Proteome-Wide Response of Dormant Caryopses of the Weed, Avena fatua, After Colonization by a Seed-Decay Isolate of Fusarium avenaceum.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

Wheat Genotype-Specific Recruitment of Rhizosphere Bacterial Microbiota Under Controlled Environments.

Plants recruit beneficial microbial communities in the rhizosphere that are involved in a myriad of ecological services, such as improved soil quality, nutrient uptake, abiotic stress tolerance, and soil-borne disease suppression. Disease suppression caused by rhizosphere microbiomes has been important in managing soil-borne diseases in wheat. The low heritability of resistance in wheat to soil-borne diseases like Rhizoctonia root rot has made management of these diseases challenging, particularly in direct-seeded systems. Identification of wheat genotypes that recruit rhizosphere microbiomes that promote improved plant fitness and suppression of the pathogen could be an alternative approach to disease management through genetic improvement. Several growth chamber cycling experiments were conducted using six winter wheat genotypes (PI561725, PI561727, Eltan, Lewjain, Hill81, Madsen) to determine wheat genotypes that recruit suppressive microbiomes. At the end of the third cycle, suppression assays were done by inoculating R. solani into soils previously cultivated with specific wheat genotypes to test suppression of the pathogen by the microbiome. Microbiome composition was characterized by sequencing of 16S rDNA (V1-V3 region). Among the growth cycling lengths, 160-day growth cycles exhibited the most distinct rhizosphere microbiomes among the wheat genotypes. Suppression assays showed that rhizosphere microbiomes of different wheat genotypes resulted in significant differences in shoot length (value of p=0.018) and had an impact on the pathogenicity of R. solani, as observed in the reduced root disease scores (value of p=0.051). Furthermore, soils previously cultivated with the ALMT1 isogenic lines PI561725 and PI561727 exhibited better seedling vigor and reduced root disease. Microbiome analysis showed that Burkholderiales taxa, specifically Janthinobacterium, are differentially abundant in PI561727 and PI561725 cultivated soils and are associated with reduced root disease and better growth. This study demonstrates that specific wheat genotypes recruit different microbiomes in growth chamber conditions but the microbial community alterations were quite different from those previously observed in field plots, even though the same soils were used. Genotype selection or development appears to be a viable approach to controlling soil-borne diseases in a sustainable manner, and controlled environment assays can be used to see genetic differences but further work is needed to explain differences seen between growth chamber and field conditions.

Plant Uptake of Lactate-Bound Metals: A Sustainable Alternative to Metal Chlorides.

Global agricultural intensification has prompted investigations into biostimulants to enhance plant nutrition and soil ecosystem processes. Metal lactates are an understudied class of organic micronutrient supplement that provide both a labile carbon source and mineral nutrition for plant and microbial growth. To gain a fundamental understanding of plant responses to metal lactates, we employed a series of sterile culture-vessel experiments to compare the uptake and toxicity of five metals (Zn, Mn, Cu, Ni, and Co) supplied in lactate and chloride salt form. Additionally, primary root growth in plate-grown Arabidopsis thaliana seedlings was used to determine optimal concentrations of each metal lactate. Our results suggest that uptake and utilization of metals in wheat (Triticum aestivum L.) when supplied in lactate form is comparable to that of metal chlorides. Metal lactates also have promotional growth effects on A. thaliana seedlings with optimal concentrations identified for Zn (0.5-1.0 µM), Mn (0.5-1.0 µM), Cu (0.5 µM), Ni (1.0 µM), and Co (0.5 µM) lactate. These findings present foundational evidence to support the use of metal lactates as potential crop biostimulants due to their ability to both supply nutrients and stimulate plant growth.

Chronic Sublethal Aluminum Exposure and Avena fatua Caryopsis Colonization Influence Gene Expression of Fusarium avenaceum F.a.1.

Fusarium avenaceum F.a.1 is a novel strain of a fungal plant pathogen capable of preferentially decaying wild oat (Avena fatua) caryopses compared with those of wheat (Triticum aestivum). Understanding the molecular mechanisms governing weed seed-pathogen interactions is crucial to developing novel weed seed suppression technologies. Additionally, wild oat often competes with wheat in regions undergoing soil acidification, which leads to increases in soluble concentrations of many metals, including aluminum (Al). There is a dearth of information regarding the gene expression responses of Fusarium species to Al toxicity, or how metal toxicity might influence caryopsis colonization. To address this, a transcriptomic approach was used to investigate molecular responses of F.a.1 during wild oat caryopsis colonization in the presence and absence of chronic, sublethal concentrations of Al (400 µM). Caryopsis colonization was associated with induction of genes related to virulence, development, iron metabolism, oxidoreduction, stress, and detoxification, along with repression of genes associated with development, transport, cell-wall turnover, and virulence. Caryopsis colonization during Al exposure resulted in the induction of genes associated with virulence, detoxification, stress, iron metabolism, oxidoreduction, and cell wall turnover, along with repression of genes associated with cell wall metabolism, virulence, development, detoxification, stress, and transcriptional regulation. Aluminum exposure in the absence of caryopses was associated with induction of genes involved in siderophore biosynthesis, secretion, uptake, and utilization, along with several other iron metabolism-related and organic acid metabolism-related genes. The siderophore-related responses associated with Al toxicity occurred concurrently with differential regulation of genes indicating disruption of iron homeostasis. These findings suggest Al toxicity is attenuated by siderophore metabolism in F.a.1. In summary, both caryopsis colonization and Al toxicity uniquely influence transcriptomic responses of F.a.1.

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.

Nanotoxicity of engineered nanomaterials (ENMs) to environmentally relevant beneficial soil bacteria - a critical review.

Deposition of engineered nanomaterials (ENMs) in various environmental compartments is projected to continue rising exponentially. Terrestrial environments are expected to be the largest repository for environmentally released ENMs. Because ENMs are enriched in biosolids during wastewater treatment, agriculturally applied biosolids facilitate ENM exposure of key soil micro-organisms, such as plant growth-promoting rhizobacteria (PGPR). The ecological ramifications of increasing levels of ENM exposure of terrestrial micro-organisms are not clearly understood, but a growing body of research has investigated the toxicity of ENMs to various soil bacteria using a myriad of toxicity end-points and experimental procedures. This review explores what is known regarding ENM toxicity to important soil bacteria, with a focus on ENMs which are expected to accumulate in terrestrial ecosystems at the highest concentrations and pose the greatest potential threat to soil micro-organisms having potential indirect detrimental effects on plant growth. Knowledge gaps in the fundamental understanding of nanotoxicity to bacteria are identified, including the role of physicochemical properties of ENMs in toxicity responses, particularly in agriculturally relevant micro-organisms. Strategies for improving the impact of future research through the implementation of in-depth ENM characterization and use of necessary experimental controls are proposed. The future of nanotoxicological research employing microbial ecoreceptors is also explored, highlighting the need for continued research utilizing bacterial isolates while concurrently expanding efforts to study ENM-bacteria interactions in more complex environmentally relevant media, e.g. soil. Additionally, the particular importance of future work to extensively examine nanotoxicity in the context of bacterial ecosystem function, especially of plant growth-promoting agents, is proposed.

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.

Silver engineered nanomaterials and ions elicit species-specific O2 consumption responses in plant growth promoting rhizobacteria.

Metal containing engineered nanomaterials (ENMs) are now commonly used in various industrial and commercial applications. Many of these materials can be transformed during waste water treatment and ultimately enter terrestrial ecosystems via agriculturally applied biosolids. It is unclear how agriculturally important soil microbes will be affected by exposure to environmentally relevant, sublethal concentrations of ENMs and their transformation products (i.e., ions, aggregates, etc.). A method was developed, which puts O2 consumption responses in terms of viability, and tested by examining the toxic effects of Ag+, Zn2+, and Ni2+ ions on the plant growth promoting rhizobacterium (PGPR) Bacillus amyloliquefaciens GB03. The method was then used to examine the toxicity of Ag+, as-synthesized polyvinylpyrrolidone-coated silver ENM (PVP-AgENMs), and 100% sulfidized AgENM on B. amyloliquefaciens GB03, and two additional PGPRs Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. S. meliloti was found to have the highest LC50 for Ag+ and PVP-AgENMs (6.6 and 207 µM, respectively), while B. amyloliquefaciens and P. putida exhibited LC50's for Ag+ and PVP-AgENMs roughly half those observed for S. meliloti. The authors observed species-specific O2 consumption responses to ENM and ion exposure. PVP-AgENMs were less toxic than ions on a molar basis, and abiotic dissolution likely explains a significant portion of the observed toxic responses. Our results suggest microbes may exhibit distinct metabolic responses to metal and ENM exposure, even when similar LC50's are observed. These findings together illustrate the importance of understanding species-specific toxic responses and the utility of examining O2 consumption for doing so.

Effects of biosolids from a wastewater treatment plant receiving manufactured nanomaterials on Medicago truncatula and associated soil microbial communities at low nanomaterial concentrations.

Concern has grown regarding engineered nanomaterials (ENMs) entering agricultural soils through the application of biosolids and their possible effects on agroecosystems, even though the ENMs are extensively transformed. The effects of exposure to biosolids containing transformation products of these ENMs at low concentrations remain largely unexplored. We examined the responses of Medicago truncatula and its symbiotic rhizobia Sinorhizobium meliloti exposed to soil amended with biosolids from WWTP containing low added concentrations of ENMs (ENM Low), bulk/dissolved metals (bulk/dissolved Low), or no metal additions (control). We targeted adding approximately 5mg/kg of Ag and 50mg/kg of Zn, and Ti. Measured endpoints included M. truncatula growth, nodulation, changes in the expression of stress response genes, uptake of metals (Ag, Zn and Ti) into shoots, and quantification of S. meliloti populations and soil microbial communities. After 30days exposure, no effects on root or shoot biomass were observed in ENM Low and bulk/dissolved Low treatments, whereas both treatments had a larger average number of nodules (5.7 and 5.57, respectively) compared to controls (0.33). There were no significant differences in either total accumulated metal or metal concentrations in shoots among the treatments. Expression of five stress-related genes (metal tolerance protein (MTP), metal transporter (MTR), peroxidase (PEROX), NADPH oxidase (NADPH) and 1-aminocyclopropane-1-carboxylate oxidase-like protein (ACC_Oxidase)) was significantly down-regulated in both bulk/dissolved Low and ENM Low treatments. However, a change in soil microbial community composition and a significant increase in total microbial biomass were observed in ENM Low relative to control. The ENM Low treatment had increased abundance of Gram-negative and anaerobic bacteria and reduced abundance of eukaryotes compared to control. The study demonstrated that although there were some subtle shifts in microbial community composition, plant health was minimally impacted by ENMs within the time frame and at the low exposure concentrations used in this study.

Toxicogenomic Responses of the Model Legume Medicago truncatula to Aged Biosolids Containing a Mixture of Nanomaterials (TiO2, Ag, and ZnO) from a Pilot Wastewater Treatment Plant.

Toxicogenomic responses in Medicago truncatula A17 were monitored following exposure to biosolids-amended soils. Treatments included biosolids produced using a pilot wastewater treatment plant with either no metal introduced into the influent (control); bulk/ionic TiO2, ZnO, and AgNO3 added to influent (bulk/dissolved treatment); or Ag, ZnO, and TiO2 engineered nanomaterials added to influent (ENM treatment) and then added to soil, which was aged in the field for 6 months. In our companion study, we found inhibition of nodulation in the ENM but not in the bulk/dissolved treatment. Gene expression profiling revealed highly distinct profiles with more than 10-fold down-regulation in 239 genes in M. truncatula roots from the ENM treatment, while gene expression patterns were similar between bulk/dissolved and control treatments. In response to ENM exposure, many of the identified biological pathways, gene ontologies, and individual genes are associated with nitrogen metabolism, nodulation, metal homeostasis, and stress responses. Expression levels of nine genes were independently confirmed with qRT-PCR. Exposure to ENMs induced unique shifts in expression profiles and biological pathways compared with bulk/dissolved treatment, despite the lack of difference in bioavailable metal fractions, metal oxidation state, and coordination environment between ENM and bulk/dissolved biosolids. As populations of Sinorhizobium meliloti Rm2011 were similar in bulk/dissolved and ENM treatments, our results suggest that inhibition of nodulation in the ENM treatment was primarily due to phytotoxicity, likely caused by enhanced bioavailability of Zn ions.

Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial Community Composition, and Result in Increased Metal Uptake Relative to Bulk/Dissolved Metals.

We examined the effects of amending soil with biosolids produced from a pilot-scale wastewater treatment plant containing a mixture of metal-based engineered nanomaterials (ENMs) on the growth of Medicago truncatula, its symbiosis with Sinorhizobium meliloti, and on soil microbial community structure. Treatments consisted of soils amended with biosolids generated with (1) Ag, ZnO, and TiO2 ENMs introduced into the influent wastewater (ENM biosolids), (2) AgNO3, Zn(SO4)2, and micron-sized TiO2 (dissolved/bulk metal biosolids) introduced into the influent wastewater stream, or (3) no metal added to influent wastewater (control). Soils were amended with biosolids to simulate 20 years of metal loading, which resulted in nominal metal concentrations of 1450, 100, and 2400 mg kg(-1) of Zn, Ag, and Ti, respectively, in the dissolved/bulk and ENM treatments. Tissue Zn concentrations were significantly higher in the plants grown in the ENM treatment (182 mg kg(-1)) compared to those from the bulk treatment (103 mg kg(-1)). Large reductions in nodulation frequency, plant growth, and significant shifts in soil microbial community composition were found for the ENM treatment compared to the bulk/dissolved metal treatment. These results suggest differences in metal bioavailability and toxicity between ENMs and bulk/dissolved metals at concentrations relevant to regulatory limits.

Morphological and genetic changes induced by excess Zn in roots of Medicago truncatula A17 and a Zn accumulating mutant.

BACKGROUND: Nutrient fluxes associated with legume-rhizobia symbioses are poorly understood and little is known regarding the influence of abiotic stresses on development and maintenance of N-fixing nodules and root system architecture (RSA). We examined effects of Zn on nodule development and structure, root architecture, and expression of nodulation-related miRNAs in Medicago truncatula and the mutant, raz (requires additional Zn). FINDINGS: Excess Zn increased root and shoot associated Zn in both genotypes, however, raz plants had lower root associated Zn than WT plants. Roots of raz plants exposed to excess Zn had less volume, surface area, and total length compared to WT plants. Raz plants had lower lateral root number than WT plants. Excess Zn was found to increase root diameter in both genotypes. The Mn Translocation Factor (TfMn) increased in response to Zn in both genotypes; this was more pronounced in raz plants. TfZn was higher in raz plants and reduced in both genotypes in response to Zn. Nodulation was not influenced by Zn treatment or plant genotype. MicroRNA166 was upregulated under excess Zn in WT plants. CONCLUSIONS: Neither the raz mutation nor Zn treatment affected nodulation, however, raz plants had altered RSA compared with WT and responded differently to Zn, implying the mutation potentially modulates RSA responses to Zn but doesn't play a direct role in nodulation. MicroRNA166 was significantly induced in WT plants by excess Zn, warranting further investigation into the potential role it plays in controlling RSA.