Salicylhydroxamic Acid Research Articles

Glucosinolate Degradation Products, Isothiocyanates, Nitriles, and Thiocyanates, Induce Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production inArabidopsis thaliana

Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.

Direct electrochemical determination of Candida albicans activity

Despite advances made in the field, rapid detection methods for the human pathogen Candida albicans are still missing. In this regard, bio-electrochemical systems including electrochemical sensors and biosensors satisfy the increasing demand for rapid, reliable, and direct microbial analyses. In this study, the bioelectrochemical characteristics of C. albicans were investigated for use in an analytical system that determines the viability of the organisms. The electrochemical responses of viable and non-viable cells of C. albicans and Saccharomyces cerevisiae were monitored. Cyclic voltammograms (CV) showed an irreversible oxidation peak at about 750mV that accounts for viable cells. The peak current increased at viable cell numbers ranging from 3×105 to 1.6×107cells/ml, indicating that the amount of viable cells can be accurately quantified. To elucidate the underlying electron transfer processes, the influence of electron transfer chain (ETC) – inhibitors on the electrochemical behavior of the two organisms were investigated. Inhibition of the function of classical respiratory chain (CRC) led to a decrease in the electrochemical response, whereas the oxidation current increased when the alternative oxidase (AOX) pathway was blocked by salicylhydroxamic acid (SHA). Blocking the AOX pathway improved the electrochemical performance, suggesting an involvement in the CRC, with cytochrome c oxidase (COX) as a relevant protein complex. Mutants, in which components of COX were deleted, showed a lower electro-activity than the wild-type strain. Particularly, deletion of subunit COX5a almost completely abolished the electrochemical signal. We believe that this work can be utilized for the development of early detection assays and opens the door for new technological developments in the field of C. albicans.

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells

N(6)-benzyladenine (BA) and N(6)-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723-1735, 2003, Plant Sci 168:389-395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N(6)-benzyladenine 7-β-D-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.

Involvement of alternative oxidase in the regulation of sensitivity of Sclerotinia sclerotiorum to the fungicides azoxystrobin and procymidone

Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.

Cell Death of Rice Roots under Salt Stress May Be Mediated by Cyanide-Resistant Respiration

Treatment with solutions containing high concentrations of NaCl (200 or 300 mM) induced cell death in rice (Oryza sativa L.) roots, as well as the application of exogenous hydrogen peroxide (H2O2). Moreover, the pretreatment with dimethylthiourea (DMTU), a scavenger of H2O2, partially alleviated the root cell death induced by 200 mM NaCl. These observations suggest that the cell death of rice roots under high salt stress is linked to H2O2 accumulation in vivo. NaCl stress increased the level of cyanide-resistant respiration to some extent and enhanced the transcript levels of the alternative oxidase (AOX) genes AOX1a and AOX1b in rice roots. High-salt-stressed (200 mM NaCl) rice roots pretreated with 1 mM salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, exhibited higher levels of cell death and H2O2 production than roots subjected to either 200 mM NaCl stress or SHAM treatment alone. These results suggest that cyanide-resistant respiration could play a role in mediating root cell death under high salt stress. Furthermore, this function of cyanide-resistant respiration could relate to its ability to reduce the generation of H2O2.

Saccharomyces cerevisiae-induced stomatal closure mainly mediated by salicylhydroxamic acid-sensitive peroxidases in Vicia faba

Saccharomyces cerevisiae induced stomatal closure in a dose-dependent manner on Vicia faba L. (cv. Daqingpi). Using pharmacological inhibitors in this study, we found that stomatal closure was completely inhibited by salicylhydroxamic acid (SHAM) and reduced glutathione (GSH), whereas slightly inhibited by diphenyleneiodonium chloride (DPI), suggesting that H2O2 was mostly produced by cell wall peroxidases. The specific NO scavenger (cPTIO), NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME) and sodium azide (NaN3; inhibitor of nitrate reductase) prevented yeast-induced stomatal closure, suggesting that NO in guard cells of V.faba is derived from both NOS-like enzyme and nitrate reductase. Results of HgCl2 and β-mercaptoethanol (ME) treatment (as a functional inhibitor of water channels and its reversing agent, respectively) suggest that water channels are involved in yeast-induced stomatal movements. CoCl2 (the blocker of calcium channel), LaCl3 (Ca(2+) antagonist) and EGTA (Ca(2+) chelator) also impaired yeast-induced stomatal closure. Thus, it is concluded that H2O2, NO, water channels and Ca(2+) are involved in yeast-induced stomatal closure.

Some properties of active and latent catechol oxidase of mushroom

Latent form of mushroom catechol oxidase was activated by O,1% sodium dodecyl sulfate (SDS). Catalytic power of the latent form, calculated from the kinetic parameters was 1,8 times higher than that of active one. Salicyl hydroxamic acid (SHAM) appeared as a powerful inhibitor for both active and latent forms of catechol oxidase. However, in the range of 150-250 μM SHAM the inhibitory effect for active catechol oxidase was significantly higher than that for the latent one. Non-competitive and irreversible characteristics of inhibition of latent and active catechol oxidase was calculated from kinetic data. Electrophoretic analysis followed by scanning of the gels was used. The spots' absorbance was determined from a computer image of the isoenzyme band patterns. It allowed us to estimate gels quantitatively. Presence of one additional clearly defined slow moving isoform of SDS-activated catechol oxidase, differed in the respect of 3 bands for the active and 4 bands for the total.

Cell Death of Rice Roots under Salt Stress May Be Mediated by Cyanide-Resistant Respiration

Treatment with solutions containing high concentrations of NaCl (200 or 300 mM) induced cell death in rice (Oryza sativa L.) roots, as well as the application of exogenous hydrogen peroxide (H2O2). Moreover, the pretreatment with dimethylthiourea (DMTU), a scavenger of H2O2, partially alleviated the root cell death induced by 200 mM NaCl. These observations suggest that the cell death of rice roots under high salt stress is linked to H2O2 accumulation in vivo. NaCl stress increased the level of cyanide-resistant respiration to some extent and enhanced the transcript levels of the alternative oxidase (AOX) genes AOX1a and AOX1b in rice roots. High-salt-stressed (200 mM NaCl) rice roots pretreated with 1 mM salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, exhibited higher levels of cell death and H2O2 production than roots subjected to either 200 mM NaCl stress or SHAM treatment alone. These results suggest that cyanide-resistant respiration could play a role in mediating root cell death under high salt stress. Furthermore, this function of cyanide-resistant respiration could relate to its ability to reduce the generation of H2O2.

Beneficiation of Ultrafine Cassiterite from a Tin Tailings by Flotation

With the continuous depletion of primary tin ores, tin tailings has become an important source for the production of tin; however, the large-scale utilization of tin tailings is disappointedly scarce, due to its leanness in valuable minerals and insufficient methods available for processing such low-grade tailings. A flotation process has been used on a laboratory scale to investigate the effects of various reagents such as salicylhydroxamic acid, lead nitrate, sodium hexametaphosphate and pine oil on tin recovery at neutral pulp. Because cassiterite is friable and a large amount of fines and slimes were generated, usually de-sliming is used to prevent slime coating and to increase the recovery of tin. However, analyses have shown that fine particles in the sample mostly contain tin, thus de-sliming was not suggested. By applying the process, ultrafine cassiterite could be efficiently recovered from the slime tailings, and the concentrate assaying 1.82% Sn with a recovery of 76.90% was obtained.

Genes encoding plant-specific class III peroxidases are responsible for increased cold tolerance of the brassinosteroid-insensitive 1 mutant.

We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1-mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA-dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA-dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahGsiz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA-responsive genes, such as PR1 and PR2. Furthermore, other SA-accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahGsiz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.

Strobilurin (QoI) Resistance in Populations of Erysiphe necator on Grapes in Michigan.

Powdery mildew, caused by Erysiphe necator, is the most common and destructive disease of grapes (Vitis spp.) worldwide. In Michigan, it is primarily controlled with fungicides, including strobilurins (quinone outside inhibitors [QoIs]). Within the United States, resistance to this class of fungicides has been reported in E. necator populations in some east coast states. Among 12 E. necator isolates collected from five Michigan vineyards in 2008, one carried the G143A single-nucleotide mutation responsible for QoI resistance. This isolate was confirmed to be resistant in a conidium germination assay on water agar amended with trifloxystrobin at 0.001, 0.01, 0.1, 1, 10, or 100 μg/ml and salicylhydroxamic acid (100 mg/liter). The mutant isolate was able to germinate on media amended with 100 μg/ml trifloxystrobin, whereas a representative wild-type isolate did not germinate at concentrations higher than 0.1 μg/ml. In 2009, 172 isolates were collected from a total of 21 vineyards (juice and wine grapes): three vineyards with no fungicide application history (baseline sites), six research vineyards, and 12 commercial vineyards. QoI resistance was defined as the effective concentration that inhibited 50% of conidial germination (EC50) > 1 μg/ml. Isolates from baseline sites had EC50 values mostly below 0.01 μg/ml, while isolates that were highly resistant to trifloxystrobin (EC50 > 100 μg/ml) occurred in five research and three commercial wine grape vineyards at frequencies of 40 to 100% and 25 to 75% of the isolates, respectively. The G143A mutation was detected in every isolate with an EC50 > 1 μg/ml. These results suggest that fungicide resistance may play a role in suboptimal control of powdery mildew observed in some Michigan vineyards and emphasizes the need for continued fungicide resistance management.

A synthetic polypeptide conjugate from a 42‐residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity

Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which is present in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innate immune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovascular diseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO, salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2 μM, was conjugated to a designed set of 42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates, and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate 4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around 400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed in competition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions of SHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests a route to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

Phylogenetic relationships, pathogenicity and fungicide sensitivity ofGreeneria uvicolaisolates fromVitis viniferaandMuscadinia rotundifoliagrapevines

Greeneria uvicolacauses bitter rot onVitis vinifera(bunch grapes) andMuscadinia rotundifolia(muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship ofG. uvicolarepresentatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–5∙8S–ITS2), 28S large subunit (LSU) nuclearrDNAand β‐tubulin‐2.Greeneria uvicolaisolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates fromV. vinifera; USA isolates fromM. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic onV. vinifera(cv. Chardonnay) berries although those originating fromM. rotundifoliawere not as aggressive as isolates fromV. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.

Temporal analysis of xylose fermentation by Scheffersomyces stipitis using shotgun proteomics

Proteomics and fermentation technology have begun to integrate to investigate fermentation organisms in bioprocess development. This is the first shotgun proteomics study employed to monitor the proteomes of Scheffersomyces stipitis during xylose fermentation under oxygen limitation. We identified 958 nonredundant proteins and observed highly similar proteomes from exponential to early stationary phases. In analyzing the temporal proteome, we identified unique expression patterns in biological processes and metabolic pathways, including alternative respiration salicylhydroxamic acid (SHAM) pathway, activation of glyoxylate cycle, expression of galactose enzymes, and secondary zinc-containing alcohol dehydrogenase and O-glycosyl hydrolases. We identified the expression of a putative, high-affinity xylose sugar transporter Xut1p, but low-affinity xylose transporters were absent. Throughout cell growth, housekeeping processes included oxidative phosphorylation, glycolysis, nonoxidative branch of the pentose phosphate pathway, gluconeogenesis, biosynthesis of amino acids and aminoacyl total RNA (tRNA), protein synthesis and proteolysis, fatty acid metabolism, and cell division. This study emphasized qualitative analysis and demonstrated that shotgun proteomics is capable of monitoring S. stipitis fermentation and identifying physiological states, such as nutrient deficiency.

Integrated Use of Pyraclostrobin and Epoxiconazole for the Control of Fusarium Head Blight of Wheat in Anhui Province of China.

Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. Carbendazim (a benzimadazole fungicide, MBC), has been extensively used for the control of FHB, resulting in severe MBC resistance in China. This article presents the baseline sensitivity of F. asiaticum and F. graminearum isolates from Anhui Province of China to fungicides pyraclostrobin (a quinone outside inhibitor) and epoxiconazole (a sterol demethylation inhibitor). In the presence of salicylhydroxamic acid, the 50% effective concentration (EC50) values for pyraclostrobin in inhibiting mycelial growth of the 126 F. asiaticum isolates and 63 F. graminearum isolates were 0.012 to 0.135 μg/ml and 0.010 to 0.105 μg/ml, and the EC50 values for pyraclostrobin in inhibiting conidium germination of the F. asiaticum and F. graminearum populations were 0.047 to 0.291 and 0.042 to 0.255 μg/ml, respectively. The EC50 values for epoxiconazole in inhibiting mycelial growth of the F. asiaticum and F. graminearum populations were 0.12 to 0.95 and 0.16 to 0.93 μg/ml, respectively. All of the baseline sensitivity curves were unimodal. This study also suggested that there was no cross-resistance between MBC and pyraclostrobin or epoxiconazole. In the protective and curative tests, pyraclostrobin and epoxiconazole applied at 200 and 300 μg/ml exhibited over 75% protective and curative control efficacy in all treatments. In field trials, both pyraclostrobin and epoxiconazole at 225 g a.i./ha provided over 80% efficacy in 2010 and 2011 at both sites where MBC resistance occurred, suggesting excellent activity against FHB. Interestingly, integrated use of pyraclostrobin + epoxiconazole applied at 150 + 150 g a.i./ha provided over 85% efficacy at both sites in 2010 and 2011. Pyraclostrobin and epoxiconazole should be good alternatives to MBC for the control of FHB, and integrated use of these two fungicides might achieve greater efficacy.

Distribution of lanthanide and actinide elements between bis-(2-ethylhexyl)phosphoric acid and buffered lactate solutions containing selected complexants

With the renewed interest in the closure of the nuclear fuel cycle, the TALSPEAK process is being considered for the separation of Am and Cm from the lanthanide fission products in a next generation reprocessing plant. However, an efficient separation requires tight control of the pH which likely will be difficult to achieve on a large scale. To address this issue, we measured the distribution of lanthanide and actinide elements between aqueous and organic phases in the presence of complexants which were potentially less sensitive to pH control than the diethylenetriaminepentaacetic (DTPA) used in the process. To perform the extractions, a rapid and accurate method was developed for measuring distribution coefficients based on the preparation of lanthanide tracers in the Savannah River National Laboratory neutron activation analysis facility. The complexants tested included aceto-, benzo-, and salicylhydroxamic acids, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and ammonium thiocyanate (NH4SCN). The hydroxamic acids were the least effective of the complexants tested. The separation factors for TPEN and NH4SCN were higher, especially for the heaviest lanthanides in the series; however, no conditions were identified which resulted in separations factors which consistently approached those measured for the use of DTPA.

First Report of Reduced Sensitivity to a QoI Fungicide in Apple Scab (Venturia inaequalis) in Virginia and Maryland.

Apple scab caused by Venturia inaequalis (Cooke) Winter continues to be a significant concern for apple growers in Virginia and Maryland. Management of scab has relied on foliar fungicides including strobilurins (QoIs) such as trifloxystrobin (TFX). In recent years, populations of V. inaequalis with reduced sensitivity to the QoIs have been reported in other apple-growing regions of the United States (1,2). Although QoIs generally remain effective in the mid-Atlantic, concerns about the development of resistance in some Virginia and Maryland orchards prompted this study. Twenty-five isolates of V. inaequalis were obtained from scabby leaves from commercial and experimental orchards in Virginia in 2010 (n = 6) and 2011 (n = 14) and from a commercial orchard in Maryland (n = 5) in 2011. Orchards had previously been treated with QoI or sterol-inhibiting (SI) fungicides. Isolates of V. inaequalis were grown on potato dextrose agar (PDA) amended with 0, 0.1, or 1.0 μg ml-1 TFX with 100 μg ml-1 salicylhydroxamic acid (SHAM) and incubated at 19°C. Colony growth was measured weekly for 4 weeks. To account for the SI use at some orchards, isolates of V. inaequalis were also evaluated on PDA amended with 0, 0.5, or 1.0 μg ml-1 myclobutanil. Fungicide sensitivities were expressed as a percentage of the difference in colony growth using a discriminatory dose of 1.0 μg ml-1 TFX with SHAM or 1.0 μg ml-1 myclobutanil at 28 days. Isolates with <25% growth suppression (GS) were classified as fully resistant, whereas those with >70% GS were classified as sensitive. Isolates with 25 to 70% GS were classified as partially resistant. Effective concentration (EC50) values (TFX concentration inhibiting colony growth by 50%) were also calculated for a subset of fully resistant and sensitive isolates. Of the 25 isolates tested, six were fully resistant to TFX (mean EC50 value greater than 10.0 μg ml-1) and 10 were sensitive (mean EC50 value of 0.04 μg ml-1 ± 0.05 μg ml-1). Nine isolates were classified as partially resistant. Some isolates showed more than a 200-fold increase in resistance to TFX, and one isolate grew almost as well on 10.0 μg ml-1 TFX as on the unamended control (GS of 3%). Current-season use of QoIs on isolate source trees was significantly associated with a lack of sensitivity Ç2 (1) = 3.72 (P < 0.06). All six fully resistant isolates originated from QoI-treated commercial orchards, which had shown control failures. Seven of 10 isolates sensitive to QoIs originated from trees that had been treated with SIs during the isolation year. Resistance to myclobutanil was not significantly associated with resistance to TFX Ç2 (1) = 1.220 (P < 0.5), and only one isolate was resistant (i.e. >25% GS) to both. Despite the long history of QoI use at the experimental orchards, no isolates fully resistant to TFX were identified there. To our knowledge, this is the first report of V. inaequalis isolates with resistance to TFX in Virginia and Maryland. Since SI resistance has been documented in Virginia (3) and resistance to both the SI and QoI chemical classes is a concern in the mid-Atlantic region (4), tank-mixing or alternating QoIs with broad-spectrum fungicides with different modes of action is recommended.

Induction of heat resistance of wheat coleoptiles by salicylic and succinic acids: Connection of the effect with the generation and neutralization of reactive oxygen species

The influence of salicylic (SaA) and succinic (SuA) acids on the generation of active oxygen forms (AOFs) and the heat resistance of wheat (Triticum aestivum L.) coleoptiles has been studied. The treatment of coleoptiles with 10 microM SaA or SuA results in the accumulation of hydrogen peroxide and enhanced formation of a superoxide anion radical. This effect is partially suppressed by both alpha-naphthol (the NADPH oxidase inhibitor) and salicylhydroxamic acid (peroxidase inhibitor). SaA and SuA cause an increase in the activity of antioxidant enzymes, such as superoxide dismutase, catalase, and soluble peroxidase, and improve the heat resistance ofcoleoptiles. Antioxidant ionol and compounds, which inhibit the NADPH oxidase and peroxidase, significantly reduce the positive influence of SaA and SuA on the heat resistance of wheat coleoptiles. AOFs are considered to be intermediates for heat resistance induction in coleoptiles, treated with SaA and SuA; enhanced AOF generation can be caused by an increased activity of the NADPH oxidase and peroxidase.

Involvement of alternative oxidase in the regulation of growth, development, and resistance to oxidative stress of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum.