Selective inhibition of Erwinia amylovora by the herbicidally active germination-arrest factor (GAF) produced by Pseudomonas bacteria.

AIMS: The germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4-formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination. METHODS AND RESULTS: Culture filtrate from Ps. fluorescens WH6 had little or no effect on 17 species of bacteria grown in Petri dish lawns, but the in vitro growth of Erwinia amylovora, the causal agent of the disease of orchard crops known as fire blight, was strongly inhibited by the filtrate. The anti-Erwinia activity of WH6 culture filtrate was shown to be due to its GAF content, and a commercially available oxyvinylglycine, 4-aminoethoxyvinylglycine (AVG), exhibited anti-Erwinia activity similar to that of GAF. The effects of GAF on Erwinia were reversed by particular amino acids. CONCLUSIONS: The biological properties of GAF include a rather specific antimicrobial activity against Erw. amylovora. This may be a general property of oxyvinylglycines as AVG exhibited similar activity. The ability of particular amino acids to reverse GAF inhibition is consistent with a potential effect of this compound on the activity of aminotransferases. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here demonstrate a novel antimicrobial activity of oxyvinylglycines and suggest that GAF and/or GAF-producing bacteria may have potential for the control of fire blight.

Mineral concentration in selected native temperate grasses with potential use as biofuel feedstock.

Stands of native grasses along roadways, in buffer strips, riparian zones and grass prairies have potential utility as feedstock for bioenergy production. The sustainability of harvesting these stands is reliant, in part, on knowledge of the mineral concentration of the harvested grasses because removal of mineral nutrients such as phosphorus (P) and potassium (K) can impact subsequent biomass production and ecosystem services associated with these stands. Mineral content of biomass, particularly that of silicon (Si), chlorine (Cl), and sulfur (S) also impacts thermochemical conversion approaches that convert grasses into bioproducts. This study quantified Cl, S, Si, P and K in Bromus marginatus, Elymus glaucus, Poa secunda, Pseudoroegneria, Elymus lanceolatus, Elymus trachycaulus, Leymus cinereus, Leymus triticoides, and Pseudoroegneria spicata collected at three growth developmental stages from four plant introduction stations located in the western US. Differences (P< or =0.05) in mineral concentrations were associated with developmental stage, species, and location. Variability was greatest in Si concentrations which ranged from 1847 to 28620 mg kg(-1), similar to those recorded in other grasses. Variability in Cl and S concentrations also occurred, but at less magnitude than that of Si. Concentrations of P and K, two mineral fertilizer components, varied approximately threefold among these grasses. Differences in mineral concentrations among these grasses were not completely dependent upon soil mineral content. Long-term evaluations of available soil mineral concentrations under contrasting management practices are needed to quantify how local conditions impact mineral cycling, and in turn, the sustainability of harvesting these stands. The data presented here establish baselines for these species in locations subject to contrasting environmental and microbiological conditions that affect mineral cycling and availability.

Genotypic variability in mineral composition of switchgrass.

Switchgrass (Panicum virgatum L.) is a warm season perennial grass with great potential as an energy crop in the USA. It is widely adapted to many regions of the country, produces large amounts of biomass, serves as a useful forage grass, and provides ecosystem services that benefit soil and water quality and wildlife. Biological and thermochemical technologies are being developed to convert herbaceous biomass, including switchgrass, to energy. The objective of this research was to determine the effect of genotype and production environment on the concentration of minerals that affect the suitability of switchgrass for thermochemical conversion and to quantify the amount of potassium (K) and phosphorus (P) removed from the production system by harvest of the aboveground biomass, a measure of the sustainability of the practice. Straw dry biomass contained from 1.3 to 6.4 kg Mg(-1) and from 6.2 to 15.8 kg Mg(-1) of P and K, respectively. Variability in aluminum (Al), calcium (Ca), chloride (Cl), K, P, silicon (Si), and sulfur (S) concentrations across locations was relatively high, ranging from twofold (Al) to eightfold (Cl). Location had a strong impact on mineral concentrations among switchgrass genotypes evaluated in this study. Latitude of origin impacted the Cl and Si concentrations measured in plant tissues, but none of the other minerals analyzed in this study. Upland and lowland cytotypes explained some of the observed differences, but populationxlocation interactions were the primary source of variability in the concentration of these minerals.

Differential accumulation of a 24-kd dehydrin protein in wheat seedlings correlates with drought stress tolerance at grain filling.

The Pacific Northwest (PNW), an important region for wheat production in the USA, is often subject to water deficits during sowing and grain filling. These deficits reduce the quality and yield of the crop. As a consequence, an important objective of breeding programs in the region is improving the genetic adaptation of wheat cultivars to drought stress. One response to dehydrative stresses is the accumulation of proteins called dehydrins, which are believed to protect membranes and macromolecules against denaturation. We characterized dehydrin accumulation in seedlings during drought stress and its correlation with stress tolerance during grain filling in seven wheat cultivars, 'Connie', 'Gene', 'TAM105', 'Rod', 'Hiller', 'Rhode', and 'Stephens'. A 24-kd dehydrin accumulated in seedlings under stress, but not in irrigated control plants. Connie, TAM105, and Gene started to accumulate dehydrins at the fourth day of stress, while the other cultivars showed dehydrins after twelve days of stress. This differential accumulation in seedlings was associated with stress tolerance at grain filling, characterized by a lower reduction in yield and in the rate of decrease in leaf water potential per day of stress. Connie, TAM105, Gene and Rod where the most tolerant cultivars. The results indicate that expression of this 24-kd dehydrin might serve as a rapid and non-destructive screening technique at the seedling stage. Even though the results are promising, selection experiments using a population segregating for stress tolerance are needed to test more conclusively whether this dehydrin can serve as a genetic marker for cultivars with tolerance to drought stress.

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.

Monoclonal antibodies against the plant cytokinin, dihydrozeatin riboside.

Three monoclonal antibodies (all IgG1) were prepared against the plant cytokinin dihydrozeatin riboside (diH[9R]Z) and characterized for suitability in the isolation and quantification of dihydrozeatin-type phytohormones. These antibodies detected as little as 5-10 pg of the homologous cytokinin when used in both competitive and noncompetitive immunoassays and each had characteristic crossreactivity with structurally-related cytokinins. One antibody was used to quantify HPLC-purified diH[9R]Z recovered from wheat tissue. The same antibody also was linked to an insoluble support for use in affinity purification of a mixture of cytokinins. These antibodies will be useful for purification and quantification of dihydrozeatin and its 9-riboside and 9-glucoside in plant tissue.

A monoclonal antibody against the plant growth regulator, abscisic acid.

Monoclonal antibodies were prepared against the plant growth regulator abscisic acid (ABA) conjugated to keyhole limpet hemocyanin through C-4. One of these antibodies was characterized for use in a competition fluorescence enzyme-linked immunosorbent assay (F-ELISA). The antibody detected femtomole quantities of ABA when used in the F-ELISA and showed minimal cross-reactivity with ABA metabolites and structural analogs. Dilution analysis suggested that the F-ELISA could be used to determine the ABA content of methanolic extracts of crude samples of wheat seeds without further purification. The F-ELISA was used to determine the effect of seed priming on ABA levels in wheat seeds. The antibody also was used in a modified noncompetitive indirect ELISA to measure ABA content of wheat caryopses. The noncompetitive ELISA was more sensitive than the F-ELISA, although the F-ELISA had a broader measuring range. When our anti-ABA antibody and a commercially available anti-ABA antibody were compared by indirect ELISA, there were no significant differences between the ABA estimates.

Monoclonal antibodies against the plant cytokinin, cis-zeatin riboside.

Three monoclonal antibodies (two IgG1 and one IgA) prepared against the plant cytokinin, cis-zeatin riboside, were characterized for use in a fluorescence competition enzyme linked immunosorbant assay (F-ELISA). Immunoassays that employed each antibody detected femtomole quantities of the homologous cytokinin and characteristic quantities of structurally-related cytokinins. One of the antibodies was used to quantify HPLC-purified cis-zeatin riboside recovered from spiked samples of wheat leaves. These are the first monoclonal antibodies against a cis- derivative of a plant cytokinin and will be useful for purification and quantification of cis-zeatin and the 9-riboside and 9-glucoside in plant samples.

Phenotypic Analysis of a Dwarf Wheat (Triticum aestivum L.) with Altered Phytochrome-Mediated Growth Responses.

The influence of the amount of red light relative to far red light (red/far red ratio) on leaf-sheath elongation, leaf length, tillering, assimilate partitioning to shoots and roots, and chlorophyll content in a dwarf wheat (Triticum aestivum L. cv Tibet Dwarf) and seven other dwarf and standard cultivars was determined. All cultivars tested showed far red-stimulated leaf and leaf-sheath elongation except Tibet Dwarf. Tibet Dwarf was also unresponsive to a brief end-of-day exposure to far red light, although the other cultivars exhibited increased leaf-sheath and leaf length. In these cultivars, the effects of an end-of-day 5-min far red exposure were reversible by a subsequent 5-min red light exposure. Shoot/root ratios were higher and tillering was suppressed by increased far red irradiance in all cultivars except Tibet Dwarf. In addition, Tibet Dwarf was less responsive to dark-induced leaf and leaf-sheath elongation (etiolation) and retained chlorophyll in dark-adapted leaves longer than control cultivars. Tibet Dwarf did not differ from the other cultivars in leaf chlorophyll content. Western blot analysis, using an antibody against phytochrome A, showed that dark-grown Tibet Dwarf shoots contained at least twice the amount of detectable phytochrome A protein present in the other wheat cultivars. These results, plus the short, thick-stemmed, dark-pigmented phenotype of Tibet Dwarf suggest the possibility of a mutation in the phytochrome/signal transduction pathway.

Hybrid hybridoma cell lines which express anti-zeatin riboside, anti-abscisic Acid, and hybrid antibodies.

Hybrid hybridoma cell lines that secreted antibodies which reacted with two distinct plant hormones, abscisic acid (ABA) and zeatin riboside (ZR) were prepared and cloned. These cell lines were derived by polyethylene glycol-mediated fusion of HAT-sensitive anti-ZR hybridoma cells with splenocytes harvested from a BALB/c mouse previously immunized with an ABA-bovine serum albumin conjugate. Chromatographic analyses indicated that these lines expressed two different isotypes, each associated with a specific immunologic reactivity, and that the populations of immunoglobulins secreted by these hybridomas included antibodies directed against each individual hapten as well as hybrid molecules which reacted simultaneously with both. Hybrid hybridomas such as these should provide antibody populations useful for simultaneous isolation of multiple plant hormones from individual plant samples.

A quantitative fluorescence enzyme immunoassay for plant cytokinins.

An enzyme-linked immunosorbent assay (ELISA) which used 4-methylumbelliferyl phosphate as an enzyme substrate was used to quantify two plant cytokinins. This assay detected as little as 0.03 pmol (approximately 10 pg) of cytokinin in microplate wells coated with a cytokinin-ovalbumin conjugate. The method measured competition between free cytokinin and the bound conjugate for reaction with monoclonal anticytokinin antibodies and used a standard curve prepared by use of known amounts of free cytokinin to quantify hormone levels in unknown samples. Standard curves which consisted of logit/log plots of fluorescence units versus picomoles of competing cytokinin measured from 0.03 to 256 pmol (approximately 10-85,000 picograms) of zeatin riboside (ZR) or isopentenyl adenosine. The fluorescence ELISA was compared with radioimmunoassay for the quantification of ZR in wheat (Triticum aestivum L., cultivar Stephens) seed samples. This fluorescence ELISA method is recommended for use in combination with a fractionation method, such as HPLC, to quantify cytokinins present in plant extracts.

Analysis of ovine colostrum to detect antibody against progressive pneumonia virus.

Immunoglobulins were isolated and purified from the colostrum and serum of progressive pneumonia virus infected sheep and also from non-infected control sheep. Four classes of immunoglobulins were isolated from sheep colostrum (IgG1, IgG2, IgA and Ig10s). Three classes of immunoglobulins were isolated from sheep serum (IgG1, IgG2 and IgM). Low levels of virus neutralizing activity were demonstrated only in the whole serum and purified serum IgG1 preparations. No complement fixing activity was detected in any of the antibody preparations from colostrum.