Performing Department
(N/A)
Non Technical Summary
Agricultural microbiomes confer many benefits for crop productivity, plant-, and soil health. However, nitrogen use efficiency in agricultural crops continues to be less than optimal. Microbial nitrification is considered as a primary cause for this and large knowledge gaps remain on how organismal interactions connect carbon and nitrogen mineralization, crop nutrition, nitrification, and nitrogen leaching in agricultural systems. This project aims to elucidate how plants and nitrifiers interact in the rhizosphere, and which chemical handoffs occur between both groups. The specific objectives are 1) to enrich representatives of globally-predominant soil ammonia-oxidizing archaea (AOA) in laboratory culture, and determine genome sequences and gene expression patterns, 2) to identify and characterize associations of AOA with the rhizosphere and mycorrhizal fungi of a diverse range of agricultural crops and other land plants, and 3) to characterize physical and chemical interactions between nitrifiers, mycorrhizal fungi, and plants in laboratory model systems. The project addresses AFRI Program Area Priority 1402 Agricultural Microbiomes in Plant Systems and Natural Resources by characterizing molecular mechanisms and signal exchange involved in microbiome assembly and interactions, as well as by functionally characterizing microbiomes and microbiome metabolites for optimization of environmental process. The project will provide a comprehensive model for N transfer within the soil-rhizosphere-plant root continuum. It will generate significant insight into activities of archaeal nitrifiers with a potential for improved management practices for increased nitrogen use efficiency and sustainability. The project will further train soil microbiologists at the undergraduate, graduate, and postgraduate levels.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The planned research for this project encompasses three main objectives:1) Enrich representatives of globally-predominant soil AOA lineages in laboratory culture and determine genome sequences and gene expression patterns. We will use a new chemostat enrichment strategy developed in the Martens-Habbena lab to enrich 36-72 representative strains of globally predominant soil archaeal lineages, obtain high-quality genome sequences from metagenomes, and study their gene expression patterns under different growth conditions. These analyses will be conducted to address major knowledge gaps on the genomic inventory, environmental adaptation of AOA, and their potential for interactions with plant hosts.2) Identify and characterize associations of AOA with the rhizosphere and mycorrhizal fungi of a diverse range of agricultural crops and other land plants. We will sample a broad range of monocot and dicot crops and wild plants across south Florida, and determine abundance and composition of AOA, and fungi in bulk soil and rhizosphere samples by combination of quantitative PCR, amplicon sequencing of marker genes (amoA, 16SrRNA, fungal ITS), as well as microscopic analysis by fluorescence in situ hybridization (FISH). These experiments will yield a comprehensive inventory of the potential physical interactions of AOA, AMF, and plant roots across monocot and dicot plants.3) Characterize physical and chemical interactions between AOA, NOB, AMF, and plants in laboratory model systems. We will use FISH and scanning electron microscopy (SEM) to characterize conditions of development of physical associations AOA, NOB, AMF, and plant roots in agricultural crops, and determine N fluxes through the multi-domain network using stable isotope tracing techniques. These studies will reveal fundamental mechanistic insights into the chemistry of these multi-domain interactions.
Project Methods
Objective 1: We will establish enrichments by varying pH (6.5, 7.0, 7.5) and temperature (28, 36, 44 ºC) with ammonia and urea as nitrogen source in a growth medium. Ammonia- and urea consumption, as well as nitrite and nitrate production will be determined every 48 hours using colorimetric assays. Gas samples will be analyzed for N2O and O2 by gas chromatography. Samples for DNA and RNA isolation will be harvested from 500 ml culture volumes. DNA and RNA will be isolated using Qiagen PowerSoil DNA and Total RNA isolation kits. RNA isolation will be followed by DNA removal using Thermo TURBO DNA-free Kit. Depletion of DNA will be confirmed by PCR. Ribosomal RNA will be removed using the QIAseq FastSelect RNA Removal kit. Purified DNA and RNA will be quantified spectrophotometrically on a NanoDrop ND-1000 instrument and using Qubit DNA and RNA quantification kits. If needed, RNA will be isolated from multiple samples and combined to obtain sufficient high-quality RNA. cDNA will be synthesized. Shotgun metagenomic and metatranscriptomic sequencing libraries will be constructed following Illumina library preparation protocols and sequencing will be performed on Illumina NovaSeq platform following standard operating procedures at Novogene, CA. with approximately 6.0 Gb sequencing depth, previously shown to be sufficient for genome reconstruction of archaeal genomes from reactor enrichments. Biomass of successful enrichments will be backed-up in freezer stocks, and enrichments will be sub-cultivated in batch cultures and subjected to dilution-to-extinction or antibiotic treatment for further purification. Depending on the success of each round of enrichments, we will conduct two rounds of enrichments each year for Year 1, Year 2, and Year3. We anticipate to obtain at least 36 unique enrichments. We will initially begin enrichments with inoculum from our previously studied soils in the Everglades Agricultural Area. For these soils we can leverage nine deeply sequenced paired metagenomes and metatranscriptomes for identification of enrichment targets and comparison of gene expression patterns in culture and in situ. Later we will begin to augment the EAA plots with rhizosphere samples from various plants sampled as part of Objective 2 as source of enrichments.Metagenomic- and metatranscriptomic analyses. Samples of DNA and RNA will be subjected to metagenome and metatranscriptome sequencing using Illumina platform at ~6.0 Mbp depth (equivalent of 1/10 of a full metagenome). Sequencing data will be quality-filtered, read-merged, assembled, and annotated using standard in-house pipelines. Briefly, we will take advantage of our established workflow using UF HiPerGator, and where necessary, online resources such as the Joint Genome Institute workflow. Paired-end reads will be processed with BBDuk (BBTools software package, http://bbtools.jgi.doe.gov) to remove contaminants, adapter sequences, as well as low quality reads (average Q score < 30 or ≤51 bp read length). BBMap (BBTools software package) will used to map and remove potential human and animal contaminant sequences. The filtered fastq files will be merged by BBMerge (BBTools software package) with least 10 overlapping bases and only reads ≥150 bp length were retained for further analysis. The resultant merged reads will be queried against NCBI RefSeq protein database for gene taxonomy and functional annotations using Diamond BLASTX. If necessary, filtered high quality reads will be further corrected by bbcms using Tadpole error-correction algorithm (mincount=2 highcountfraction=0.6). The corrected reads will be assembled using metaSPAdes assembler, and binned using metaBAT2 or maxBin. Functional annotation of genome bins will be performed against COG, arCOG, Pfam, Tigrfam and KEGG databases, and manually curated against AOA reference genomes.Genome-wide gene expression analyses of enriched AOA will be conducted simultaneously or separately, depending on biomass yield of the enrichments. Quality-filtered meta-transcriptomic data will be mapped to the reconstructed genomes using bowtie2, and quantified using edgeR and limma. Final transcript abundances will be normalized by gene length and mapped read number.Objective 2We will conduct three sampling campaigns to capture the diversity of agricultural crops and approximately 100 most common native plant genera in South Florida. Sampling areas will include the Everglades Agricultural Area, the Deluca Preserve (a UF-owned nature preserve with limited agricultural management), as well as unmanaged areas in South Florida. Replicate samples of each crop and native plant species will be collected. Bulk soil samples will be analyzed for inorganic nutrients (NH4+, NO2-, NO3-, urea, and PO42-), pH, and TOC content using standard wet chemical assays. DNA will be isolated using Qiagen PowerSoil DNA isolation kits. Sample DNA concentration will be normalized for downstream sequencing analyses. Bacterial and archaeal 16S rRNA genes, archaeal amoA genes, and fungal ITS genes will subsequently quantified by qPCR, and community composition of 16S rRNA genes, fungal ITS genes, and archaeal amoA genes will be determined by amplicon sequencing.Based on qPCR and amplicon sequencing analyses, plant specieswill be selected for metagenomic- and metatranscriptomic sequencing of bulk soil and rhizosphere samples, as well as detailed microscopic characterization using Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) following standard protocols. Metagenome-assembled genomes will be assembled, binned, and annotated for bulk soil and rhizosphere samples. RNA-seq reads will be mapped onto assembled and binned MAGs or assembled genes, if MAG representation is poor for AOA.Objective 3We will study interactions of AOA and plant hosts to identify key carbon- and nitrogen compound hand-offs. Initial interaction experiments will be carried out in hydroponic culture. Sorghum and corn will be germinated from sterilized seeds and initially grown in semi-sterile hydroponic culture. Seeds for market varieties of sorghum and corn will be commercially obtained. Plants will be grown in groups of 8 plants for 6-8 weeks total in the absence of AOA (control), or presence of AOA only, or AOA+NOB (Nitrospira moscoviensis). We will initially test Nitrososphaera viennensis, Ca. Nitrosocosmicus franklandianus, and Nitrosocosmicus sp. R2. The experiments will be monitored for phenotypic characteristics of plant growth. Representative plant roots will be sectioned and investigated by light microscopy. Presence of AOA and NOB will be evaluated by CARD-FISH using probes targeting the 16S rRNA of Archaea and Nitrospirota. Inorganic N species will be determined, and root exudate composition and free amino acids will be analyzed by HPLC. Root sections will be fixed and further characterized by CARD-FISH and SEM imaging.We will use a stable isotope approach to trace the activities of microbes and plants. Distribution of tracer in the liquid medium and plant biomass will be analyzed by GC-isotope ratio mass spectrometry (GC-IRMS) and elemental analyzer - isotope ratio mass spectrometry (EA-IRMS) analysis.