Location: Grain Legume Genetics Physiology Research
Project Number: 2090-21600-040-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Jul 31, 2028
Objective:
Autumn-sown food-grade winter pea (WP) varieties represent an exciting new crop for growers in the Pacific Northwest and Northern Plains (PNW). Several new food-grade WP cultivars are in the process of being released or already in production, including cultivars originating from USDA ARS Grain Legume Genetics Physiology Research Unit. However, little is known about the ability of these new cultivars to establish symbiotic associations with rhizobia native to the PNW soils or to improve soil nitrogen availability. This is a four-year project, with the following objectives: (1) Define native components of WP root microbiomes and ability for native rhizobia to compete with commercial inoculants in dryland production systems; (2) Isolate and characterize bacteria from nodule microbiomes; (3) Evaluate symbiotic and plant growth-promoting performance of native WP symbionts; and (4) Assess competitiveness of isolates in the PNW soils.
Approach:
The approach for Objective 1 is as follows: The diversity and structure of WP-associated microbiomes and plant symbiotic preferences will be evaluated for newly released food-grade WP cultivars across multiple PNW locations. In total, nine sites including low pH soils, fields with and without a history of commercial inoculation, as well as locations with low precipitation and/or with no known history of WP rotation will be tested. During sample collection, we will evaluate the nodulation rate of WP grown with and without commercial inoculum to examine symbiotic competitiveness of native rhizobia. The sites with strong nodulation, where commercial inoculation did not improve WP nodulation, will be the source of native rhizobia for isolation. The plots, which did not receive inoculation will be used for rhizobial isolation. For nodule microbiome analysis, the 16S rRNA V6-V8 and nodD regions will be amplified using appropriate. Root fungal microbiomes will also be analyzed using short read ITS2 amplicon sequencing. Based on amplicon sequencing data, such as the effect of plant genotype, location, or inoculation, the subsets of samples/conditions representing the most distant microbiomes for metagenome analysis will be selected.
The approach for Objective 2 is as follows: Microbiomes associated with productive symbiotic performance with WP across the PNW. Based on this information, the isolation protocols for bacterial groups of interests will be targeted. A complex (rich) and defined (minimal) media will be used to select both copiotrophic and oligotrophic bacteria. Identification will be done using PacBio Sequel 2 for full-length 16S amplicon sequencing. Additionally, nodC, nodD and gyrB gene regions will be used to identify Rhizobium leguminosarum at species level.
The approach for Objective 3 is as follows: The isolated bacteria will be tested in the laboratory and greenhouse individually and as synthetic communities (SynComs) for their ability to form effective symbiosis with food-grade WP cultivars and their ability to protect against Fusarium root rot. The isolated bacteria will be tested for PGP functions and antagonism substances to plant pathogens as described below.
The approach for Objective 4 is as follows: Rhizobial strains selected in Objective 3 will be assessed for their ability to compete with native microbiomes for nodulation using reported plasmids that allow measurement of rhizobial competitiveness and N fixation. The titer of native rhizobial populations in the soils will be estimated. The nodules formed by labeled rhizobia will be detected by GFP fluorescence. The nodule DNA will be isolated, and the index region of the plasmid sequenced to identify rhizobia in each nodule. This technique will allow us to identify competitive N-fixing rhizobia to be used as inoculum in the PNW.
