Core and Differentially Abundant Bacterial Taxa in the Rhizosphere of Field Grown Brassica napus Genotypes: Implications for Canola Breeding.

Modifying the rhizosphere microbiome through targeted plant breeding is key to harnessing positive plant-microbial interrelationships in cropping agroecosystems. Here, we examine the composition of rhizosphere bacterial communities of diverse Brassica napus genotypes to identify: (1) taxa that preferentially associate with genotypes, (2) core bacterial microbiota associated with B. napus, (3) heritable alpha diversity measures at flowering and whole growing season, and (4) correlation between microbial and plant genetic distance among canola genotypes at different growth stages. Our aim is to identify and describe signature microbiota with potential positive benefits that could be integrated in B. napus breeding and management strategies. Rhizosphere soils of 16 diverse genotypes sampled weekly over a 10-week period at single location as well as at three time points at two additional locations were analyzed using 16S rRNA gene amplicon sequencing. The B. napus rhizosphere microbiome was characterized by diverse bacterial communities with 32 named bacterial phyla. The most abundant phyla were Proteobacteria, Actinobacteria, and Acidobacteria. Overall microbial and plant genetic distances were highly correlated (R = 0.65). Alpha diversity heritability estimates were between 0.16 and 0.41 when evaluated across growth stage and between 0.24 and 0.59 at flowering. Compared with a reference B. napus genotype, a total of 81 genera were significantly more abundant and 71 were significantly less abundant in at least one B. napus genotype out of the total 558 bacterial genera. Most differentially abundant genera were Proteobacteria and Actinobacteria followed by Bacteroidetes and Firmicutes. Here, we also show that B. napus genotypes select an overall core bacterial microbiome with growth-stage-related patterns as to how taxa joined the core membership. In addition, we report that sets of B. napus core taxa were consistent across our three sites and 2 years. Both differential abundance and core analysis implicate numerous bacteria that have been reported to have beneficial effects on plant growth including disease suppression, antifungal properties, and plant growth promotion. Using a multi-site year, temporally intensive field sampling approach, we showed that small plant genetic differences cause predictable changes in canola microbiome and are potential target for direct and indirect selection within breeding programs.

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors. The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Tracking stable isotope enrichment in tree seedlings with solid-state NMR spectroscopy.

Enriching plant tissues with (13)C and (15)N isotopes has provided long-lasting, non-reactive tracers to quantify rates of terrestrial elemental fluxes (e.g., soil organic matter decomposition). However, the molecular location and level of isotope enrichment may differ among plant tissues. This factor is central to the integrity and interpretation of tracer data, but is seldom considered in experiments. We propose a rapid, non-destructive method to quantify molecular isotope allocation using solid-state (13)C and (15)N nuclear magnetic resonance spectroscopy. With this method, we tracked and quantified the fate of multiple pulses of (13)CO(2)(g) and K (15)NO(3)(l) in boreal tree seedling roots and leaves as a function of time. Results show that initial preferential (13)C carbohydrate enrichment in the leaves was followed by redistribution to more complex compounds after seven days. While (13)C allocation within the roots was uniform across molecules, (15)N results indicate an initial enrichment of amine molecules after two hours.

The influence of condensed tannin structure on rate of microbial mineralization and reactivity to chemical assays.

We examined how tannin structure influences reactivity in tannin assays and carbon and nitrogen mineralization. Condensed tannins from the foliage of ten tree and shrub species and from pecan shells (Carya illinoensis) had different proportions of: (a) epicatechin (cis) and catechin (trans) isomers, (b) procyanidin (PC) and prodelphinidin (PD) monomers, and (c) different chain lengths. The response of each tannin to several widely used tannin assays was determined. Although there was some variation in response to proanthocyanidin (butanol/HCl) and Folin Ciocalteu assays, we did not deduce any predictable relationship between tannin structure and response to either assay. There was little variation in protein precipitation among the different tannins. To assess biological activity, six of the tannins were incubated with forest humus for 22 days. We determined that, while PC-based tannins remained at least partly extractable for the duration of the incubation, tannins with a high proportion of PD subunits rapidly became unextractable from soil. There was a positive correlation between net nitrogen mineralization and cis chemical structure. Carbon mineralization was enhanced initially by the addition of tannins to humus, but after 22 days, a negative correlation between the proportion of cis subunits and respiration was determined. Overall, we were not able to demonstrate consistent effects of structure on either microbial mineralization or reactivity to chemical assays; such relationships remain elusive.