Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

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.     

Correlation between sensitivity of barley to Pyrenophora teres toxins and susceptibility to the fungus

Detached leaves of 25 barleys, ranging from highly susceptible to highly resistant to Pyrenophora teres f. teres and Pyrenophora teres f. maculata, were tested for their reaction to three phytotoxins isolated from cultures of the fungus: toxin A [ L, L - N -(2-amino-2-carboxyethyl)aspartic acid], toxin C (aspergillomarasmine A) and toxin B (anhydroaspergillomarasmine A). 0.75 m M toxin A caused mainly dark yellow chlorotic symptoms but little necrosis, whereas leaves treated with 0.25 m M toxin C developed distinct necrotic symptoms and zones of light yellow chlorosis. Toxin B is only weakly toxic, and toxin B and control solutions containing aspartic acid in the concentration of 0.75 m M did not cause any symptoms. The best differentiation between the barleys was obtained by scoring chlorosis after 120 h, and the optimal toxin concentrations for this differentiation were 0.75 m M toxin A and 0.25 m M toxin C, respectively. Results with different toxin concentrations inducing distinct variation in symptom expression indicate that the two toxins have different potencies as phytotoxins. The reaction of the barleys to toxins A and C correlated well with their reaction to infection by P. teres f. teres and P. teres f. maculata, suggesting that toxins A and C may be used to select resistant barley lines in the early stages of a breeding programme.     

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.     

Chlorpyrifos induced alterations in the levels of hydrogen peroxide,nitrate and nitrite in rat brain and liver

Organophosphate pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests. Chlorpyrifos (CPF) is among the leading organophosphate (OP) pesticides used extensively throughout the world including India. Besides being potent anticholinesterase compounds, these OP pesticides are known to generate oxidative stress. Present study was carried out to see the level of reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), nitrate (NO₃⁻) and nitrite (NO₂⁻) in different parts of rat brain and liver after giving different doses of CPF intramuscularly for 3 days or for 1 month. Results of the present study clearly revealed that levels of H₂O₂, NO₃⁻ and NO₂⁻ were increased significantly in all the three parts of rat brain i.e., fore-, mid- and hind-brain as well as in liver of rats given for 3 days or 1 month due to exposure of different doses of CPF. The study clearly demonstrated that CPF generated oxidative stress in all the three brain regions as well as liver of rats and the ROS were accumulated. Accumulation of ROS in all the regions of brain and other tissues may disturb the normal physiological functions aggravating the toxicity symptoms of CPF.     

Pathogenic and non‐pathogenic Agrobacterium tumefaciens,A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on root surfaces

Pathogenic and non‐pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains growing in minimal liquid medium adhered to different abiotic surfaces, forming biofilms at initial stages of development. Agrobacterium tumefaciens and A. vitis strains were able to attach to both polystyrene and polypropylene materials, whereas the A. rhizogenes strains only bound to polystyrene surfaces. Strains of the three species were also able to form biofilms on borosilicate coverslips. It is concluded that their ability to adhere to and form nascent biofilms on abiotic surfaces is dependent on the Agrobacterium species (biovar), surface material and growth conditions. Furthermore, tumorigenic A. tumefaciens and A. vitis strains, and the biological control agent A. rhizogenes strain K84, bound tightly to and formed complex biofilms on the surface of tomato root tips ex planta. More importantly, in planta assays confirmed that all three Agrobacterium spp. strains efficiently colonized tomato seedlings and also formed biofilms on roots. These complex structures, as revealed by scanning electron microscopy, were composed of numerous bacterial cells arranged in different ways: either dense and continuous carpets, large aggregates embedded in extra‐cellular material or globular mushrooms traversed internally by channels. Confocal laser scanning microscopy, using GFP‐marked derivative strains, corroborated the presence of live, three‐dimensional and thick green fluorescent structures attached to plant material. This study illustrates that besides A. tumefaciens, strains of the species A. rhizogenes and A. vitis are also able to build biofilms on abiotic as well as on root surfaces.     

Role of sucrose in the development of Fusarium wilt in lupine embryo axes

The response of embryo axes of Lupinus luteus L. cv. Polo to invasion by Fusarium oxysporum Schlecht f.sp. lupini was studied 84, 96 and 108 h after inoculation and under various trophic conditions. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). The exogenous sucrose caused an accumulation of sucrose, glucose and fructose in +Sn embryo axes. Pathogen invasion generally resulted in a sharp decline in soluble carbohydrate content of +Si embryo axes. Symptoms of Fusarium wilt included growth inhibition of the embryo axes and a decrease in their fresh weight, which was particularly strong in −Si axes. While the addition of exogenous sucrose to the medium 24 h prior to inoculation significantly alleviated the disease, its application 48 h after inoculation did not restrict disease development. In +Si embryo axes, the activity of peroxidases (EC 1.11.1.7), covalently and ionically bound to the cell walls, increased considerably, mainly up to 96 h after inoculation. This increase was significantly higher than that seen in +Sn embryo axes. These results are in contrast to those of an earlier study [Morkunas I, Marczak Ł, Stachowiak J, Stobiecki M. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005;43:363–73], which showed a pronounced decrease in the flavonoid glycoside content of +Si tissues. Furthermore, starting 96 h after inoculation, a decrease in phenylalanine ammonialyase activity (PAL, EC 4.3.1.5) was observed in inoculated tissues, in comparison to non-inoculated tissues. The decrease was especially pronounced in +Si tissues. However, a significant increase in the free isoflavone aglycones content of +Si embryo exes, i.e. genistein, 2′-hydroxygenistein, wighteone and luteone. Simultaneously, a strong increase in the activity of β-glucosidase (EC 3.2.1.21), hydrolyzing isoflavone glycosides, was observed in inoculated tissues. The activity of these enzymes was much higher in −Si embryo axes than in +Si embryo axes. The results suggest that soluble carbohydrates may play a role in the restriction of Fusarium wilt development, as they may be related both to intensified lignification and to the antimicrobial role of free aglycones, which may be products of the hydrolysis of isoflavone glycosides generated prior to the invasion stage.     

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.     

The role of salicylic acid in defense response of tomato to root-knot nematodes

Salicylic acid (SA) is involved in hypersensitive reactions of plants to incompatible pathogens and in systemic acquired resistance (SAR) after the attack of necrosis-inducing pests. The possible involvement of SA in defense responses of tomato to root-knot nematodes (Meloidogyne spp., RKNs) was investigated. SA was found not to be responsible for the inhibition of catalase (CAT) detected in the early stages of Meloidogyne-tomato incompatible interactions. CAT extracted from leaves was inhibited only after treatment of the seedlings with SA concentrations as high as 4 mM. Most of the amount of free SA found in plants after SA treatment was detected in the leaves. SA (0.2 mM) was found to cause a competitive inhibition of CAT only at high substrate (H₂O₂) concentrations. Under different conditions it did not affect, or even enhanced, the enzyme activity. Therefore, it is suggested that SA-mediated CAT inhibition does not operate early in resistance against RKN in tomato, although it might have a role in the consequent lesion formation. Plant uptake of SA was detected by immersion of roots of 1-month-old seedlings in aqueous solutions of SA and SA plus a soil humic acid. Considering the low level of free SA retained by roots, the capacity of exogenously provided SA to act as an elicitor of resistance to root pests is considered unlikely.     

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.     

Effect of Vitis vinifera L. cv. Chardonnay xylem fluid on cecropin B activity against Xylella fastidiosa

Cecropin B (CB) is a very efficient antimicrobial agent against Xylella fastidiosa. CB activity decreased after incubation with xylem fluid from Vitis vinifera cv. Chardonnay. SDS-PAGE demonstrated the existence of protein(s) in xylem fluid of V. vinifera cv. Chardonnay that interact with CB and inhibit CB activity. The antimicrobial activity of CB was dependent on incubation time and colony size of X. fastidiosa. X. fastidiosa colonies in periwinkle wilt medium plus (PW⁺), and chemically defined media (3G10-R and CHARD2) showed different patterns in colony size and colony number in these media. After 1 h of incubation in 10 μM CB, only large-sized colonies were observed in the three media. These results suggested that most of the bacterial cells or small aggregates in suspension were killed by CB. Aggregation of X. fastidiosa cells may serve as a mechanism of protection against CB.     

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.     

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.     

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.     

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.     

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.     

Evaluation of molluscicidal activities of Arisaema tubers extracts on the snail Oncomelania hupensis

Four extracts of Arisaema erubescens tubers by acetic acetal (AAE), benzinum (BZE), n-butanol (NBE) and chloroform (CFE) were obtained to evaluate their molluscicidal activities against the snail Oncomlania hupensis. The responses of choline esterase (ChE), alkaline phosphatase (ALP), esterase (EST), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to the extracts (NBE) were also investigated. In the four extracts (AAE, BZE, NBE and CFE), NBE showed the highest toxicity on the snails after 48 h exposure. NBE also showed the time- and concentration-dependent effect, for example, the LC₉₀ values of the NBE were decreased from 365.5 mg/L (24 h) to 36.4 mg/L (96 h). At the end of exposure to NBE (LC₅₀ concentration), the activities of ChE and ALP in snail tissues (cephalopodium and liver) decreased significantly. Isozyme electrophoresis profiles indicated that responses of isozymes (EST, SOD and GSH-Px) to NBE were more intense in liver than in cephalopodium. After 72 h exposure to NBE, the EST activity in snail liver decreased and some enzyme bands (EST1 and EST4) disappeared. But the activities of SOD 1 and GSH 2 in liver increased after 48 h exposure. The results indicated that NBE was the highest toxic component in the four extracts. The decline of the detoxification ability and the oxidative damage in snail tissues might be the main reason for the molluscicidal activities.     

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.     

Purification of an infection-related acidic peroxidase from pearl millet seedlings

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE–Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS–PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27–60 °C). The K_m for H₂O₂ and V_max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl₂ dependent and both MgCl₂ and ZnCl₂ showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.     

Retraction notice to “Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus” [Pesticide Biochemistry and Physiology 94 (2009) 5-9]

A total of 185 hexanic, dichloromethanic, ethanolic and hydroethanolic extracts from 24 species of Cerrado plants, were tested against Zabrotes subfasciatus, Acanthoscelides obtectus, and human saliva α-amylases. Twelve crude extracts presented inhibition rates greater than 80% against digestive α-amylases of the insect pest Z. subfasciatus, at a concentration of 1 mg mL⁻¹. These extracts were also tested against A. obtectus and human saliva α-amylases to verify their affinity and specificity of action. The hydroethanolic Kielmeyera coriacea stem bark extract presented a strong inhibitory potential, with IC₅₀ values of 110 μg mL⁻¹ for Z. subfasciatus and 272.12 μg mL⁻¹ for A. obtectus, in addition to a 97.09% reduction in enzyme activity of human saliva α-amylases at 125 μg mL⁻¹. The hexanic Aspidosperma macrocarpon root wood extract totally inhibited the activity of Z. subfasciatus α-amylases, reduced the enzyme activity of A. obtectus by 14.69% at 1 mg mL⁻¹, but did not alter the activity of human saliva α-amylases, thus characterizing greater inhibition affinity and specificity. The results suggest that the application of plant extracts against insect α-amylases represent a promising biotechnological tool for development of new insect pest control strategies, with noticeable affinity and specificity of action against different target enzymes.