rhizosphere, and water samples before and after exposure to water quality treatments. Functional community changes were determined using Biolog™ EcoPlates which assess community-level carbon source utilization profiles. Wetland mesocosm removal of inorganic nutrients (N, P) and fecal coliforms was also determined, and compared among treatments. Treatment-specific effects were assessed using a repeated measures restricted maximum likelihood (REML) analysis. Structural and functional characteristics of rhizoplane microbial communities were significantly influenced by the interaction between plant species and water treatment (P = 0.04, P = 0.01). Plant species-specific effects were observed for rhizosphere structural diversity (P = 0.01) and wetland water community metabolic diversity (P = 0.03). The effect of water treatment alone was significant for structural diversity measurements in wetland water communities (P = 0.03). The effect of plant species, water quality treatment, and the interaction between the two is dependent on the microhabitat type (rhizoplane, rhizosphere, or water). Rhizoplane communities appear to be more sensitive to water quality-specific environmental changes and may be a good candidate for microbial community-based monitoring of wetland ecosystems.

E151 (sym15), a pleiotropic mutant of pea (Pisum sativum L.), displays low nodule number, enhanced mycorrhizae, delayed lateral root emergence, and high root cytokinin levels.

In legumes, the formation of rhizobial and mycorrhizal root symbioses is a highly regulated process which requires close communication between plant and microorganism. Plant mutants that have difficulties establishing symbioses are valuable tools for unravelling the mechanisms by which these symbioses are formed and regulated. Here E151, a mutant of Pisum sativum cv. Sparkle, was examined to characterize its root growth and symbiotic defects. The symbioses in terms of colonization intensity, functionality of micro-symbionts, and organ dominance were compared between the mutant and wild type. The endogenous cytokinin (CK) and abscisic acid (ABA) levels and the effect of the exogenous application of these two hormones were determined. E151 was found to be a low and delayed nodulator, exhibiting defects in both the epidermal and cortical programmes though a few mature and functional nodules develop. Mycorrhizal colonization of E151 was intensified, although the fungal functionality was impaired. Furthermore, E151 displayed an altered lateral root (LR) phenotype compared with that of the wild type whereby LR emergence is initially delayed but eventually overcome. No differences in ABA levels were found between the mutant and the wild type, but non-inoculated E151 exhibited significantly high CK levels. It is hypothesized that CK plays an essential role in differentially mediating the entry of the two micro-symbionts into the cortex; whereas it would inhibit the entry of the rhizobia in that tissue, it would promote that of the fungus. E151 is a developmental mutant which may prove to be a useful tool in further understanding the role of hormones in the regulation of beneficial root symbioses.

Standardized mapping of nodulation patterns in legume roots.

Optimizing nodulation in legumes is a target for crop improvement, and the spatial control of nodulation is just beginning to be unravelled. However, there is currently no method for standard phenotyping of nodulation patterns. Here we present a method and software for the quantitative analysis of nodulation phenotypes. Roots of nodulated peas (Pisum sativum), wild-type and two mutants, were photographed. Data from the photographs were extracted using custom image and data analysis software. The software makes it possible to extract each nodule's position along primary and lateral roots, and to represent the nodulated root system in a standardized way independent of the way roots are arranged in the soil. A wide variety of nodulation and root variables are calculated, and average spatial nodulation patterns can be computed from multiple samples. Standardized spatial analysis of nodulation patterns opens the way for comparative analyses among genotypes of a single legume species, as here in pea. This approach could also be used to compare nodulation patterns among crops, among plants grown under different environmental conditions, or among plants exposed to different pharmacological treatments. The proposed method should therefore prove useful for studies on nodule organogenesis and nodule physiology and for optimizing nodulation in crops.

Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea.

Pea has lagged behind other model legumes in the molecular study of nodulation and mycorrhizae-formation because of the difficulty to transform its roots and its poor growth on agar plates. Here we describe for pea 1) a transformation technique which permits the complementation of two known non-nodulating pea mutants, 2) a rhizobial inoculation method which allows the study of early cellular events giving rise to nodule primordia, and 3) a targeted fungal inoculation method which allows us to study short segments of mycorrhizal roots assured to be infected. These tools are certain to advance our knowledge of pea root symbioses.