Gasified Grass and Wood Biochars Facilitate Plant Establishment in Acid Mine Soils.

Heavy metals in exposed mine tailings threaten ecosystems that surround thousands of abandoned mines in the United States. Biochars derived from the pyrolysis or gasification of biomass may serve as a valuable soil amendment to revegetate mine sites. We evaluated the ability of two biochars, produced by gasification of either Kentucky bluegrass seed screenings (KB) or mixed conifer wood (CW), to support the growth of plants in mine spoils from the abandoned Formosa and Almeda Mines in Oregon. To evaluate the potential for plant establishment in mine tailings, wheat was grown in tailings amended with biochar at rates ranging from 0 to 9% (w/w). Both KB and CW biochars promoted plant establishment by increasing soil pH, increasing concentrations of macro- and micronutrients, and decreasing the solubility and plant uptake of heavy metals. Formosa tailings required at least 4% biochar and Almeda soil required at least 2% biochar to promote healthy wheat growth. A complimentary experiment in which mine spoils were leached with simulated precipitation indicated that biochar amendment rates ≥4% were sufficient to neutralize the elution pH and reduce concentrations of potentially toxic elements (Zn, Cu, Ni, Al) to levels near or below concern. These findings support the use of gasified biochar amendments to revegetate acid mine soils.

Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor.

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Pseudomonas fluorescens SBW25 produces furanomycin, a non-proteinogenic amino acid with selective antimicrobial properties.

BACKGROUND: Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors' laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid. RESULTS: P. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2'R,5'S)-2-amino-2-(5'methyl-2',5'-dihydrofuran-2'-yl)acetic acid]. CONCLUSIONS: The identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.

Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

Chemical characterization of chars developed from thermochemical treatment of Kentucky bluegrass seed screenings.

Seed mill screenings would be a considerable biofeedstock source for bioenergy and char production. Char produced from the gasification of residues resulting from cleaning of grass seed and small grains could be recycled to a cropping system as a soil amendment if chemical characterization determined that the gasification process had not produced or concentrated deleterious chemical or physical factors that might harm the environment, crop growth or yield. Previous reports have shown that char derived from the pyrolysis of a variety of biomass feedstocks has potential to enhance soil quality by pH adjustment, mineral amendment, and improved soil porosity. The objective of this research was to characterize char produced from Kentucky bluegrass seed mill screenings (KBss) by a small-scale gasification unit, operated at temperatures between 600 and 650°C, with respect to polycyclic aromatic hydrocarbons, selected heavy metals, as well as other physical and chemical characteristics, and determine its suitability for agricultural application as a soil amendment. We utilized KBss as a model for seed and grain-cleaning residues with the understanding that chemical and physical characteristics of char produced by gasification or other cleaning residues may differ based on soil and environmental conditions under which the crops were produced. Our results support the hypothesis that KBss char could be applied in a cropping system without toxic environmental consequences and serve multiple purposes, such as; recycling critical plant macro- and micro-nutrients back to existing cropland, enhancing soil carbon sequestration, managing soil pH, and improving water holding capacity. Crop field trails need to be implemented to further test these hypotheses.

Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis.

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6.

BACKGROUND: Pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. RESULTS: We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system. CONCLUSIONS: The improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

4-Formylaminooxyvinylglycine, an herbicidal germination-arrest factor from Pseudomonas rhizosphere bacteria.

A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over 20 other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The germination-arrest factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine on the basis of NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally active rhizosphere bacteria in our collection.

Fatty acid methyl ester analysis to identify sources of soil in surface water.

Efforts to improve land-use practices to prevent contamination of surface waters with soil are limited by an inability to identify the primary sources of soil present in these waters. We evaluated the utility of fatty acid methyl ester (FAME) profiles of dry reference soils for multivariate statistical classification of soils collected from surface waters adjacent to agricultural production fields and a wooded riparian zone. Trials that compared approaches to concentrate soil from surface water showed that aluminum sulfate precipitation provided comparable yields to that obtained by vacuum filtration and was more suitable for handling large numbers of samples. Fatty acid methyl ester profiles were developed from reference soils collected from contrasting land uses in different seasons to determine whether specific fatty acids would consistently serve as variables in multivariate statistical analyses to permit reliable classification of soils. We used a Bayesian method and an independent iterative process to select appropriate fatty acids and found that variable selection was strongly impacted by the season during which soil was collected. The apparent seasonal variation in the occurrence of marker fatty acids in FAME profiles from reference soils prevented preparation of a standardized set of variables. Nevertheless, accurate classification of soil in surface water was achieved utilizing fatty acid variables identified in seasonally matched reference soils. Correlation analysis of entire chromatograms and subsequent discriminant analyses utilizing a restricted number of fatty acid variables showed that FAME profiles of soils exposed to the aquatic environment still had utility for classification at least 1 wk after submersion.

Regulation of flooding tolerance of SAG12:ipt Arabidopsis plants by cytokinin.

A SAG12:ipt gene construct, which increases cytokinin biosynthesis in response to senescence, was introduced into Arabidopsis plants to delay senescence induced by flooding stress. Two forms of flooding stress, including total submergence and root waterlogging, were applied to SAG12:ipt (IPT) and wild-type (WT) plants for 1, 3, and 5 d. A separate experiment compared the recovery of WT and IPT plants subjected to flooding stress. Biomass accumulation, carbohydrate and chlorophyll contents, and cytokinin and abscisic acid were quantified to compare genotypic responses to flooding stress and post-flooding recovery. Real-time RT-PCR studies were performed to quantify ipt and SAG12 gene expression. IPT plants exposed to waterlogging accumulated greater quantities of cytokinins more rapidly than WT plants or those exposed to total submergence. Cytokinin accumulation was accompanied by phenotypic adaptations, including chlorophyll retention and increased biomass and carbohydrate content relative to WT plants. Abscisic acid accumulated rapidly in WT and IPT plants under waterlogging stress but remained low in all genotypes exposed to total submergence. IPT plants showed improved recovery after waterlogging stress was removed. Expression of ipt in submerged plants did not result in cytokinin accumulation until submergence stress was removed. At that point, IPT plants accumulated greater quantities of cytokinin and recovered to a greater extent than WT plants. This study established the relationship between flooding tolerance and cytokinin accumulation in IPT plants and suggested that translation of ipt transcripts and subsequent cytokinin accumulation were delayed under submergence stress.

An affinity probe for isolation of abscisic acid-binding proteins.

An affinity probe has been developed for isolation of receptor proteins that bind the plant hormone abscisic acid (ABA). The structural features required for biological activity have been preserved, and the probe has been demonstrated to bind to known ABA-binding proteins.

Microplate quantification of plant leaf superoxide dismutases.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose-response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50 units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60 min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD.

Overproduction of cytokinins in petunia flowers transformed with P(SAG12)-IPT delays corolla senescence and decreases sensitivity to ethylene.

Plant senescence is regulated by a coordinated genetic program mediated in part by changes in ethylene, abscisic acid (ABA), and cytokinin content. Transgenic plants with delayed senescence are useful for studying interactions between these signaling mechanisms. Expression of ipt, a cytokinin biosynthetic gene from Agrobacterium tumefaciens, under the control of the promoter from a senescence-associated gene (SAG12) has been one approach used to delay senescence. We transformed petunia (Petunia x hybrida cv V26) with P(SAG12)-IPT. Two independently transformed lines with extended flower longevity (I-1-7-22 and I-3-18-34) were used to study the effects of elevated cytokinin content on ethylene synthesis and sensitivity and ABA accumulation in petunia corollas. Floral senescence in these lines was delayed 6 to 10 d relative to wild-type (WT) flowers. Ipt transcripts increased in abundance after pollination and were accompanied by increased cytokinin accumulation. Endogenous ethylene production was induced by pollination in both WT and IPT corollas, but this increase was delayed in IPT flowers. Flowers from IPT plants were less sensitive to exogenous ethylene and required longer treatment times to induce endogenous ethylene production, corolla senescence, and up-regulation of the senescence-related Cys protease phcp1. Accumulation of ABA, another hormone regulating flower senescence, was significantly greater in WT corollas, confirming that floral senescence was delayed in IPT plants. These results extend our understanding of the hormone interactions that regulate flower senescence and provide a means of increasing flower longevity.

Wheat dehydrin accumulation in response to drought stress during anthesis.

The winter wheat (Triticum aestivum L.) producing region of the US Pacific Northwest (PNW) is often subject to water deficits at sowing and during grain filling. Improved genetic adaptation of wheat cultivars to drought stress is one objective of breeding efforts in the region. Consequently, there is interest in identifying molecular markers associated with drought tolerance. Dehydrins, a family of proteins that accumulate in response to dehydrative stress, may provide a suitable marker for use in breeding programs. Seven cultivars (Connie, Gene, TAM105, Rod, Hiller, Rhode and Stephens) were evaluated in two experiments in which dehydrin accumulation and their association to stress tolerance during grain filling were characterized during progressive drought stress. A24-kDa dehydrin was present in leaves at each sampling date in all seven cultivars. Quantitative differences in accumulation of this protein were observed between cultivars on the third sampling date (4 d of stress). This differential accumulation was associated with stress tolerance characterized by a lower yield reduction and a lowered rate of decrease in leaf water potential in Connie, TAM105 and Gene. In contrast to leaves, an increased number of dehydrins were observed in grains under stress and non-stress treatments. Despite the number of dehydrins detected, there was no apparent association between drought stress and dehydrin expression in grains. Although the specific role of these proteins remains unknown, their association with stress tolerance suggests that dehydrins have utility in improving adaptation to drought and as markers for drought tolerance.