Improving the Rotational Value of Field Pea as a Legume Cover Crop.

Field Site: The experiment took place at the University of Vermont's’ Horticulture Research and Education Center located at 65 Green Mountain Drive, South Burlington, VT 05403 (Appendix, Figure 1).

Field pea material: 15 field pea accessions were used in this experiment (Appendix, Figure 2A). All accessions were requested from the USDA NPGS and then amplified in Burlington, Vermont. Eight of the accessions are wild material from Georgia. The other accessions are cultivated lines originating in the United States. The use of wild material that has not undergone the genetic bottlenecks of domestication and breeding provided more genetic variation to this experiment.

Objective 1: Determine the extent of variation of beneficial crop rotational traits (e.g. nitrogen fixation, nutrient mobilization, organic matter deposition) within multiple field pea accessions.

Experiment 1: Testing for variation of rotational traits: nitrogen fixation, nutrient and mineral mobilization, and organic matter deposition.

To test for variation of rotational traits, 15 field pea accessions and two controls (no cover crop and no cover crop with fertilizer) were grown or administered in a randomized block design in one continuous field (Appendix, Figure 2). There was four replicates of each accession and control, for a total of 92 2m2 plots. Before the field pea was planted, “pre-planting” soil core samples approximately 30 cm (or 12 inches) deep were taken from the direct center of each plot using a 5-inch diameter hand-held auger. Once soil samples were collected, 50 field peas were hand sown in each plot and grown for 40 days. 50 plants in each plot simulated the recommended field pea density of 180 lbs/ac (NDSU, 2002, Stepanovic, 2017). Two days before field pea harvesting, “post-planting” soil samples were taken using the same methods as listed previously. Soil samples were only collected from the center of plots to avoid edge effects of neighboring plots. All soil samples (182 soil samples) were sent to the University of Vermont Agricultural and Environmental Testing Laboratory where they tested for pH, organic matter, available nitrate, phosphorus, potassium, aluminum, boron, calcium, copper, iron, magnesium, manganese, sulfur, and zinc.

A two-way repeated-measures analysis of variance (ANOVA) test with a Tukey's honestly significant difference (HSD) post hoc test was used to analyze the measurements reported by the testing laboratory. The two factors used in the analysis were accession and block. The analysis determined if there is variation in rotational traits in field pea and if this variation is due to genetic variation or placement in the field.

Objective 2. Characterize the effect of field pea rotational trait variation on a subsequently grown crop under Northeastern U.S. conditions.

Experiment 2: How is the subsequently grown corn affected by the genetic variation of the previously planted field pea?

To test how the variation of rotational traits in field pea affects a subsequently grown crop, a nutrient-intensive (“heavy feeder”) subsequent crop of corn was sown. Due to the late planting in June, the early sweet corn variety “Sugar Buns” was used. “Sugar Buns” is an early maturing corn that is harvestable 70 days after sowing. The corn was hand-planted according to the manufacturer's specification, of 2 seeds a foot (30.48 cm) in rows 36 inches (91.44 cm) apart. Fertilizer was added to the previously specified “no cover crop with fertilizer” control plots according to the recommendations from the UVM soil testing laboratory. Thirty-five days after sowing, chlorophyll content was recorded using a SPAD 502 Plus Chlorophyll Meter. The youngest fully developed leaf of the three plants closest to the center of each plot was sampled. Once the corn was ready to be harvested, plant height, dry aboveground biomass, and yield was measured for the same three plants. Plants closest to the direct center of the plot was only sampled to avoid edge effects from neighboring plots.

A two-way repeated-measures ANOVA test with a Tukey's HSD post hoc test was used to analyze chlorophyll content, plant height, dry aboveground biomass, and yield, with accession and block as the two factors. The analysis determined if there is variation in corn performance and if this variation is due to genetic variation of the previously planted field pea or the location of the corn in the field.

Objective 3: Characterize the variability and functional activity of the shifted microbiome communities in the soil/rhizosphere between field pea accessions.

Experiment 3: Extracting microbial DNA from soil samples, sequencing 16s rRNA, and analyzing the sequences for diversity and functional activity.

To test for microbial community shifts in the soil, microbial DNA was extracted from pre-planting soil samples and rhizosphere samples. A small portion, ~10 g, of the pre-planting soil samples was separated and stored at -80C. The rhizosphere is the soil still attached to the roots after the plant has been uprooted. Rhizosphere microbial samples were collected two days before the field pea is harvested. The center-most field pea plant in each plot was uprooted and the soil still clinging to the roots was removed and collected in the field and stored at -80C. The centermost field pea plant was sampled to avoid edge effect of neighboring plots.184 DNA samples (92 pre-planting, 92 rhizosphere samples) were extracted from all soil samples using the QIAGEN DNeasy PowerSoil Kit.

DNA extracts were sent to LC Science, a biotechnology sequencing company. Samples were processed for DNA library preparation in order to sequence 16s rRNA. Specifically, Phusion polymerase was used to amplify the 16s rRNA region of the DNA samples for 25-25 PCR cycles. After a single cycle of PCR, sequencing adapters and barcodes was added to each sample before further amplification. After amplification, an Illumina cBot system was used to generate clusters for sequencing using a next-generation MiSeq sequencer. Sequencing data was “cleaned” by trimming the barcodes and adapters, merging paired ends reads into single sequence tags, excluding tags with more than 5% ambiguous bases, and lastly excluding tags with more than 20% of low-quality bases (a Phred Score of < 10). The clean data, which is usually 300-400 bp in length, was then analyzed for operational taxonomic units (OTU) (analogous to species identification for microbial groups) using CD-HIT, a nucleotide clustering, and comparison program. To determine an OTU, the sequence similarity had to be greater than 97% within clusters. In addition to OTUs, LC Science provided species accumulation curves, alpha diversity, Shannon index (H), Simpson index, Chao1 index, rank-abundance curves, beta diversity, and a principal coordinate analysis (PCA). These analyses provided a measure of microbial diversity and species abundances for each submitted soil sample. In addition to diversity analysis, LC Science determined the taxa of the OTUs, by mapping each OTUs’ representative tag in the Ribosomal Database Project (RDP, version 11.3), Greengeens Database, and the NCBI 16S Microbial Database.