Optimization of in vitro growth conditions of Pyrenophora teres for production of the phytotoxin aspergillomarasmine A

In liquid cultures of Pyrenophora teres, three phytotoxins may be found: L, L - N -(2-amino-2-carboxyethyl) aspartic acid (toxin A), anhydroaspergillomarasmine A (toxin B) and aspergillomarasmine A (toxin C). In particular, toxins A and C cause chlorotic and necrotic symptoms in detached barley leaves, toxin C being the most damaging, whereas toxin B is only weakly phytotoxic. When P. teres is grown in liquid modified Fries medium, toxin B is the main toxin accumulated, possibly due to a ring closure of toxin C at the low pH value of the medium. The amount of toxin B produced by 11 isolates of P. teres was compared in modified Fries medium. Generally, the most virulent isolates of P. teres produced higher amounts of toxin B than the less virulent isolates. During growth, the pH of the media decreased from 6.7 to about 3.0–3.5, followed by a slight increase to about 3.5–4.0. All isolates, except one, produced toxin B, whereas only two isolates produced toxin C and toxin A. Maintaining the pH at about 6.5 by sterile titration with 1 M NaOH resulted in a shift in toxin accumulation from toxin B to toxin C. The addition of tris or phosphate buffer to the media resulted in higher pH during the growth period, an increase in the total amount of toxins produced, and a shift in toxin accumulation from toxin B to toxin C. The higher pH value probably prevented the conversion of toxin C into toxin B. No toxins were produced in two routinely used media, potato glucose broth and grass broth. Toxin B and toxin C were purified by ion exchange chromatography and precipitation with HCl.     

Agrobacterium induces plant cell death in wheat (Triticum aestivum L.)

The symptoms of gall or hairy root do not occur in the interactions between wheat (Triticum aestivum L.) and other monocotyledonous plants, with Agrobacterium tumefaciens or Agrobacterium rhizogenes. However, both bacteria colonized wheat root surfaces at similar levels (2.0 × 10⁷ colony forming U g⁻¹ root) and grew without inhibition in suspension with intact or wounded wheat embryos or root segments present. Suspension-cultured wheat embryo cells grown in 7.4 m M O₂ displayed 23% cell death after 1 h exposure to Agrobacterium cells, while the extent of cell death with 2.1 m M O₂ averaged 8%. Cultured wheat embryo and root cells rapidly produced hydrogen peroxide (H₂O₂) when contacted with A. tumefaciens or A. rhizogenes. Production of H₂O₂ was lower at 2.1 m M O₂ than 7.4 mM O₂. Browning and autofluorescence of epidermal cells of callus derived from wheat embryos and wheat roots was observed after inoculation with Agrobacterium. An increase in ferulic acid was detected in the walls of roots exposed to Agrobacterium. However, neither lignin nor callose was detected by diagnostic staining methods. These findings suggest that Agrobacterium induced a resistance-like response in wheat that may reduce the efficacy of transformation and limit the normal symptom formation.     

In vivo growth and pathotoxin production by the maize pathogen Cochliobolus carbonum

Conidia of Cochliobolus carbonum secrete a toxin (HC-toxin) during appressorium formation on maize leaves. Plasma desorption mass spectrometry revealed that approximately 70 ng of toxin per 10⁶ conidia were secreted during the first 16 h of morphogenesis. Growth of the fungus was monitored microscopically. Extensive fungal growth occurred in the susceptible interaction by 24 h. In spite of a substantial amount of HC-toxin, the fungus failed to become established in the resistant host even after 36 h. Results suggest that the resistance conditioned by Hm1, which encodes a toxin reductase, causes inactivation of the toxin early in the interaction.     

Effect of miconazole,an antifungal agent,on allene oxide synthase: Inhibition,kinetics, and binding

We investigated the inhibition of allene oxide synthase (AOS), a key enzyme in jasmonic acid biosynthesis, by miconazole. Kinetic analysis indicated that miconazole was a mixed-type inhibitor of AOS with a K_i value of approximately 8.4 ± 0.2 μM. Analysis of the interactions between miconazole and AOS by optical difference spectroscopy revealed that miconazole binding induces type II binding spectra with a K_d value of approximately 6.0 ± 0.2 μM.     

A soluble carbohydrate elicitor from Blumeria graminis f. sp. tritici is recognized by a broad range of cereals

Wheat plants rapidly recognize pathogenic and non-pathogenic conidia of the powdery mildew fungusBlumeria (syn. Erysiphe)graminis on their leaf surfaces. This suggests that a chemical signal emanates from conidia at the pre-penetration stage of infection. Conidia of B. graminis f. sp. tritici were found to contain an elicitor that was easily washed off their surface. The elicitor activity is heat stable and could not be removed by phenol extraction. By contrast, elicitor activity is sensitive to periodate oxidation and partial acid hydrolysis suggesting that the elicitor activity resides in a carbohydrate moiety. Analysis of carbohydrates revealed mostly glucose, with smaller amounts of xylose and mannose. The glucosyl residues of the B. graminis elicitor were found to be linked (1 → 2)-, (1 → 4), and (1 → 6)-, with (1 → 4, 1 → 6)- branch point residues, and no 3-linked glucose residues were detected. As treatment with β -mannanase significantly reduced elicitor activity, mixed-linkage (1 → 4), (1 → 6)-mannosyl residues appeared to be important for elicitor activity. The B. graminis elicitor induced the expression of all defence-related genes tested in wheat and also induced resistance to subsequent attack by B. graminis f. sp. tritici. In contrast, a hypersensitive response was not induced by the elicitor in the absence or the presence of a challenging inoculum of B. graminis f. sp. tritici. The elicitor also induced the accumulation of thaumatin-like proteins in barley, oat, rye, rice and maize, but did not induce necrosis in any of these species. This suggests that the B. graminis elicitor represents a host non-specific determinant of non-self recognition in cereals activating general defence responses other than the hypersensitive reaction.     

Thaxtomin A: evidence for a plant cell wall target

Thaxtomins are unique 4-nitroindol-3-yl containing dioxopiperazines that cause dramatic plant cell hypertrophy and seedling stunting. This family of phytotoxins is produced by Streptomyces species that cause diseases of root and tuber crops; its members are essential for pathogenicity. The symptoms produced by thaxtomin A suggest several potential plant cell targets including the plasma membrane, various components of the cytoskeleton and the cell wall. Dramatic increases in cell volume in onion seedling hypocotyls, radish seedling hypocotyls and tobacco suspension cultures, in response to 0.05–1.0 μM thaxtomin A, suggested that this phytotoxin is interacting with one or more conserved plant cell targets. Onion root tip cells treated with thaxtomin A concentrations at or below that which inhibited onion root growth were binucleate or had abnormal cell plates. Thaxtomin A (1.0–3.0 μM) inhibited normal cell elongation of tobacco protoplasts in a manner that suggested an effect on primary cell wall development. In summary, these data suggest that thaxtomin A alters, either directly or indirectly, the deposition or composition of monocot and dicot plant cell walls in ways that affect the wall integrity and the ability of the cell to progress normally through cytokinesis.     

Ethylene production by Colletotrichum musae in vitro

Seven isolates of the pathogen Colletotrichum musae (Berk & Curt.) v. arx. were isolated from banana fruit. These isolates produced ethylene to varying degrees in methionine-amended Czapek Dox liquid medium as both shake and static cultures. Rates of ethylene production by C. musae were positively associated with the concentration of methionine in the growth medium. C. musae did not produce ethylene on basal medium containing l-glutamate, α -ketoglutarate or l-cysteine. Isolate CM 100 produced the highest cumulative amount of ethylene (2·27 μm g⁻¹ dry wt) over 12 days on 35 mm methionine-amended shake cultures of basal medium. In the presence of methionine, ethylene biosynthesis by C. musae occurred via 2-keto-4-methylthiobutyric acid (KMBA). The capacity of C. musae to produce ethylene may have a role in its pathogenicity on climacteric banana fruit.     

Induction of disease resistance against Botrytis cinerea by heat shock treatment in melon (Cucumis melo L.)

The present study investigated resistance against Botrytis cinerea after heat shock treatment in melon plants. Heat shock at 50 °C for 20 s 0–24 h before inoculation resulted in maximal B. cinerea symptom reduction and peroxidase gene expression, which peaked 12 and 72 h post-treatment and decreased 24–48 h post-treatment, suggesting pathogenesis-related protein expression priming. Hot water dipping did not directly inhibit mycelia growth. Plants treated with 2-benzisothiazol-3(2H)-one 1,1-dioxide, which induces systemic acquired resistance, demonstrated higher peroxidase gene expression but no B. cinerea resistance, indicating possible involvement of additional novel mechanisms in heat shock-activated resistance of melon against B. cinerea.     

Oxygen consumption and ammonia excretion of the freshwater crab Trichodactylus borellianus exposed to chlorpyrifos and endosulfan insecticides

The effects of lethal and sublethal concentrations of chlorpyrifos and endosulfan on oxygen consumption and ammonia excretion rate of the crab Trichodactylus borellianus were evaluated. Oxygen consumption and energy expenditure had significant effect in relation to exposure times. Regarding endosulfan, a significant difference in consumption among times of exposure was registered in 625 μg L⁻¹. Moreover, at the highest concentration, energy expenditure rate was observed stabilized during 1–3 h. A significant increase in ammonia excretion was evidenced in 150 and 300 μg L⁻¹ of chlorpyrifos. The O:N ratio showed a decrease in chlorpyrifos and in 2500 μg L⁻¹ of endosulfan. This indicated a shift towards protein primary metabolism. An increment in the O:N ratio was observed in the lower endosulfan solutions. The relation oxygen:nitrogen showed a shift towards lipid and carbohydrate primary metabolism. This work indicated the complexity of the metabolism in the freshwater crab affected by xenobiotic elements.     

Biochemical,physiological and morfological responses in common carp (Cyprinus carpio L.) after long-term exposure to terbutryn in real environmental concentration

The effects of terbutryn at concentrations of 0.02 (reported concentration in Czech rivers), 0.2, and 2.0 μg l^?1 were assessed in one-year-old common carp (Cyprinus carpio L.) exposed for 90 days. Influence on biometric parameters, hematology, biochemistry, histology, and oxidative stress was investigated. Exposure to 0.02, 0.2 and 2.0 μg l^?1 showed significant differences oxidative stress biomarkers compared to controls but exposure to 0.2 and 2.0 μg l^?1 significantly affected biochemical and hematological profiles. Long-term exposure of terbutryn in carp resulted in slight alterations in internal organs and increased reactive oxygen species formation, resulting in oxidative damage to lipids and proteins and inhibition of antioxidant capacities.     

Effects of trichlorfon and sodium dodecyl sulphate on antioxidant defense system and acetylcholinesterase of Tilapia nilotica in vitro

The effects of organophosphorus insecticide trichlorfon, surfactant sodium dodecyl sulphate (SDS), and the mixture of trichlorfon and SDS on the antioxidant defense system and acetylcholinesterase (AChE) in Tilapia nilotica were assessed in vitro. Various concentrations of trichlorfon (0, 0.0001, 0.001, 0.01, 0.1 and 1 g/L) and SDS (0, 0.0625, 0.125, 0.25, 0.5, 1 g/L) were incubated with homogenate of liver and muscle, respectively, at 25 °C for 0, 30, 60 and 90 min. Two concentrations of mixture of trichlorfon and SDS (0.0001 g/L trichlorfon + 0.5 g/L SDS, 0.1 g/L trichlorfon + 0.5 g/L SDS) and 0.0001 g/L trichlorfon, 0.1 g/L trichlorfon, 0.5 g/L SDS and control, were incubated simultaneously with homogenate of liver and muscle, respectively, at 25 °C for 60 min. After incubation, the content of reduced-glutathione (GSH) and the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in homogenate of liver were determined, and the activities of AChE in homogenate of muscle were also measured.Treatment with trichlorfon caused a significant concentration-dependent and time-related inhibition of AChE activity at all treatment concentrations and times since trichlorfon is a cholinesterase inhibitor. For the same trichlorfon treatment, an apparent decrease in GSH content was found in concentration of 0.01, 0.1, 1 g/L, whereas no significant alteration in antioxidant enzyme activity were found at all experiment concentrations and times, which might indicate that antioxidant enzymes have not involved in the metabolism of trichlorfon. The depletion of GSH might indicate that ROS could be involved in the toxic effects of trichlorfon. Exposure of SDS can inhibit activities of AChE, GST and CAT at concentrations of 0.5 and/or 1 g/L, which could be due to the denaturing process of SDS to the enzymes. For the mixture exposure of trichlorfon and SDS, the effect of the mixture of 0.0001 g/L trichlorfon and 0.5 g/L SDS on inhibition of AChE shows synergistic other than simple additive of trichlorfon and SDS. The combined effects of chemicals and detergents deserve to be particularly noted. It should be noted that the toxicity experiments were made in tissue homogenates instead of whole organisms. The responses against the toxic compounds will not be the same in both systems.     

An integrated multivariate approach to net blotch of barley: Virulence quantification,pathotyping and a breeding strategy for disease resistance

Knowledge of pathotype diversity and virulence in local populations of Pyrenophora teres is a prerequisite to screening for durable resistance to net blotch. The current study aimed to quantify the virulence level of Moroccan isolates, identify and designate existing pathotypes, and select resistant genotypes. We developed a method for virulence quantification of P. teres isolates based on a conversion of infection responses into frequencies for use in correspondence analysis. Coordinates of the first axis of this analysis had a virulence spectrum and ranked isolates from virulent to avirulent. Mixed model analysis was also devised for virulence quantification. Coordinates of the first dimension of correspondence analysis were linearly correlated to BLUPs (Best Linear Unbiased Predictors) of the mixed model. A genotype by genotype by environment model (GGE) coupled with cluster analysis differentiated P. teres isolates into ten and nine pathotypes for net- and spot-forms respectively. Populations of these two forms were dissimilar in terms of classes of virulence. For P. teres f. maculata, avirulent, moderately virulent and highly virulent isolates represented one-third of the population, whereas 90% of P. teres f. teres population was composed of avirulent to moderately avirulent isolates. Barley differential sets were subsequently reduced to two new sets that simplified pathotyping through a key code based on resistant or susceptible reactions. Dendrograms of cluster analysis based on GGE analysis depicted the stability of a genotype’s reactions across all isolates, and using only resistant cultivars as sources of resistance to control net blotch disease would, based on this analysis, fail to control all pathotypes. Therefore, we propose an alternative breeding strategy to control net blotch effectively.     

Toxic effects of acephate on Paramecium caudatum with special emphasis on morphology,behaviour, and generation time

The continuous increase in the number of new chemicals as well as the discharges of solid and liquid wastes triggered the need for simple and inexpensive bioassays for routine testing. In recent years, there has been increasing development of methods (particularly rapid tests) for testing environmental samples. This paper describes the quick toxic evaluation of an organophosphorus insecticide, acephate (O,S-dimethyl acetylphosphoramidothioate) on Paramecium caudatum for acute and sub-acute toxicity studies with reference to morphology, behaviour, and its generation time. The lethal concentrations for 10 min and 2 h were determined by probit method, as 500 mg L⁻¹ and 300 mg L⁻¹, respectively. Higher concentrations of 10 min exposure caused cell lysis with disintegration of cell membrane and precipitation of protoplasm. Combination of conventional light microscopy and computerized video tracking systems were used to study the locomotor behaviour of paramecia. The test organism was under stress and exhibited an initial increase and subsequent decrease in the swimming speed when exposed to 1/4, 1/2, 3/4, and LC₅₀ concentrations for 10 min (125, 250, 375, and 500 mg L⁻¹, respectively). Similar changes were also noticed when paramecia were exposed to LC₅₀ for 2 h. In a separate set of experiments, the number of generations and generation time in 24 h was evaluated with respect to the different sub-lethal concentrations (30, 60, 120, and 240 mg L⁻¹). The number of generations decreased and generation time extended significantly in a concentration dependent manner. The results indicate that the Paramecium toxicity assay could be used as a complimentary system to rapidly elucidate the cytotoxic potential of insecticides. The major advantages associated with these tests are: they are inexpensive, simple, user-friendly, space saving, and seem to be attractive alternatives to conventional bioassays.     

Nile tilapia (Oreochromis niloticus), liver morphology,CYP1A activity and thyroid hormones after Endosulfan dietary exposure

Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g⁻¹ of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g⁻¹ for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g⁻¹, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g⁻¹ of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant.     

Physiological and molecular mechanisms of glyphosate tolerance in an in vitro selected cotton mutant

We have selected an upland cotton (Gossypium hirsutum L.) cell line (R1098) that is highly tolerant to glyphosate. This cell line was developed by in vitro selection with gradually increasing glyphosate concentrations, and its mechanisms conferring glyphosate tolerance were studied. Based on a whole-plant dose–response bioassay, R1098 plants were tolerant to glyphosate at a concentration of 1500 g ae ha⁻¹ glyphosate (1.5× the recommended field rate) whereas the control plants (Coker 312) were unable to survive at 150 g ae ha⁻¹ glyphosate. Coker 312 accumulated 13.1 times more shikimate in leaves at 5 days after glyphosate treatment (1500 g ae ha⁻¹) than that of R1098. Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), EPSPS-1 and EPSPS-2, were isolated from R1098. Both cDNAs were 97.7% identical within the common protein-coding region and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other known higher plants. In comparison to the glyphosate-susceptible cotton Coker 312, sequence analysis of the EPSPS-1 gene indicated that R1098 has an alanine insertion at nucleotide position 1216 resulting in frameshift. It leads to two copy functional EPSPS genes in R1098. There was no difference between R1098 and Coker 312 in EPSPS mRNA levels before glyphosate treatment. However, its treatment caused a 2–4 times increase in the basal EPSPS mRNA level in R1098.     

Stage specificity of catalase isoform activity in Exserohilum turcicum

Exserohilum turcicum is the fungal agent that causes northern leaf blight disease in maize. Spores of E. turcicum can germinate in water containing up to 15 m M hydrogen peroxide. Initially the catalase isoform activities from E. turcicum cultured on artificial medium were analysed by polyacrylamide gel electrophoresis. Seven different catalase isoform activities were detected during a 72-hour time course from spore germination to young hyphal formation. During the time period studied the activity levels of each isoform varied independently. In two-week-oldE. turcicum mycelia, only the activities of catalase isoforms #2 and #3 were detected. During a compatible interaction on maize leaves, plant catalases were suppressed with E. turcicum isoform #3 being detected from 72 h after inoculation onwards. E. turcicum #2 was the predominant isoform detected in necrotic lesions. Salicylic acid was not found to effect fungal or maize catalase activities. E. turcicum isoform #3 activity was found to be strongly induced by hydrogen peroxide and by the herbicide 3-amino-1,2,4-triazole (AT), while the activity of all other isoforms was suppressed by AT.     

Neurophysiological alterations in Caenorhabditis elegans exposed to dichlorvos,an organophosphorus insecticide

AChE inhibition is widely regarded as a good biomarker of exposure to organophosphorus insecticides (OPIs), suggesting increase in the cholinergic transmission and consequent accumulation of acetylcholine in the organism. This might lead to behavioral changes and create widespread disturbances in the normal physiology of an organism. In the present study we have employed the model organism Caenorhabditis elegans to evaluate the biochemical and behavioral alterations induced by dichlorvos, a well known OPI. Exposure of worms to dichlorvos (at sublethal concentrations: 5, 50, 100, 150 and 200 μM) induced a concentration and time dependent AChE inhibition, and accumulation of acetylcholine. Further, we also observed cessation in feeding (by 72%), shutting of pharyngeal pumping, inhibition of egg laying (34–55%), contraction of nose (45%) and significant paralysis (50%) after 4 h of exposure. Significant correlation was observed between biochemical effects and behavioral parameters clearly suggesting the implications of sublethal concentrations of dichlorvos on non-target invertebrate organism such as C. elegans. These data further suggest that assessment of subtle neurophysiological parameters may serve as useful indicators of OPI exposure.     

Biochemical aspects of reactive oxygen species formation in the interaction between Lycopersicon spp. and Oidium neolycopersici

Three Lycopersicon spp. accessions differing in the level of resistance to Oidium neolycopersici L. Kiss (tomato powdery mildew) were studied. Defence reactions occurring in tissue of Lycopersicon esculentum cv. Amateur (susceptible control), Lycopersicon hirsutum f. glabratum (LA 2128) (highly resistant) and Lycopersicon chmielewskii (LA 2663) (moderately resistant) were investigated during the first 120 h post-inoculation (hpi). A hypersensitive reaction was detected after 48 hpi in both resistant tomato accessions. Changes in accumulation of hydrogen peroxide and enzymes involved in its metabolism (catalase, peroxidases, superoxide dismutase) were monitored. In resistant accessions, intensive H₂O₂ production correlated with increased activity of cytosolic guaiacol peroxidase, syringaldazine peroxidase and ascorbate peroxidase. Catalase activity increased especially in moderately resistant L. chmielewskii. A similar degree of lipid peroxidation occurred in all Lycopersicon accessions. An increase in the concentration of free phenols but no change in the level of cell-wall-bound phenols were observed during the first 120 hpi in all Lycopersicon spp. accessions. Spermidine represented the major part of the total polyamine content. Pathogen-induced lignification was not observed in any of the studied accessions.