Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity

Intensified Pulse Rotations Buildup Pea Rhizosphere Pathogens in Cereal and Pulse Based Cropping Systems

The association of plants and microbial communities is crucial for crop production, and host plants influence the composition of rhizosphere microbiomes. Pulse crops play an important role in the development of sustainable cropping systems, and producers in the Canadian prairies often increase the frequency of pulses in their cropping systems. In this study, we determined the shifts in the fungal community of pea (Pisum sativum L.) rhizosphere, as influenced by the frequency of pulses in rotation, using high throughput sequencing. Six cropping systems containing pea (P), lentil (Lens culinaris Medik., L), hybrid canola (Brassica napus L., C), wheat (Triticum aestivum L., W), and oat (Avena sativa L., O) in different intensities were tested. The fungal communities were assessed at the flowering stage in the fourth and fifth year of the 4-year rotations. Cropping system had a significant impact on the composition of the rhizosphere fungal community, and the effect of crop rotation sequence was greater and explained more of the variation than the effect of previous crops. The rotation with consecutive pulses (WPLP) decreased fungal evenness and increased the proportion of pathotrophs. Fusarium was a dominant and ubiquitous pathotrophic genus. Olpidium virulentus, Botrytis cinerea, Fusarium solani, F. graminearum, and Alternaria eichhorniae were generally more abundant in pulse intensive rotations (WPLP, WLOP, and WPOP), the exception being F. solani which was not promoted by lentil. Reads of O. virulentus and B. cinerea were most abundant in pea preceded by lentil followed by the reads of Mortierella elongata in pea preceded by wheat. Pea consistently had higher grain yield when grown in diversified rotations including wheat, canola/lentil, and oat than rotations with two repeated crops (canola or pea). Cropping system affected the soil physicochemical properties, and soil pH was the main driver of fungal community shift. No evidence of beneficial microorganisms involvement in plant productivity was observed, but the high abundance of pathotrophs in pulse intensified rotations suggests the possibility of pathogen buildup in the soil with increasing pulse frequency. Diversifying rotation sequences minimized disease risk and increased pea production, in this study. Careful selection of plant species appears as a strategy for the management of rhizosphere fungal communities and the maintenance of crop production system’s health

Long-Term Land Use Affects Phosphorus Speciation and the Composition of Phosphorus Cycling Genes in Agricultural Soils

Agriculturally-driven land transformation is increasing globally. Improving phosphorus (P) use efficiency to sustain optimum productivity in diverse ecosystems, based on knowledge of soil P dynamics, is also globally important in light of potential shortages of rock phosphate to manufacture P fertilizer. We investigated P chemical speciation and P cycling with solution 31P nuclear magnetic resonance, P K-edge X-ray absorption near-edge structure spectroscopy, phosphatase activity assays, and shotgun metagenomics in soil samples from long-term agricultural fields containing four different land-use types (native and tame grasslands, annual croplands, and roadside ditches). Across these land use types, native and tame grasslands showed high accumulation of organic P, principally orthophosphate monoesters, and high acid phosphomonoesterase activity but the lowest abundance of P cycling genes. The proportion of inositol hexaphosphates (IHP), especially the neo-IHP stereoisomer that likely originates from microbes rather than plants, was significantly increased in native grasslands than croplands. Annual croplands had the largest variances of soil P composition, and the highest potential capacity for P cycling processes based on the abundance of genes coding for P cycling processes. In contrast, roadside soils had the highest soil Olsen-P concentrations, lowest organic P, and highest tricalcium phosphate concentrations, which were likely facilitated by the neutral pH and high exchangeable Ca of these soils. Redundancy analysis demonstrated that IHP by NMR, potential phosphatase activity, Olsen-P, and pH were important P chemistry predictors of the P cycling bacterial community and functional gene composition. Combining chemical and metagenomics results provides important insights into soil P processes and dynamics in different land-use ecosystems

Allelopathic influence of tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea maculosa Lam.) on arbuscular mycorrhizal fungi

Many exotic weeds interfere with other species by releasing allelochemicals into the environment that have a negative effect on their growth and distribution. Allelochemicals can have a direct influence on plant growth and/or indirect influence by disrupting interactions between plants and soil organisms, such as arbuscular mycorrhizal (AM) fungi. The goal of this research was to explore the allelopathic influences of the exotic weeds tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea maculosa Lam.). The allelopathic potential of tall hedge mustard was assessed using aqueous root and shoot extracts in seed germination and seedling growth bioassays. Aqueous tall hedge mustard root and shoot extracts strongly inhibited germination and growth of bluebunch wheatgrass, Idaho fescue, and spotted knapweed. Glucosinolate (GSL) analysis of tall hedge mustard tissues revealed the presence of two major GSLs (isopropyl GSL and sec-butyl GSL) and four indolylic GSLs. The degradation products of the two major GSLs (isopropyl isothiocyanate [ITC] and sec-butyl ITC) were identified in tall hedge mustard aqueous root and shoot extracts. Commercially available isopropyl ITC and sec-butyl ITC inhibited seed germination and seedling growth, suggesting their role in the allelopathic influence of tall hedge mustard. Tall hedge mustard aqueous extracts and ITCs incorporated into an agar medium inhibited Glomus intraradices Shenck & Smith spore germination and hyphal growth. Tall hedge mustard aqueous extracts strongly inhibited spore germination and hyphal growth of G. intraradices. Isopropyl ITC and sec-butyl ITC inhibited spore germination and hyphal growth, with the former exhibiting a stronger effect. Tall hedge mustard infestations were also found to reduce the A M inoculum potential of soil. The A M colonization and total biomass was reduced in bluebunch wheatgrass and spotted knapweed plants growing in tall hedge mustard infested compared to noninfested soil. Spotted knapweed is known to produce two major allelochemicals, (±)-catechin and cnicin. Both allelochemicals inhibited Glomus intraradices spore germination, and cnicin also inhibited the hyphal growth, suggesting that these allelochemicals may be involved in the inhibitory effect spotted knapweed has on A M fungi. Results of this study show that tall hedge mustard and spotted knapweed both produce allelochemicals that have the potential to directly and/or indirectly inhibit the growth of neighboring species and their A M fungal associates.Land and Food Systems, Faculty ofGraduat

Soil 16S DNA sequence data and corresponding soil property and wheat yield data from a 72-plot field experiment involving pulses and wheat crops grown in rotations in the semiarid prairie

The soil bacteria diversity and corresponding environmental data made available here are from a 72-field plot experiment testing the effect of pulse frequency in nine wheat-based rotation systems, in the semiarid prairie. The data include sequences of the V6–V8 regions of bacterial 16S rDNA from soil and root extracts, generated using Roche GS FLX Titanium technology, and associated environmental data, specifically levels of soil organic carbon, total carbon, total nitrogen, total phosphorus, pH, electrical conductivity, and extractible sulfate sulfur, copper, iron, manganese, zinc, potassium, nitrate nitrogen, phosphate phosphorus, calcium, and magnesium in the 0–15 cm soil layer, and mineral nitrogen and phosphate in the 0–120 cm soil layer. The grain yield of wheat in the last (4th) phase of the crop rotation systems is also given. The data can be used in meta-analyses of the effect of pea, lentil and chickpea in wheat-based cropping systems on soil bacterial diversity or for monitoring the evolution of soil bacteria communities in cultivated prairie soils in the context of climate change. Samples were collected between 2012 and 2014

Water stress history and wheat genotype modulate rhizosphere microbial response to drought

International audienceDifferent crop genotypes and soils with different water stress histories are known to harbour different microorganisms, but their relative effect on the response of plant-associated microbes to water stress is not known. In a pot experiment, four wheat genotypes (two with recognized drought resistance and two without) were grown in semi-arid soils with different irrigation histories (irrigated and non-irrigated soils) and exposed to four levels of soil water content (ranging from high to low). After one month of exposure to different soil water contents, we examined plant biomass as well as a general (CO2 production) and a specialized (soil uptake of atmospheric H2) functional processes in the rhizosphere. We further measured the abundance of bacteria and fungi in the rhizosphere using real-time PCR. Wheat shoot biomass was lower when growing in non-irrigated soils under low water content. In contrast, under moderate water contents wheat grown in non-irrigated soils had a significantly higher root biomass compared with those grown in irrigated soils. CO2 production did not differ between genotypes and soil irrigation histories under low soil water content. However, we found significantly higher H2 oxidation rates under low water content in the rhizosphere of plants growing in formerly irrigated soil as compared to those grown in formerly non-irrigated soils, although the intensity of the change was genotype-specific. Bacterial abundance was more sensitive to decreasing soil water content than fungal abundance and was mainly influenced by soil water stress history. Taken together, our results highlight that wheat breeding history and soil water stress history differentially influence crop growth performance, a specialized and a general rhizosphere processes, and rhizosphere bacterial and fungal abundance in the face of decreasing soil water content

Soil sample storage conditions impact extracellular enzyme activity and bacterial amplicon diversity metrics in a semi-arid ecosystem

The analysis of microbiological and metabolic features of soils is an important aspect of soil ecology, but the results can be heavily impacted by sample storage conditions. Inconsistencies in storage methods and length of storage across studies reduce the ability to accurately collect field-based measures and compare results between projects. In this study, we examined the effects of various storage conditions and storage time on results of subsequent bacterial 16S rRNA amplicon sequencing and on activities of extracellular enzymes, using soil samples collected from three different land use types (annual cropping system, native rangeland, and riparian forest) in a semi-arid region of the Canadian Prairies. We found that when comparing enzyme activities and bacterial communities across different land use types, storage conditions may not have a significant impact. However, storage conditions were found to be important within a single land use type. Air-drying of soil samples caused significant shifts in enzyme activity and β-diversity from the controls. Storage at −80 °C was best for maintaining consistent enzyme activity and microbial α- and β-diversity compared to controls (i.e., freshly collected soil) across all three land use types and storage times. The results from this study provide useful information about the impact of sample storage conditions for researchers in similar climates and encourage further consideration and discussion of the impacts of sample storage, as well as the reporting of storage conditions used in future studieISSN:0038-0717ISSN:1879-342

Presentation_1_Long-Term Land Use Affects Phosphorus Speciation and the Composition of Phosphorus Cycling Genes in Agricultural Soils.pdf

<p>Agriculturally-driven land transformation is increasing globally. Improving phosphorus (P) use efficiency to sustain optimum productivity in diverse ecosystems, based on knowledge of soil P dynamics, is also globally important in light of potential shortages of rock phosphate to manufacture P fertilizer. We investigated P chemical speciation and P cycling with solution ³¹P nuclear magnetic resonance, P K-edge X-ray absorption near-edge structure spectroscopy, phosphatase activity assays, and shotgun metagenomics in soil samples from long-term agricultural fields containing four different land-use types (native and tame grasslands, annual croplands, and roadside ditches). Across these land use types, native and tame grasslands showed high accumulation of organic P, principally orthophosphate monoesters, and high acid phosphomonoesterase activity but the lowest abundance of P cycling genes. The proportion of inositol hexaphosphates (IHP), especially the neo-IHP stereoisomer that likely originates from microbes rather than plants, was significantly increased in native grasslands than croplands. Annual croplands had the largest variances of soil P composition, and the highest potential capacity for P cycling processes based on the abundance of genes coding for P cycling processes. In contrast, roadside soils had the highest soil Olsen-P concentrations, lowest organic P, and highest tricalcium phosphate concentrations, which were likely facilitated by the neutral pH and high exchangeable Ca of these soils. Redundancy analysis demonstrated that IHP by NMR, potential phosphatase activity, Olsen-P, and pH were important P chemistry predictors of the P cycling bacterial community and functional gene composition. Combining chemical and metagenomics results provides important insights into soil P processes and dynamics in different land-use ecosystems.</p