Experimental Design
To investigate N acquisition in rapeseed (Brassica napus), a greenhouse pot study was conducted at the Colorado State University Plant Growth Facility in Fort Collins, CO (40.5717° N, 105.0812° W) from June to September 2016. Ten rapeseed varieties were selected from the 51 founder lines of the Parkin, Vail, and Robinson 2017 project, that were selected for development of a germplasm resource to dissect complex traits in Brassica napus. This project collected diverse rapeseed lines from around the world to make a nested association mapping (NAM) population that could be used to introduce new diversity into breeding germplasm (Parkin, Vail, and Robinson 2017). The ten varieties for this study were selected using 12,612 single nucleotide polymorphisms (SNP) markers to capture the widest possible general genetic diversity based on genetic distance, and geographic locations of the markers while controlling for common flowering time (Table 1, Fig. 1).
The rapeseed plants were grown in 3.8 Liter pots with a water catch tray. A non-soil mixture of 2/8 sand, 3/8 calcined clay, and 3/8 vermiculite by volume was homogenized. A high organic matter field soil was added to the non-soil mixture to create up to a 6-fold difference in soil organic matter levels across the four treatments. The field soil was a fine loamy Aridic Argiustoll with 6.7% organic matter (3.7% C, 0.38% N) collected from a farm near Fort Collins, Colorado, with a history of organic vegetable production. The field topsoil (0-10cm) was collected and sieved to 8-mm. Soil mixtures were homogenized using a clean cement mixer. A 15N enriched N fertilizer solution using 98% 15N enriched dual labeled NH4NO3, diluted down to 8% 15N enrichment, was applied weekly to obtain the specified total N additions for the high and low fertilizer rates as outlined in Table 2 (Damon, Osborne, and Rengel 2007; Balint and Rengel 2008). Fertilizer rates of 50 mg N/pot for low N treatments and 150 mg N/pot for high N treatments were chosen to provide sufficient N through vegetative growth based on the Balint and Rengel 2008 study. The SOM levels were selected by assuming less than 1-2% of total N in SOM would be mineralized during the short time period of the study, resulting in mineralization of approximately 50-100 mg N in high SOM and 10-20 mg N in low SOM treatments.
The plants were planted in randomized complete blocks with restricted randomization design with 5 blocks of each of the 4 treatments with each of the 10 varieties for a total of 200 pots. Each of the blocks were divided to have 1 plant of each variety and treatment in a randomly assigned block design in the greenhouse. Each block was planted 1 week apart for 5 weeks.
Four seeds were planted into each pot and at one week they were thinned to one plant per pot. Fertilizer treatments were initiated at 2 weeks after planting when the first true leaves were beginning to emerge. Once per week, 50ml of N-free Hoagland’s nutrient solution was applied to each pot to ensure that nutrients other than N were not limiting across all treatments. Based on the treatment, supplemental 15N enriched fertilizer was applied weekly to achieve desired N rates. The first week 100 ml of N-fertilizer solution was applied, and 50 ml was applied in all subsequent weeks for a total of 5 fertilizer applications. Any liquid that ran through the pot was caught in the trays below the pot and added back into the pot to eliminate N loss by leaching.
The pots were watered with a drip emitter irrigation system starting at week three. The irrigation system watered for two minutes each day fertilizer treatments were not applied. A moisture probe was used in the pots twice a week to measure pot moisture and the irrigation amounts were adjusted to equalize moisture levels between treatments and blocks.
Because our research question was about N acquisition from the soil environment and not internal N translocation patterns, we sampled plants at peak biomass. Each block was destructively harvested when about 75% of the plants in a block were at the elongation stage before flowering, around 6 weeks. Each individual pot in the block was photographed and weighed. The plant was clipped at the base of the stem. The clipped plant shoot was put in a paper bag and dried at 55℃ and weighed for dry shoot biomass.
The pot of soil was turned upside down in a clean tub. The loose soil was gently brushed off leaving the root ball and the rhizosphere soil surrounding the roots. The rhizosphere soil and root ball and the bulk soil were placed in separate zip lock bags and placed in a cooler with ice until they were put in cold storage for further processing. A subsample of the bulk soil from each pot was weighed and dried at 105℃ to determine soil gravimetric water content.
Enzyme activity
The activity of four soil enzymes involved in SOM decomposition and soil nutrient cycling were measured using fluorescence-based enzyme activity assay (Table 3). Rhizosphere soil samples from each pot were analyzed using the microplate enzyme assay using fluorescence-based MUB (4-methylumbelliferone) and MUC (7-amino-4-methylcoumarin) substrate protocol (Bell et al. 2013). Briefly, the day after the plants were harvested, 1.1-1.3 g of soil was weighed from the rhizosphere soil sample. The soil was blended to homogenize sample with a 50mM sodium acetate buffer solution, that had been adjusted to the average soil pH of 7.5 to make a soil slurry. Soil slurry was pipetted into black, 96-well microplates with compound-specific fluorescing substrates. Samples were analyzed using a Tecan Infinite M200 plate reader (Tecan Austria GmbH, Salzburg, Austria).
Inorganic nitrogen
A sample of the rhizosphere soil from each pot was extracted with 100 mL of a 2 M Potassium chloride (KCl) solution to analyze levels of extractable ammonium (NH4+) and nitrate (NO3) in the soil at the time of harvest using the microplate colorimetric method (Sims, Ellsworth, and Mulvaney 1995).
The Vanadium (III) Chloride (VCl3) protocol was used to determine soil NO3, where 30μL of the KCl extracted sample was pipetted into microplates with VCl3 solution (Doane and Horwáth 2003). The salicylate-hypochlorite method was used to determine soil NH4+, where 70μL of KCl extracted sample was used in each of the microplate wells. Both assay reactions were read on a microplate reader (BioTek Instruments, Winooski, VT). Inorganic N values are not dependent on dry plant biomass so, all 5 blocks of data are used in analysis
Nitrogen Source Analysis
Isotopic values of the dried plant shoots were analyzed to determine the relative contributions of the fertilizer and SOM to plant N uptake. Dried plant samples were ground to 2 mm in a Wiley Mill and then roller ground until the sample was homogenized. All samples were analyzed for total C, total N and 15N at EcoCore Analytical Services Lab, at Colorado State University, Fort Collins, CO, using an Elemental-Analyzer – Isotope Ratio Mass Spectrometry (Costech, Valencia, CA).
The contributions of the N from the labeled inorganic 15N and the organic N acquired from the SOM were calculated by applying the isotopic mixing model (Hauck and Bremner 1976). The fraction of fertilizer-derived N (ffertilizer) was calculated using the equation:
ffertilizer = (δ15N sample – δ15N soil) / (δ15N fertilizer- δ15N soil)
Where δ15N sample, δ15N soil, and δ15N fertilizer represent the atom % 15N of the total sample, natural abundance of the soil mixture, and fertilizer (8 atom% 15N) respectively. The value for δ15N sample was the sample value output from EA-IRMS analysis. The δ15N Soil was the natural abundance of the soil mixture, 0.3681 atom% 15N for low SOM and 0.3699 atom% 15N for High SOM treatments. The contribution of soil derived N was calculated using the equation fSOM = 1- ffertilizer. The REN was calculated using the equation: Total Plant N/Applied N Fertilizer.
Root Biomass
The root biomass was obtained by washing the growth media away from the bulk and rhizosphere samples of blocks 1, 2 and 3. The washed roots were dried at 55℃ and weighed for dry biomass. The samples were a mixture of roots, vermiculite, and particulate organic matter. The root samples were homogenized in a ball grinder. A subsample of each root sample was analyzed for organic content using the ash correction protocol to obtain an estimated root biomass for each sample (Sparks et al. 1996). Briefly, a subsample of the homogenized sample was weighed in tin weigh boats and placed into a 105℃ oven for at 24 hours. The sample was weighed again and then placed into a cold muffle furnace and baked at 450℃ for 4 hours. Once samples were cooled to at least 200℃, they were weighed again. The difference in the sample weights were used to correct root weights for inorganic compounds and get an ash corrected estimated root biomass for each sample (Harmon, Nadelhoffer, and Blair 1999). The root-shoot ratio was calculated as the ash corrected root biomass estimate divided by the dry shoot weight. Only blocks 1 and 3 were included in the root-shoot ratio estimate.
Data Analysis
The data were analyzed in R, using a mixed model approach (R Core Team 2017). Due to a data loss of block 2 aboveground dry plant weights, only four of the five blocks were used in analyses that relied on plant biomass. Block was included as a random variable, while the fixed predictor variables were rapeseed variety and treatment. The response variables were the four different enzyme activities, N uptake, SOM and Fertilizer N uptake, percent N from SOM and fertilizer, total soil inorganic N, soil NH4+, NO3⁻, dry root and shoot biomass, and root-shoot ratio. The data were not normally distributed, so the lme() function in the nlme package to allow for unequal variances (Pinheiro et al. 2018). The exceptions are REN was analyzed with the lemr() function, and for NH4+ and NO3⁻, the data were transformed by taking the square root and then analyzed using lemr() function of the Lme4 package (Bates et al. 2015). Due to near zero nitrate levels in some samples, some samples had negative values after subtracting sample blanks. In this case, a constant was added to make all values positive and then were square root transformed. These values were then analyzed with the lme() function from the nlme package (Pinheiro et al. 2018). A type three analysis of variance Anova() from the with Kenward-Roger approximation for degrees of freedom was used from the car package (Fox et al. 2018). The emmeans function, from the emmeans package, was used to make pairwise comparisons of significant predictors (Russell Lenth 2019).