Postinfection Activity of Synthetic Antimicrobial Peptides Against Stemphylium vesicarium in Pear.

Brown spot of pear is a fungal disease of economic importance caused by Stemphylium vesicarium that affects the pear crops in Europe. Due to the characteristics of this disease and the moderate efficacy of available fungicides, the effectiveness of control measures is very limited; however, synthetic antimicrobial peptides (AMPs) may be a complement to these fungicides. In the present study, 12 AMPs of the CECMEL11 library were screened for fungicidal activity against S. vesicarium. In vitro experiments showed that eight AMPs significantly reduced the germination of conidia. The most effective peptides, BP15, BP22, and BP25, reduced fungal growth and sporulation at concentrations below 50 µM. Leaf assays showed that preventive application of BP15 and BP22 did not reduce infection; however, when the peptides were applied curatively, infection was significantly reduced. The use of a BP15 fluorescein 5-isothiocyanate conjugate revealed that the peptide binds to hyphae and germ tubes and produces malformations that irreversibly stop their development.

Differential effect of transient global ischaemia on the levels of γ-aminobutyric acid type A (GABA(A)) receptor subunit mRNAs in young and older rats.

AIMS: This study has investigated how global brain ischaemia/reperfusion (I/R) modifies levels of mRNAs encoding γ-aminobutyric acid type A (GABA(A)) receptor α1, ß2 and γ2 subunits and glutamic acid decarboxylase 65 (GAD65) in an age- and structure-dependent manner. Gene expression in response to treatment with the anti-inflammatory agent meloxicam was also investigated. METHODS: Global ischaemia was induced in 3- and 18-month-old male Sprague-Dawley rats. CA1, CA3, and dentate gyrus (DG) hippocampal areas, cerebral cortex (CC) and caudate putamen (C-Pu) from sham-operated and I/R-injured animals were excised 48 h after the insult and prepared for quantitative polymerase chain reaction assays. Following I/R, meloxicam treatment was also carried out on young animals. RESULTS: Data revealed significant decreases in the levels of all GABA(A) receptor subunit transcripts in the hippocampus of both young and older injured animals compared with sham-operated ones. In contrast, there was either an increase or no change in GAD65 mRNA levels. GABA(A) receptor subunit transcript decreases were also observed in the CC and C-Pu in young injured animals but not in the CC of the older injured ones; interestingly, significant increases were observed in the C-Pu of older injured animals compared with controls. Meloxicam treatment following the insult resulted in a diminution of the previously described I/R response. CONCLUSIONS: The data indicate that I/R results in the modification of the levels of several gene transcripts involved in GABAergic signalling in both the pre- and postsynaptic components, of this neurotransmitter system, in an age- and structure-dependent manner.

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

Characterization of root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia.

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR--RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.

Electrochemically reduced graphene oxide on CoCr biomedical alloy: Characterization, macrophage biocompatibility and hemocompatibility in rats with graphene and graphene oxide.

Electrochemically reduced graphene oxide (ErGO) films on a biomedical grade CoCr alloy have been generated and characterized in order to study their possible application for use on joint prostheses. The electrodeposition process was performed by cyclic voltammetry. The characterization of the ErGO films on CoCr alloys by XPS revealed sp2 bonding and the presence of CO and CO residual groups in the graphene network. Biocompatibility studies were performed with mouse macrophages J774A.1 cell cultures measured by the ratio between lactate dehydrogenase and mitochondrial activities. An enhancement in the biocompatibility of the CoCr with the ErGO films was obtained, a result that became more evident as exposure time increased. Macrophages on the CoCr with the ErGO were well-distributed and conserved the characteristic cell shape. In addition, vimentin expression was unaltered in comparison with the control, results that indicated an improvement in the CoCr biocompatibility with the ErGO on the material surface. The in vivo response of graphene and graphene oxide was assessed by intraperitoneal injection in wistar rats. Red blood cells are one of the primary interaction sites so hemocompatibility tests were carried out. Rats inoculated with graphene and graphene oxide showed red blood cells of smaller size with a high content in hemoglobin.

A kinematic comparison of the locomotor pattern of horses sedated with detomidine alone and in combination with low doses of butorphanol.

BACKGROUND: Chemical restraint is often used to perform diagnostic and minor surgical procedures; α2 -adrenoceptor agonists are the most commonly used drugs; however, the combination with an opiate can induce a profound sedation. There is a lack of kinematic studies examining the effects of the combination of these drugs on locomotor patterns. OBJECTIVES: The objective of the study was to evaluate the duration of the effects of sedation with detomidine and detomidine combined with a low dose of butorphanol on the movement patterns of horses. STUDY DESIGN: The study was a controlled, randomised, blinded and crossover experiment. METHODS: Each of six horses was injected intravenously with saline (0.9%) solution (10 mL), detomidine diluted in saline solution (0.01 mg/kg bwt) or a combination of detomidine (0.01 mg/kg bwt) and butorphanol (0.02 mg/kg bwt) diluted in saline solution, in a random order. A single accelerometer positioned at the sacrum was used for gait assessment 15 min before (baseline) and 5, 15, 30, 45, 60, 75, 90, 105 and 120 min after each injection. Eight variables were measured, including speed, stride frequency, stride length, regularity, dorsoventral power, propulsive power, mediolateral power and total power; force of acceleration and the three components of power were calculated. The degree of sedation was measured by the ground-to-lip distance. RESULTS: There were significant differences among groups, with shorter effects after the injection of the combination of drugs, for most parameters. MAIN LIMITATIONS: A small number of horses were involved in the study. CONCLUSIONS: The combination of detomidine and butorphanol produces a shorter effect on almost all accelerometric parameters, probably due to the excitement produced by the opioid drug causing a quicker return to normal values. Accelerometry offers a method of objectively monitoring gait abnormalities in walking sedated horses.

Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stress.

Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS-scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated. Sediments of the metal-polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP-OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm-forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively. Metal excess induced SOD (five-fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal-resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non-exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation. The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75- and five-fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

Study of overall and local electrochemical responses of oxide films grown on CoCr alloy under biological environments.

The interaction of the physiological medium and living tissues with the implant surfaces in biological environments is regulated by biopotentials that induce changes in the chemical composition, structure and thickness of the oxide film. In this work, oxide films grown on CoCr alloys at 0.5 V vs Ag/AgCl and 0.7 V vs Ag/AgCl have been characterized through overall and localized electrochemical techniques in a phosphate buffer solution and 0.3% hyaluronic acid. Nanopores of 10-50nm diameter are homogeneously distributed along the surface in the oxide film formed at 0.7 V vs Ag/AgCl. The distribution of the Constant Phase Element studied by local electrochemical impedance spectroscopy showed a three-dimensional (3D) model on the oxide films grown at 0.5 V vs Ag/AgCl and 0.7 V vs Ag/AgCl. This behaviour is especially noticeable in oxide films grown at 0.7 V vs Ag/AgCl, probably due to surface inhomogeneities, and resistive properties generated by the potentiostatic growth of the oxide film.

Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance.

There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity. Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant-growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na+ in its shoots, thus improving sodium phytoextraction capacity. Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils.

Isolation of plant-growth-promoting and metal-resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation.

Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain that can tolerate and accumulate heavy metals. A total of 48 bacteria (30 endophytes and 18 from the rhizosphere) were isolated from A. macrostachyum growing in the Odiel River marshes, an ecosystem with high levels of contamination. All the isolates exhibited plant-growth-promoting (PGP) properties and most of them were multiresistant to heavy metals. Although the presence of heavy metals reduced the capability of the isolates to exhibit PGP properties, several strains were able to maintain their properties or even enhance them in the presence of concrete metals. Two bacterial consortia with the best-performing endophytic or rhizospheric strains were selected for further experiments. Bacterial inoculation accelerated germination of A. macrostachyum seeds in both the absence and presence of heavy metals. These results suggest that inoculation of A. macrostachyum with the selected bacteria could ameliorate plant establishment and growth in contaminated marshes.

Glutamate receptor and transporter modifications in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation: the contribution of cyclooxygenase-2.

Meloxicam is a non-steroidal anti-inflammatory drug which has been reported to lessen the ischemic transcriptional effects in some of the glutamatergic system genes as well as to decrease the infarct volume in in vivo assays. In this study, we show how the presence of meloxicam decreases cell mortality in assays of oxygen-glucose deprivation (OGD) in rat organotypic hippocampal slices culture. Mortality was measured using propidium iodide. Transcript levels of some glutamatergic system genes, including vesicular and membrane glutamate transporters (VGLUT1, VGLUT2, GLAST-1A, GLT-1, and EAAC-1) and some glutamatergic receptor subunits (NMDA receptor, GluN1, GluN2A and GluN2B subunits and AMPA receptor, GluA1 and GluA2 subunits) were measured by real-time PCR (qPCR). The transcription of vesicular glutamate transporters and glutamatergic receptor subunits, but not membrane glutamate transporters, was modified by the presence of meloxicam. The study demonstrates the neuroprotective role of meloxicam in organotypic hippocampal slice cultures and shows how meloxicam is able to selectively increase or decrease the OGD-induced changes in the expression of the different glutamatergic system genes studied here. We suggest that the neuroprotective role of meloxicam could be due to a modification in the balance of the expression of some glutamatergic receptor subunits, leading to a different stoichiometry of receptors such as NMDA or AMPA. Thus, meloxicam would decrease the excitotoxicity induced by OGD.

The use of low doses of acepromazine as an aid for lameness diagnosis in horses: An accelerometric evaluation.

OBJECTIVES: The aim of the present study was to quantify by accelerometry the trotting pattern of adult horses sedated with two different doses of acepromazine, in order to assess the use of this drug in equine lameness evaluations. METHODS: Seven mature horses were used and three treatments were administered to each horse: saline solution, acepromazine (0.01 mg/kg), and acepromazine (0.02 mg/kg). The portable gait analyzer used consisted of three orthogonal accelerometers that measure accelerations along the dorsoventral, longitudinal, and lateral axes. Baseline values were obtained and after treatment, accelerometric recordings were repeated every five minutes during the first 20 minutes after the injection and then every 10 minutes thereafter for two hours. Ground-to-lip distance was also measured. RESULTS: Administration of acepromazine decreased some of the variables investigated and differences between doses were observed. Speed, stride frequency, and stride length were significantly reduced following treatments. For coordination parameters, no significant differences among values were observed. Energetic variables suffered only weak reductions whereas ground-to-lip distance values were significantly decreased up to 120 minutes after treatment. CLINICAL SIGNIFICANCE: Acepromazine produces significant alterations in the gait pattern with differences between doses, but it does not affect coordination variables in normal unexcited horses, and at a dose of 0.01 mg/kg may be the tranquilizer of choice for evaluating lameness in this setting.

Scouting contaminated estuaries: heavy metal resistant and plant growth promoting rhizobacteria in the native metal rhizoaccumulator Spartina maritima.

Spartina maritima is a native endangered heavy metal rhizoaccumulator cordgrass naturally growing in southwest coasts of Spain, where is used as a biotool to rehabilitate degraded salt marshes. Fifteen bacterial strains were isolated from the rhizosphere of S. maritima growing in the estuary of the Tinto River, one of the most polluted areas in the world. A high proportion of bacteria were resistant towards several heavy metals. They also exhibited multiple plant growth promoting (PGP) properties, in the absence and the presence of Cu. Bacillus methylotrophicus SMT38, Bacillusaryabhattai SMT48, B. aryabhattai SMT50 and Bacilluslicheniformis SMT51 were selected as the best performing strains. In a gnobiotic assay, inoculation of Medicago sativa seeds with the selected isolates induced higher root elongation. The inoculation of S. maritima with these indigenous metal-resistant PGP rhizobacteria could be an efficient method to increase plant adaptation and growth in contaminated estuaries during restoration programs.

Cholinergic modulation of spontaneous hypothalamic-pituitary-adrenal activity and its circadian variation in man.

Controversy still exists regarding the role of cholinergic pathways in the regulation of the hypothalamic-pituitary-adrenal axis in man. We studied the effects of the administration of placebo, pyridostigmine (PD); 120 mg, orally), and the combination of PD and pirenzepine (PZP; 100 mg, orally) on ACTH, cortisol, and GH secretion at 0730 and 2230 h in seven normal males. PD induced a clear decrease in ACTH levels at both times of the day compared to treatment with placebo, producing higher suppression in the nocturnal period (34.4 +/- 5.8% vs. 21.8 +/- 10.7%). The combination PD and PZP prevented the inhibitory action of PD on ACTH secretion in the morning, but not in the evening, when ACTH values showed a decrease similar to that seen after giving PD alone (38.1 +/- 5.6% vs. 34.4 +/- 5.8%, respectively). Cortisol values declined only when the association PD plus PZP was given in the evening. GH levels had a significant increase after PD administration in the morning (4.1 +/- 1.2 ng/mL) and in the evening (10.2 +/- 1.6 ng/mL), confirming that cholinergic stimulation was taking place, whereas the addition of PZP to PD induced a significant attenuation of these responses. It is concluded that cholinergic pathways have a inhibitory role in ACTH secretion in man. M1 muscarinic receptors seem to be involved in the diurnal inhibition of PD, whereas our observations are consistent with the mediation of another type of cholinergic receptors as an explanation for the nocturnal effect of PD on ACTH secretion. PD did not alter the circadian variation in the hypothalamic-pituitary-adrenal axis, whereas the association of PD and PZP increased the differences between diurnal and nocturnal ACTH values, suggesting a modulatory effect of the cholinergic system on the circadian rhythm of ACTH secretion.

Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes.

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, lPR, fused to luc and lucOR as reporter genes. We have demonstrated that the systems cI857-lPR::luc and cI857-lPR::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.

Effect of Relative Humidity and Interrupted Wetness Periods on Brown Spot Severity of Pear Caused by Stemphylium vesicarium.

ABSTRACT Field observations in four pear orchards during 5 years from April to October indicated that days with uninterrupted wetness of variable length represented 83.9% of the total days studied. However, days with surface wetness interruptions and with high relative humidity (RH) (>/=90%) without wetness occurred with a frequency of 7.1 and 6.2%, respectively. Accordingly, the effect of interruption of 24-h wetness periods by dry periods of high or low RH on infections caused by Stemphylium vesicarium on pear was determined. Pear plants inoculated with conidia of S. vesicarium were exposed to a 12-h wet period followed by a dry period of variable length (0, 3, 6, 12, 18, or 24 h) and a second wet period of 12 h. The dry period consisted either of low (60%) or high (96%) RH. The infection process was irreversibly stopped under low RH during dry periods between wetness, but continued at high RH. The effect of high RH on disease severity in the absence of wetness was also determined. Pear plants inoculated with S. vesicarium were exposed to periods of variable length (3 to 24 h) either at high RH (96%) in the presence of wetness or at high RH (96%) without wetness. No infections were observed on plants incubated under high RH without wetness, indicating that conidia of S. vesicarium require the presence of a water film in the plant surface to develop infections on pear.

Transformation of floral organs with GFP in Medicago truncatula.

A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108-1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants.

In-process control of midazolam synthesis by HPLC.

A high-performance liquid chromatographic assay coupled with UV detection (239 nm) has been developed for the determination of midazolam and its synthesis precursors. The separation of the analytes was performed on a Kromasil C8 column (15 cm x 4.6 mm i.d., 5 microm) at 30 degrees C. The mobile phase [ammonium chloride (pH 5.5, 1 g l(-1))-methanolacetonitrile (45:22:33, v/v/v)] was pumped at a flow-rate of 1.5 ml min(-1). This method is rapid (less than 11 min), sensitive (limit of detection (LOD) ranged between 0.05 and 0.5 mg l(-1)) and selective for the determination of midazolam, and it could be used for monitoring different synthetic routes.

Development and Field Evaluation of a Model to Estimate the Maturity of Pseudothecia of Pleospora allii on Pear.

The effect of temperature and of two levels of relative humidity (RH) on maturity of pseudothecia of Pleospora allii (teleomorph of Stemphylium vesicarium) was studied under controlled environmental conditions at temperatures of 5, 10, 15, 20, 25, and 30°C and high (≥98%) and low (≈60%) RH. Pseudothecia developed only at high RH, and the optimum temperature was between 10 and 15°C. A regression model of the form ln(1/1 - y) = 0.12550 + 0.005048x, which related the proportion of mature pseudothecia (y) to cumulative degree-days (x), was developed with data from two controlled environment experiments. Maturation of pseudothecia also was studied in leaf debris in pear orchards affected by brown spot of pear in seven field trials during 4 years. Asci with mature ascospores were observed in leaf debris from mid-January to May. A significant linear relationship was observed between the predicted values according to the monomolecular model and observed values of the percentage of mature pseudothecia in the field trials (r 2 = 0.91, intercept=8.718, and slope=0.903). This model can be used to predict the onset of ascospore discharge and for determining the initiation of fungicide applications to control the primary inoculum and prevent primary infections.

Evaluation of BSPcast Disease Warning System in Reduced Fungicide Use Programs for Management of Brown Spot of Pear.

A forecasting model (BSPcast) developed for prediction of brown spot (Stemphylium vesicarium) of pear was evaluated as an advisory system for reduced fungicide use in disease management programs. Eleven trials were performed during 1995, 1996, and 1997 in five orchards in two different climatic areas in Catalunya (Spain) and Emilia-Romagna (Italy). Values of 3-day cumulative daily infection risk (CR) provided by the model were used to determine risk periods during the growing season of pear and were taken as thresholds to schedule fungicide sprays. The fungicide application programs tested using the model consisted of guided schedules with CR action thresholds of 0.4, 0.5, or 0.6, and fixed sprays following a standard commercial schedule. In nine out of 10 trials, no significant differences were observed in disease incidence on fruit at harvest between the fixed-spray commercial schedule and guided sprays using thresholds of 0.4 or 0.5. The average savings in number of fungicide sprays applied using BSPcast compared with the fixed-spray schedule were 20 to 70% when using fungicides with a 15-day protection period (kresoxim-methyl or procymidone) and ranged from 20 to 50% when using a fungicide with a 7-day protection period (thiram).