High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

Comparative Genomics, Siderophore Production, and Iron Scavenging Potential of Root Zone Soil Bacteria Isolated from 'Concord' Grape Vineyards.

Iron (Fe) deficiency in crop production is a worldwide problem which often results in chlorosis in grapevines, particularly in calcareous soils. Siderophores secreted by microorganisms and Strategy II plants can chelate Fe and other metals in soil solution, and siderophore-Fe complexes can then be utilized by plants and microbes. Plants may also shift rhizosphere conditions to favor siderophore-producing microbes, which can increase plant available Fe. Between-row cover crops (barley, rye, wheat, wheat/vetch) were planted as living mulch to address grapevine chlorosis by enhancing soil health in two vineyards in central Washington. The objectives of the current study were to (1) enrich for siderophore-producing organisms from within the indigenous rooting zone community of 'Concord' grapevines, and (2) perform comparative genomics on putative siderophore producing organisms to assess potentially important Fe acquisition-related functional domains and protein families. A high-throughput, chrome azurol S (CAS)-based enrichment assay was used to select siderophore-producing microbes from 'Concord' grapevine root zone soil. Next-generation whole genome sequencing allowed the assembly and annotation of ten full genomes. Phylogenetic analysis revealed two distinct clades among the genomes using the 40 nearest neighbors available in the public database, all of which were of the Pseudomonas genus. Significant differences in functional domain abundances were observed between the clades including iron acquisition and metabolism of amino acids, carbon, nitrogen, phosphate, and sulfur. Diverse mechanisms of Fe uptake and siderophore production/uptake were identified in the protein families of the genomes. The sequenced organisms are likely pseudomonads which are well-suited for iron scavenging, suggesting a potential role in Fe turnover in vineyard systems.