Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750
Performing Department
(N/A)
Non Technical Summary
The root nodule microbiome represents an untapped resource to maximize symbiotic nitrogen fixation and plant health in sustainable agriculture. While nitrogen-fixing rhizobia (the primary inhabitants of nodules) have been studied for decades, the rest of the root nodule microbiome remains understudied. The plant microbiome is now widely recognized for its role in nutrient acquisition and resilience to biotic and abiotic stressors.Manipulating microbiota from the root nodule could maximize symbiotic nitrogen fixation in legume crops through direct interactions with rhizobia, or through indirect interactions such as enhancing the resilience of the nodule to stress. Our overall goal is to unlock the root nodule microbiome as a tool for sustainable agriculture and contribute to the broader understanding of how plants assemble their microbial communities. We will take the first steps toward accomplishing this by elucidating rules that govern the assembly and colonization of the nodule microbiome. This will involve 1) investigating the relative strengths of host, rhizobia, soil and drought stress as drivers of nodule microbiome composition, 2) establishing a culture collection of nodule microbiota to help develop it as a simplified model for plant microbiome assembly, and 3) using synthetic communities to directly test several hypotheses related to how the nodule microbial community is established in the context of host-microbiome interactions, microbe-microbe interactions with rhizobia, and drought stress. This project contributes to understanding multipartite plant-microbe interactions, fills a knowledge gap regarding how plant microbiomes are assembled, and in the long run leads to reduced reliance on chemicals in agriculture. ?
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Ourlong-term goalsare to 1) enable the manipulation of the nodule microbiome to enhance sustainable agriculture and 2) contribute to the broader understanding of how plants assemble their microbial communitiesbyelucidating the rules that govern the assembly of the nodule microbiome.This project will support these goals throughObjective 1)use a top-down approach to elucidate the strength of the drivers of thenodule microbiome composition,Objective 2)develop a culture collection from the nodule microbiome to enable bottom-up experiments to investigate mechanisms of nodule microbiome assembly, andObjective 3)perform a series of experiments to investigate the "rules" that govern nodule microbiome assembly through the manipulation of nodule microbiome members with synthetic communities.
Project Methods
Objective 1.The experimental design will consist of setting up a 2x2x2x2 factorial design in pots in the greenhouse. The factors in the experimental design are 2 legumes (pea and lentil), 2 soils from different locations in ND (Williston and Carrington), 2 barcodedRhizobium leguminosarumbv.viciaeand2 irrigation watering treatments (well-watered vs drought inducedFor each legume there will be 2 soils (from Williston, ND and Carrington, ND) tested. The experiment will also consider 2 different strains ofRhizobium leguminosarumbv.viciae, which is both the pea and lentil symbiont. Finally, drought will be tested with two distinct watering regimes. In total there will be 16 treatments with 20 plants each, the sampling will consist of collecting 10 plant samples per treatment. Shoot length, shoot dry weight, nodule number and nodule color will be recorded to assess if the composition of the nodule microbiome impacts plant health.DNA will be extracted from all samples and 16s rRNA gene amplicon sequencing performed. Alpha and Beta diversity analyses will be performed to assess diversity and compositional changes among the different treatments.A Principal Component Analyses (PCoA) with vector fitting will be performed and will provide evidence of which communities havemaximum correlation with the corresponding treatment and which treatment has the biggest influence. We will also performPERMANOVAs to compare samples based on their centroids or dispersion and assess statistically their differences. Differential analysis will also be used to distinguish which taxa are statistically enriched in each of the treatments.Objective 2.Nodule isolation will be performed using a limiting-dilution method for unbiased culturing form the greenhouse experiment performed in Objective 1. The protocol consists of preparing a nodule slurry and diluting it to extinction into 45 replicate 96-well plates. Based on literature and on our previous results this will yield approximately 300 unique isolates. After twelve days the dilution yielding ~40% growth will be separated into two parts, one for 16s rRNA sequencing and another for glycerol stocks. The library preparation for 16s rRNA gene identification will involve a 2-step barcoding PCR.The first PCR barcodes the well in each plate and the second PCR barcodes each plate and adds the Illumina adaptors used during MiSeq sequencing. After sequencing and identification of unique isolates, the wells with the highest purity and number of reads will be selected from the glycerol stocks and struck onto petri dishes. These dishes will then be purified several times and whole 16s rRNA gene will be Sanger sequenced for taxonomic identification. After the confirmation of taxonomy,cryogenic glycerol stocks will be prepared and stored in -80°C.Objective 3a.We will assemble two taxonomically paired 50-member SynComs (SC) from the broader collection from Obj. 2. that contain similar phylogenetic compositions representative of non-rhizobial root nodule microbiota, but are composed of unique ASVs distinguishable by amplicon sequencing. A double-pot split-root assembly will be constructed for pea and lentil (Pot-A and Pot-B). Three days after planting, Pot A will be inoculated with SC-A (root endosphere inoculant) which we will allow to colonize the plant systemically over two weeks. Pot B will be inoculated with SC-B andRhizobium leguminosarumbv.viciae3841 to stimulate nodule development (nodule invasion inoculant). Nodules will be allowed to develop for one week, and then the roots from Pot-A and Pot-B and the nodules from Pot-B will be destructively sampled for DNA extraction and amplicon sequencing of microbial communities. As control inverse inoculations of the SynComs will also be performed (SC-B into Pot-followed by SC-A + Rhizobium into Pot-B), and all conditions will include five independent replicates. In total we will sequence 64 samples; roots from both pots and nodules from one x 5 replicates x 2 plant types x 2 swapped communities = 60 and inocula for each = 4.Objective 3b.We will utilize 2 phylogenetically representative, taxonomically paired 50-member SynComs (SC-Pea, SC-Lentil) derived from ASVs that show a preference for each plant (pea and lentil) based on differential enrichment observed in Obj. 1. Plants will be grown in N-free rooting solution with sand/vermiculite as above. The two SynComs will be allowed to compete for colonization of pea and lentil nodules by mixing at equal ratios and inoculating together onto 3-day germinated pea and lentils derived from surface sterilized seeds germinated in sterile conditions. Controls inoculated with rhizobia alone will also be included to evaluate any benefit derived by the plants from the SynComs. After 6 weeks we will extract DNA from 50 pooled root nodules from each plant and characterize microbial community composition by amplicon sequencing.Objective 3c.We we will co-inoculate a diverse population of rhizobia with a single 100-member SynCom and compare the composition of nodule microbiomes occupied by a specific strains. The population of rhizobia will be co-inoculated onto peas grown with a 100-member SynCom selected to represent the taxonomic composition of the nodule microbiomes from Obj. 1.After 6 weeks, nodules will be harvested and individual nodules will be separated into 96-well plates (95 nodules per replicate x 5 replicates). The DNA from individual nodues will be used for amplicon sequencing of rhizobia barcodes to identify the Rhizobium strain in each well. After identification, we will pool DNA from 3 nodules from each replicate of the 12 most competitive strains (we anticipate from experience that 12 strains will occupy at least 3 nodules from the 95 based on preliminary Plasmid ID assays of these strains) and perform 16S amplicon sequencing to analyze microbiome composition (5 reps x 12 strains = 60 samples)Objective 3d.As the barcoded rhizobia/ synthetic community mixture (Obj. 3c) will be inoculated onto pea plants, we will concurrently inoculate an additional set of plants with rhizobia alone (no microbiome), and compare the outcomes of symbiosis at an individual strain level using barcode sequencing. As above, after 6 weeks of growth, nodules from plants without SynCom inoculation will be harvested and separated into 96-well plates (95 nodules per replicate x 5 replicates). We will compare the relative abundance of each rhizobia from the 95-member collection when inoculated with the SynCom to the relative abundance when inoculated without the SynCom to determine the impact of the nodule microbiome on rhizobia competitiveness.Objective 3e.We will construct two SynComs (one that includes members of drought-associated taxa identified in Obj. 1: SC-drought, and one that excludes drought associated taxa: SC-normal). We will then perform successive inoculation of peas under watered and drought conditions to evaluate prior-establishment effects and the contribution of drought to the incorporation of the drought-associated ASVs in the nodule microbiome. Two sets of water agar germinated pea seedlings will either be inoculated first with SC-drought or SC-normal along with the same rhizobium, and then one set will be allowed to shift to drought conditions and will be maintained at that state throughout the remainder of the experiment (10 replicates x 2 inoculation orders x 2 conditions = 40). After 3 weeks the plants will be inoculated with the second community (SC-normal for SC-drought and inverse). Plants will be allowed to grow for a further 3 weeks beforeharvest. We will sequence nodule microbiomes from each, and determine the alpha-diversity to test for increased diversity in drought microbiomes under these conditions. Next, we will determine the aggregated relative abundance of drought-associated ASVs under each condition.