Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: plant and animal production, protection, and health; sustainable agriculture; biotechnology Plants require a source of reduced nitrogen for growth and development. This can be provided in the form of organic or inorganic fertilizer or, for some plants, from plant-associated microbes capable of carrying out biological nitrogen fixation. Corn is a major user of inorganic nitrogen fertilizer and does not typically associate with nitrogen fixing microbes. This project will examine indigenous landraces of corn that appear to associate nitrogen fixing microbes in order to understand the mechanisms of this association and whether an association with nitorgen fixing microbes can be transferred to modern corn varieties.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Plants grow in close association with microbial communities that influence plant traits related to nutrient acquisition, plant development, plant defenses, and abiotic stress responses. The root-associated microbiota of Arabidopsis thaliana has been characterized and shown to be much less complex than the microbiota of the surrounding soil, being enriched in Proteobacteria, Bacteroidetes, and Actinobacteria. These microbes are selected in part by plant cell wall features and metabolic cues from host cells. Characterization of the rhizosphere microbiome associated with 27 modern maize inbred lines also indicated substantial differences in relative abundance of microbial taxa between bulk soil and the rhizosphere with the maize genotype contributing a small but significant influence on the rhizosphere selectivity. Similarly, the maize seed-associated microbiota in wild maize ancestors (teosinte) and geographically diverse modern maize was shown to have a phylogenetic-dependent community composition overlaid upon a core microbiota conserved across evolutionary and geographical boundaries. Collectively, these studies suggest that the plant microbiome is determined, at least in part, by the genetics of the host plant. Nitrogen-fixing microbial associations with non-legumes, especially cereals, have been a topic of intense interest for several decades. Nitrogen-fixing endophytes contribute to the nitrogen nutrition of sugarcane in some environments, but there is little evidence for the occurrence of efficient associations in other cereals. Triplett (1996) suggested that to identify maize diazotrophic endophytes, it may be interesting to survey primitive maize landraces from many locations including the areas of maize origin. Estrada et al. (2002) followed this suggestion and examined a landrace of maize in the Sierra Juarez region of Oaxaca, Mexico. This group successfully isolated a nitrogen-fixing endophyte from the resident maize landrace and tentatively identified the isolate as a new species of Burkholderia but did not demonstrate that atmospheric dinitrogen (N2) contributed to the nitrogen economy of the plant. Interestingly this group also reported the isolation of a similar endophyte from field grown teosinte plants and speculated that the Burkholderia strain might have formed a primitive symbiosis with teosinte that persisted during domestication of maize. We also learned of isolated indigenous landraces of maize in the Sierra Juarez region of Oaxaca that were reportedly grown using traditional practices with little or no fertilizer and speculated that these might have evolved unique microbial community associations not found in maize that had been cultivated and selected under high nutrient conditions. This indigenous maize landrace is characterized by an extensive development of aerial roots that produce large amounts of mucilage. The mucilage associated with maize underground roots has been previously described and it has been suggested that root exudates play a significant role in structuring rhizosphere microbial communities. Indeed, it has been shown that pea root mucilage can serve as a sole carbon source for some rhizosphere bacteria, including Rhizobium sp., Burkholderia sp. and Pseudomonas sp. In this project we will 1) examine the microbiota associated with Sierra Juarez indigenous maize and determine whether the mucilage supports a diazotrophic microbial community, 2) assess the contribution of diazotrophic activity to maize N nutrition and 3) characterize resident microbes and their ability to promote growth of other crops.
Project Methods
Plant Material Sierra Juarez maize seeds will be obtained in Sierra Juarez region of Oaxaca, Mexico (N 17° 15.772′; W 096° 01.758′), from an open pollinated population. Zea mays subsp. mexicana (Teosinte), LH123HT, Mo17, PHG39, LH82, PH207, and B73 seeds were obtained from USDA National Plant Germplasm System (NPGS) (accessions Ames 8083, PI601079, PI558532, PI600981, PI601170, PI601005, and PI550473, respectively). Maize line Hickory King was obtained from Victory Seeds (accession 3140041). Sample CollectionRhizosphere and plant tissues that include stem, leaf, aerial roots, underground roots and mucilage of Sierra Juarez maize will be sampled in Sierra Juarez. For plant endophyte analysis, tissues will be surface sterilized by rinsing with mqH2O, shaken gently in 70% ethanol for 5 minutes, placed into 1% hypochlorite bleach gently stirred for 10 minutes, rinsed three times in mqH20 and dried in a laminar flow cabinet. Roots and stems will also dissected to remove epidermal tissues before extraction. For seed endophyte analysis, embryo and endosperm of Sierra Juarez, Hickory King, and B73 will be withdrawn from the seeds by hand using a razor blade in a laminar flow cabinet and collected. Rhizosphere will be defined as a layer of soil covering the outer surface of the root system that could be washed from roots in a buffer/detergent solution. Plant GrowthFor experiments in the greenhouse, seeds of Sierra Juarez maize and conventional varieties will be surface sterilized and germinated. After one week, seedlings will be transplanted in 40-liter pots filled with a mix of sand and perlite (v:v) and grown in a high ceiling greenhouse. Plants were watered twice a day for two minutes with half-strength of Hoagland solution3. For experiments in the field, three independent plots of 20 plants per genotype will be planted with three border rows (B73) between each genotype. Measurements of the Sierra Juarez maize will be taken for 135 days post-planting. Mucilage Glycosyl CompositionGlycosyl composition analysis of the mucilage will be performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. Methyl glycosides will first be prepared from dry mucilage samples by methanolysis in 1 M HCl in methanol at 80°C (18-22 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples will then be per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80°C (0.5 hours) and GC/MS analysis of the TMS methyl glycosides performed on an HP 6890 GC interfaced to a 5975b MSD, using an All Tech EC-1 fused silica capillary column (30m 0.25 mm ID). Metagenome AnalysisDNA (80-150ngμl?1) will be extracted from 100 mg of the rhizosphere, plant tissues using a DNA isolation kit (Mo Bio Laboratories, Inc, USA). The PCR control for microbial DNA isolation will be performed on 16S rDNA genes. PCR will be performed using eubacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Amplification will be carried out with 1 ?M of each primer in 3 mM MgCl2, 20 μM of each dNTP, 1.25 units of Taq polymerase (Promega) in a total volume of 20 μl of 1× reaction buffer (Promega). The products obtained will be purified with a NucleoSpin Gel extraction kit (Clontech, Palo Alto, USA).Illumina-based 16S rRNA Gene Sequencing16S rDNA PCR and sequencing of rhizosphere and plant tissues will be carried out using the Caporaso protocol4, extended to include a dual barcode scheme for each sample and replacing the Golay barcodes with a different set of Illumina-compatible barcodes designed to balance base composition and tolerate up to four sequencing errors in barcode sequences. The barcodes will be designed to allow pooling of multiple samples within a single MiSeq run. Ten cycles of PCR with barcoded primers will be performed at low annealing temperature (55°C); samples were then pooled and cleaned using a Qiagen column to remove the unincorporated primers. At this stage, an additional 10 or 20 cycles of PCR will be performed on the pool using the Illumina paired-end flowcell primers with a higher annealing temperature (65°C). The resulting PCR product will be subjected to QC with an Agilent Bioanalyzer and estimated concentration using KAPA Biosystems qPCR kit. The samples will be diluted to the appropriate loading concentration for a MiSeq run, spiked with 25% phiX control library, and sequenced using an Illumina MiSeq instrument with the manufacturer's standard 150 nucleotides paired-end dual-index sequencing protocol and the custom sequencing primers. Alpha and Beta diversity metrics will be generated using the Phyloseq R package.Illumina sequencing libraries from the same DNA extractions as above will be made using an adaptation of the Nextera transposase-based library construction method with multiplex barcoding. Samples will then be submitted to sequencing using the MiSeq and HiSeq instruments. Illumina sequences will then be demultiplexed and trimmed using Trimmomatic (ver 0.33) with the following parameter: Illuminaclip 2:30:10, Headcrop:15, Leading:20, Trailing:20, Sliding window:4:20, and Minlen:100. Nitrogenase SearchPeptide sequences of the six core nif genes (nifH, nifD, nifE, nifK, nifN, nifB) and alternate nitrogenase (anfG, vnfG) from known diazotrophs as previously published will be retrieved from GenPept as a reference. A blast search (E-value < 0.001) of six frame-translated metagenomic reads will be conducted against these reference sequences and the hits aligned against the multiple sequences alignment of reference using clustal-Omga followed by generation of phylogenetic trees for every individual nif gene using Fasttree2.1 with WAG model of amino acid evolution and gamma20 likelihood. The counts of nif genes can be normalized by recA counts ( determined using RecA TIGRFAM HMM with HMMER3 and an e-value cutoff of e-10).Acetylene Reduction Assay (ARA)For ARA with the mucilage, 2 ml of freshly collected mucilage from one or two aerial roots (Sierra Juarez) or several plants (teosinte) grown in the field will be introduced into sealed 14.5 ml vials (Wheaton). Then, 850 μl of acetylene (Airgas) will be injected into each vial and ethylene quantified by injecting 1 ml of the air phase, sampled after 72 hours, on a Gas Chromatography (GC-2010 Shimadzu) equipped with a Rt-Alumina BOND/KCL column (Restek).Mucilage 15N2 Assimilation The enrichment of mucilage in 15N atom will be achieved by removing 4 ml of headspace gas and replacing it with 4 ml of either 15N2 or 14N2 nitrogen gas directly into a vial containing 1.0 mL of mucilage. Mucilage samples will be incubated at 37°C for 0 and 70 hours in the presence of 15N2. 15N2 assimilation will be stopped by freezing the mucilage samples at -20°C, the samples freeze-dried and weighed and 15N analysis in the mucilage samples performed at the UC Davis Stable Isotope Facility (Davis, CA).15N Stable Isotope Analysis The proportion (%) of nitrogen derived from biological nitrogen fixation (%Ndfa) will be estimated from the 15N natural abundance (expressed in delta units, ‰) of the Sierra Juarez maize (δ15Nfixing plant) and that of the reference plant species (δ15Nref). Individual maize samples and reference plant samples, representing 8-10 species of non-nitrogen-fixing plants will be analyzed.