Lipofuscins and sclerotial differentiation in phytopathogenic fungi

Lipofuscins of lipidic and proteinaceous origin were identified by their excitation and emission spectra in phytopathogenic fungal representatives of different sclerotial differentiation types. Lipofuscin pigments in Sclerotium rolfsii, Rhizoctonia solani, Sclerotinia minor and Sclerotinia sclerotiorum showed similar excitation and emission maxima (ex-em 330–450, 330–450, 330–470 and 3307–470 nm, respectively). Sclerotial differentiation of these fungi was proceeded by a 4.2, 2.5, 2.7, 2.5 and 6, 2.9, 3.8, 3.1 fold increase of lipofuscin accumulation (per lipid and protein content), per respective fungus, as compared to their undifferentiated stage. Lipofuscin levels were higher in older than in younger mycelia and this phenomenon was more profound in S. rolfsii. Since lipofuscins are considered as indicators of oxidative stress, these data are in accordance with the hypothesis that suggests oxidative stress to be a common underlying factor in sclerotial differentiation of sclerotia-forming filamentous phytopathogenic fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Superoxide radical is involved in the sclerotial differentiation of filamentous phytopathogenic fungi: identification of a fungal xanthine oxidase

This study shows that the direct indicator of oxidative stress superoxide radical (O·??) is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor. The production rate of O·?? and the antioxidant enzyme superoxide dismutase (SOD) levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their non-differentiating counterpart strains, which strongly suggests that the oxidative stress of the sclerotium differentiating fungi is higher than that of the non-differentiating ones. Xanthine oxidase (XO), which was detected for the first time in fungi in general, was localized in the cytoplasmic membrane. The contribution of XO in the overall O·??production was very significant, reaching 30-70% among the strains, especially in the transition developmental stage between the undifferentiated and the differentiated state, suggesting a sclerotium triggering and a phytopathogenic role of XO during plant infection. The additional finding that these fungi secrete extracellular SOD can be related to their protection from the response of plants to produce O·?? at infection sites.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Localisation of hydrogen peroxide and peroxidase in gametophytes of Ceratopteris richardii (C-fern) grown in the presence of pathogenic fungi in a gnotobiotic system

The endogenous localisation of peroxidase and hydrogen peroxide (H₂O₂) was detected when gametophytes of the fern, Ceratopteris richardii, were exposed to the plant pathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum and Phytophthora infestans, an oomycete, in a gnotobiotic system. This was accomplished by light microscopy using 3,3′‐diaminobenzidine, guaiacol and H₂O₂ and starch potassium iodide (KI) staining procedures, which facilitated the observation of the reaction in vivo and in situ, without physically damaging the tissues. All three staining methods promoted staining at the rhizoid regions. Although most of the cells were destroyed when gametophytes were exposed to S. rolfsii and S. sclerotiorum, there was staining where mycelial growth was confluent with cell walls. A qualitative test confirmed that the colour change in starch KI agar medium, as well as in the histochemical test with starch KI, was because of H₂O₂ secreted by S. rolfsii or S. sclerotiorum and not because of oxalic acid. When gametophytes were exposed to P. infestans, no infection occurred, but localisation of H₂O₂ and peroxidase was detected irrespective of staining methods tested. Based on the observation on gametophytes grown in presence of P. infestans, it is possible that the peroxidase in plants coupled with H₂O₂ may prevent the invasion of nonpathogens by functioning as a barrier. This fern–pathogen model system has potential for application as a tool to study the host–parasite interaction in a gnotobiotic system.     

Thiol redox state and related enzymes in sclerotium-forming filamentous phytopathogenic fungi

Thiol redox state (TRS) reduced and oxidized components form profiles characteristic of each of the four main types of differentiation in the sclerotiogenic phytopathogenic fungi: loose, terminal, lateral-chained, and lateral-simple, represented by Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor, respectively. A common feature of these fungi is that as their undifferentiated mycelium enters the differentiated state, it is accompanied by a decrease in the low oxidative stress-associated total reduced thiols and/or by an increase of the high oxidative stress-associated total oxidized thiols either in the sclerotial mycelial substrate or in its corresponding sclerotium, indicating a relationship between TRS-related oxidative stress and sclerotial differentiation. Moreover, the four studied sclerotium types exhibit high activities of TRS-related antioxidant enzymes, indicating the existence of antioxidant protection of the hyphae of the sclerotium medulla until conditions become appropriate for sclerotium germination.     

The role of ascorbic acid role in the differentiation of sclerotia in Sclerotinia minor

Sclerotinia minor in culture produces ascorbic acid in levels dependent on oxidative growth conditions and stage of development. During differentiation reduced/oxidized ascorbate ratio decreased by 12 and 6 fold at high and low oxidative stress, respectively. Exogenous ascorbate caused a concentration-dependent decrease of oxidative stress (lipid peroxidation), inhibition of sclerotial differentiation (up to 100%) and delay of differentiatlon (up to 10 days). Ascorbic acid may be produced to help the fungus reduce oxidative stress during growth. The data of this study support our theory proposing that oxidative stress is the inducing factor of sclerotial differentiation in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

A two‐component histidine kinase Shk1 controls stress response,sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H₂O₂‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia.     

Effect of thiol redox state modulators on oxidative stress and sclerotial differentiation of the phytopathogenic fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

This study showed that sclerotial differentiation in the filamentous phytopathogenic fungus Rhizoctonia solani is directly related to oxidative stress and thiol redox state (TRS). Sclerotial differentiation is modulated by the availability of non-cytotoxic −SH groups as was shown by the inhibition of sclerorial differentiation by the TRS modulator N-acetyl cysteine (AcCSH), and not necessarily with those of the TRS reduced components glutathione (GSH) and its precursor cysteine (CSH) as indicated by the GSH-biosynthesis inducer and inhibitor l-2–oxo-thiazolidine-4-carboxylate and l-buthionine-S,R-sulfoximine, respectively. Moreover, inhibition of sclerotial differentiation was accompanied by decrease of the high oxidative stress indicators, lipid peroxidation and DNA damage in the mycelial substrate where sclerotia initials are formed, which suggests that this phenomenon is related to oxidative stress as it is predicted by our theory on sclerotial differentiation.     

Effect of glutathione biosynthesis-related modulators on the thiol redox state enzymes and on sclerotial differentiation of filamentous phytopathogenic fungi

In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia, was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general TRS modulator Ν-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators l-oxo-thiazolidine-4-carboxylate (OTC), and l-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly related with the antioxidant –SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated stage due to the substitution of their antioxidant function by the antioxidant potential of the –SH group providers AcCSH and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress.     

Electrophoretic and Immunological Comparisons of Developmentally Regulated Proteins in Members of the Sclerotiniaceae and Other Sclerotial Fungi

The fungal stroma is a distinct developmental stage, a compact mass of hyphal cells enveloped by a melanized layer of rind cells which is produced from vegetative mycelium. Two types of stromata that are characteristic of members of the Sclerotiniaceae but are also produced in a wide range of other fungi, i.e., the determinate tuberlike sclerotium and the indeterminate platelike substratal stroma, were compared in these studies. Developmental proteins found in determinate sclerotial and indeterminate substratal stromata, but not in mycelia, were characterized and compared in 52 isolates of fungi, both ascomycetes (including 18 species in the Sclerotiniaceae and 5 species of Aspergillus) and the basidiomycete Sclerotium rolfsii. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mycelial, stromatal initial, and stromatal extracts demonstrated that all members of the Sclerotiniaceae produced proteins unique to stromatal extracts within a molecular weight range of 31,000 to 39,500 which composed 13 to 58% of the total protein in stromata. Proteins unique to the sclerotial stage were also produced in Sclerotium rolfsii and the Aspergillus species but within a generally lower-molecular-weight range of 11,000 to 30,000. The proteins were then characterized by two-dimensional electrophoresis to determine the number and isoelectric point of polypeptides composing each protein. Polyclonal antibodies were raised to the major 36-kDa sclerotial protein of Sclerotinia sclerotiorum (Ssp). Immunoblots demonstrated that all sclerotial proteins from species in the Sclerotiniaceae cross-reacted with anti-Ssp antibodies, while no cross-reaction was observed with proteins from substratal stromatal species in the Sclerotiniaceae, sclerotial species of Aspergillus, or Sclerotium rolfsii. Results of discriminant analysis of the data from competitive inhibition enzyme-linked immunosorbent assays were consistent with the results of immunoblotting. Three groupings, sclerotial species in the Sclerotiniaceae, substratal stromatal species in the family, and sclerotial species outside the family, were delimited on the basis of relative decreasing ability to compete for anti-Ssp antibody. These data demonstrate that stromatal proteins differ among different taxonomic groups of fungi and suggest that the Sclerotiniaceae may include two distinct lineages of genera.     

Cadmium-induced responses in duckweed <Emphasis Type="Italic">Lemna minor</Emphasis> L.

Although duckweed Lemna minor L. is a known accumulator of cadmium, detailed studies on its physiological and/or defense responses to this metal are still lacking. In this study, the effects of 10 μM CdCl₂ on Lemna minor were monitored after 6 and 12 days of treatment, while growth was estimated every 2 days. Cadmium treatment resulted in progressive accumulation of the metal in the plants and led to a decrease in the growth rate to 54% of the control value. The metal also considerably impaired chloroplast ultrastructure and caused a significant reduction in pigment content, i.e., at day 12, by 30 and 34% for chlorophylls a and b, and by 25% for carotenoids. During cadmium treatment, the contents of malondialdehyde and endogenous H₂O₂ progressively increased (rising 77 and 46% above the controls by day 12), indicating that cadmium induced considerable oxidative stress. On the other hand, higher activities of pyrogallol peroxidase (PPX), ascorbate peroxidase (APX) and catalase (CAT), as well as the induction of a new APX isoform, in cadmium-treated plants, clearly showed activation of an antioxidative response. At day 6, only PPX activity was significantly above the controls (15%), while, at day 12, PPX, APX and CAT activities were increased (74, 78 and 63%). Cadmium also led to accumulation of the heat shock protein 70 (HSP70) and induced an additional isoform of this protein. The obtained results suggest that cadmium (10 μM) is phytotoxic to Lemna minor, inducing oxidative stress, and that antioxidative enzymes and HSP70 play important roles in the defense against cadmium toxicity. M. Tkalec and T. Prebeg contributed equally to this work     

Impact of salinity stress on soil-borne fungi of sugarbeet

The pathogenicity of Egyptian and German isolates of soil-borne root rotting fungi to seedlings of three cultivars of sugarbeet in presence or absence of different concentrations of either NaCl or CaCl₂ were studied under greenhouse conditions. In the absence of salt treatments Egyptian isolates ofRhizoctonia solani were most virulent on all sugarbeet cultivars followed bySclerotium rolfsii andFusarium oxysporum f. sp.betae, the latter proved to be a weak pathogen. The results also revealed that the German isolates ofSclerotinia sclerotiorum were pathogenic to all sugarbeet cultivars studied, whileBotrytis cinerea was only a weak pathogen. However, the presence of salts, NaCl or CaCl₂, in different concentrations seemed to cause alterations in such pathogenicity.     

Synthesis of novel fenfuram-diarylether hybrids as potent succinate dehydrogenase inhibitors

Twelve novel fenfuram-diarylether hybrids were designed, synthesized and characterized by ¹H NMR and MS. Their in vitro antifungal activities were evaluated against five phytopathogenic fungi by mycelial growth inhibition method. Most compounds showed significant antifungal effect on Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 1c exhibited the most potent antifungal effect on R. solani with an EC₅₀ value of 0.242 mg/L, superior to the commercial fungicide boscalid (EC₅₀ = 1.758 mg/L) and the lead fungicide fenfuram (EC₅₀ = 7.691 mg/L). Molecular docking revealed that compound 1c featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 2-chlorophenyl group of compound 1c formed a π-π stacking with D/Tyr-128 and a Cl-π interaction with B/His-249, which made compound 1c more active than fenfuram against SDH.     

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

Examination of Antioxidative System’s Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant <Emphasis Type="Italic">Reaumuria soongorica</Emphasis> (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H₂O₂), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H₂O₂ in the early phase of drought and enzymes of ascorbate–glutathione cycle scavenge H₂O₂ in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H₂O₂ under drought stress.     

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

Metabolic activities of the sugarbeet pathogens Fusarium solani (Mart.) Sacc. and Sclerotium rolfsii Sacc. under pyramin stress

Effects of different concentrations of active ingredient of the herbicide pyramin on metabolic activities of Fusarium solani and Sclerotium rolfsii were examined. High concentrations of this herbicide (1000 and 2000 g mL^-1 for F. solani and 100 and 200 g mL^-1 for S. rolfsii) had inhibitory effects on the metabolic activities of both fungi. These were demonstrated by significant decreases in growth, and increases in rates of CO₂ evolved, O₂ consumed and keto acids produced. These were accompanied by increased rates of sugar, nitrate and inorganic phosphorus absorption as well as lowered rates of synthesis of carbohydrates and insoluble nitrogenous (including protein) and phosphorus (including RNA-P and DNA-P) compounds. In addition, rates of excretion of both nitrogen and phosphorus fractions by the mycelial mats were increased.A concentration of 25 g mL^-1 exerted little or no effect on the metabolic activities of these fungi, although S. rolfsii was somewhat sensitive to this concentration.     

Enzymes Associated with Defence against Toxic Oxygen Species in PCNB-Tolerant and Sensitive Soil Fungi

The specific activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR), which are involved in protection against toxic species of oxygen, were determined in mycelia extracts of pentachloronitrobenzene (PCNB)-tolerant and susceptible soil fungi. The organisms assayed were the highly PCNB-sensitive Rhizoctonia solani and Rhizopus arrhizus; Sclerotium rolfsii and Trichoderma harzianum, which are moderately susceptible to PCNB, and the fungicide-tolerant Fusarium oxysporum f. sp. melonis and Pythium aphanidermatum. No GPOX activity was detected in the six examined fungi. Significant differences in the specific activities of the other enzyme systems among the fungi were evident. Remarkably low levels of CAT activities were measured in R. solani. Except for T. harzianum, no meaningful differences regarding SOD, CAT and GR activities with age of the fungi cultures were observed. The electrophoretic patterns of SOD and CAT displayed dissimilarities among the fungi under study. P. aphanidermatum is more polymorphic with respect to both SOD and CAT enzyme systems as compared to the other fungi. The SOD of F. oxysporum f. sp. melonis, R. arrhizus and T. harzianum is a cuprozinc enzyme, while the mangano-SOD species was detected in S. rolfsii, R. solani and T. harzianum.