Changes in Peroxidase Activity during Potato Ring Rot Infection

The effects of the ring rot causal agent Clavibacter michiganensis subsp. sepedonicus (a virulent strain 5369) on the peroxidase activity of various tissues of potato plants grown under axenic conditions were studied. Root infection enhanced peroxidase activity in all plant tissues (roots, leaves, and stems). In the resistant cv. Lugovskoi, peroxidase activity was much higher than in the susceptible cv. Luk'yanovskii. Co-culturing of the suspension cells of these potato cultivars with the bacterial pathogen also activated peroxidase in the cells of the resistant cultivar; in the cells of the susceptible cultivar, peroxidase activation was less pronounced. Treating suspension cell with exopolysaccharides secreted by the pathogen enhanced the activity of extra- and intracellular peroxidases, and the degree of this enhancement differed in the two potato cultivars.     

Enzyme Changes Associated with Host-Parasite Interactions between Pearl Millet and Downy Mildew Fungus

During the pathogenesis of pearl millet by the downy mildew (Sclerospora graminicola [Sacc.] Schroet.), the activities of peroxidase (PO) and indoleacetic acid oxidase (IAAO) and their isozyme pattern determined by isoelectric focusing on polyacrylamide gels, were observed in extracts of leaves and ears at different stages of development. The PO activity in extracts from infected plants of a susceptible cultivar was found to be higher than in healthy plants. The higher activity was most probably due to the acceleration of host senescence by the pathogen. Quantitative differences in the isozyme patterns of PO and IAAO were found. In inoculated plants of the resistant cultivar, no symptoms developed under the conditions used for infection of the susceptible cultivar and the changes in enzyme activities after inoculation were not significant. The results indicated that different proteins are synthesized in the two cultivars.     

Peroxidase Activity in Leaves of Wheat Cultivars Differing in Resistance to Erysiphe graminis DC.

The peroxidase activities in leaves from resistant and susceptible cultivars of wheat infected and non-infected by Erysiphe graminis DC were studied. In non-infected wheat, soluble and ionic bound peroxidase activity level was found to be higher in the resistant cultivar than that in the one susceptible to Erysiphe graminis DC. After infecting wheat leaves with Erysiphe graminis DC a remarkable increase in the activity of soluble and ionic bound peroxidases was detected 5 days after inoculation only in the resistant cultivar. In the susceptible cultivar a high increase in the activity of the soluble and ionic bound peroxidases occurred only 15 days after inoculation. Using ion exchange chromatography four peroxidase fractions were obtained from infected susceptible and resistant cultivars as from non-infected ones. The fraction II in non-inoculated resistant cultivars was much higher than that in the susceptible one. This fraction increased after inoculation in both cases reaching a higher level in resistant cultivars. Fraction I was higher in the susceptible cultivar. Electrofocusing profiles of peroxidase from the susceptible and resistant cultivar differed from one another. New peroxidase bands after inoculation appeared only in the resistant cultivar.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H₂O₂). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis‐related proteins chitinase (CHT) and β‐1, 3‐glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H₂O₂, resulting in altered cellular redox status, accumulation of pathogenesis‐related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.     

The activity of antioxidative enzymes,contents of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and of ascorbate in tomato leaves of cultivars more or less sensitive to infection with <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

The aim of the present studies was to compare H₂O₂ and ascorbate contents as well as peroxidase (PO) and catalase (CAT) activities in leaves of less susceptible cultivar Perkoz and more susceptible Corindo after B. cinerea infection. Increase in H₂O₂ contents in both Perkoz and Corindo cytosol was observed, however, it appeared earlier in the less susceptible cultivar. The increase in PO activity in the cytosol fraction was observed 48 hours after infection in both cultivars but it was greater in the less susceptible Perkoz. No significant differences between the tested cultivars were observed in ascorbate peroxidase (APX) activity and in reduced and oxidated ascorbate contents. PO activity was thoroughly analyzed in the apoplast fraction. It was measured with syringaldazine (S), tetramethylbenzidine (TMB) and ferulic acid (FA)—substrates characteristic of isoenzymes involved in lignification and stiffening of a cell wall. Increase in PO activity with these substrates was observed earlier in cultivar Perkoz than in cultivar Corindo. Similarly, increase in PO activity with NADH appeared significantly earlier in cultivar Perkoz. Apoplastic PO was separated with DEAE Sepharose and two fractions binding and non-binding were obtained. Binding PO fraction was significantly more active especially with S, TMB and NADH after B. cinerea infection. The increase in the enzyme activity was mostly observed in cultivar Perkoz. Binding PO was separated by electrophoresis on acrylamide gel and revealed six enzymatic forms from which three were much more active after infection in cultivar Perkoz. The obtained results suggest that cell wall strengthening mediated by apoplast PO is a key factor responsible for different resistance of tomato cultivars Perkoz and Corindo to B. cinerea infection.     

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.     

Role of Pseudomonas fluorescens and INA against Black Rot of Cabbage

Cabbage (Brassica oleracea var. capitata) is an important vegetable crop among crucifers. It is affected by a bacterial disease known as black rot. Black rot is caused by Xanthomonas campestris pv. campestris a disease of worldwide importance. The present study highlights the effect of biotic inducer—Pseudomonas fluorescens—and an abiotic inducer—2,6‐dichloro‐isonicotinic acid—in combating black rot, followed by their effect on the seed treatment and disease incidence, role of antioxidant enzymes followed by validation of the defence‐related genes by quantitative real‐time PCR. The resistant (Pusa mukta) and the highly susceptible (NBH boss) cabbage cultivars were analysed for defence‐related enzymes such as peroxidase and superoxide dismutase. An increase in total peroxidase and superoxide dismutase activity was observed upon inoculation with X. campestris pv. campestris. The activity was greater in resistant cultivar when compared to susceptible ones. Both enzyme activity assays and qPCR analyses for the expression of the defence genes in susceptible and resistant cultivars demonstrated that the peroxidase gene was up‐regulated in resistant cultivar compared to susceptible cultivar. The present study proved that P. fluorescens‐induced resistance against X. campestris pv. campestris in cabbage seedlings is more efficient as compared to the use of INA—abiotic inducer.     

Necrotic Lesions as Unusual Symptoms of Ring Rot in the Potato Leaves

Test-tube plants and suspension cell cultures of two cultivars of the potato (Solanum tuberosum L.) differing in their resistance to ring rot caused by Clavibacter michiganensis subsp. sepedonicus and six strains of this bacterium were used to test the relationship between the virulence, the leaf ability to adsorb bacteria, and the symptoms of the disease. In addition to chlorosis and drying, heavy inoculation with virulent strains caused unusual symptoms, such as leaf necrotic lesions. In the resistant cultivar, the necrotic lesions were predominantly local, whereas in the susceptible cultivar, they expanded. Unlike the susceptible cultivar, suspension cells of the resistant cultivar weakly adhered bacteria of the tested strains. Bacteria entered the plants through the leaf stomata. The sorption and penetration were much more pronounced in the susceptible cultivar. It was concluded that strain virulence varies depending on the conditions of inoculation, and uncharacteristic symptoms (necrotic lesions) arise. The local necrotic lesions are considered a hypersensitive response, and exopolysaccharides of the pathogen as the factors of virulence.     

Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina

Pathogenesis-related (PR) proteins are induced in response to pathogen attack. In the present study, the induction of PR proteins in response to the fungal pathogen Macrophomina phaseolina was investigated in 15-day- and 1-month-old plants of Vigna aconitifolia with resistant and susceptible cultivars. Inoculation of the fungal pathogen resulted in the enzyme activity gradually increased throughout the experimental period of 168 h compared to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in the resistant FMM-96 cultivar as compared to susceptible RM0-40 and CZM-3 cultivars. Furthermore, the western blot analysis revealed the presence of 33- and 30-kDa bands of β-1,3-glucanase and chitinase in induced moth bean plants, respectively. The possible implications of these findings as part of the general defense response of moth bean plants against the fungal pathogen (M. phaseolina) have been discussed.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.     

Comparison of transcript profiles in different life stages of the nematode Globodera pallida under different host potato genotypes

The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second‐stage juveniles (J2s) and sedentary parasitic‐stage J2s (SJ2); (ii) a comparison of SJ2s at 8 days after inoculation (DAI) in the susceptible cultivar (Desirée) and two partially resistant lines. The results showed differential expression of G. pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines; the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30 DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30 DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.     

Cross adaptation of potato plants to low temperatures and potato cyst nematode infestation

Effect of short-term (2 h a day) and long-term (6 days) exposure to low temperature (5°C) on cold tolerance was investigated in two cultivars of potato (Solanum tuberosum L.): resistant (Sudarynya) and susceptible (Nevskii) to potato cyst nematode (Globodera rostochiensis Woll.). The extent of their infestation and changes in the expression of the genes of resistance to nematode (H1 and Gro1-4) were also analyzed. In both cultivars, exposure to low temperature enhanced cold resistance of potato plants. Enhancing cold resistance of cv. Sudarynya induced by a short-term exposure to chilling did not affect the extent of nematode infestation, whereas in susceptible cv. Nevskii, the extent of infestation decreased by almost three times. The level of expression of H1 gene in the leaves of the susceptible cultivar rose almost twofold both after short-term and long exposure to chilling, while in the resistant cultivar, gene expression increased only after a short-term effect of cold. The level of Gro1-4 gene expression increased after both temperature treatments only in the resistant cv. Sudarynya. Thus, the expression of genes for potato resistance to nematode infestation became more active in the susceptible cultivar as regards the gene H1 and in the resistant cultivar, regarding the gene Gro1-4. In the nematode-susceptible cv. Nevskii, the level of infestation decreased and cold resistance increased, apparently indicating cross adaptation to two factors of different nature.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Visualising the ionome in resistant and susceptible plant–pathogen interactions

At the morphological and anatomical levels, the ionome, or the elemental composition of an organism, is an understudied area of plant biology. In particular, the ionomic responses of plant–pathogen interactions are scarcely described, and there are no studies on immune reactions. In this study we explored two X-ray fluorescence (XRF)-based ionome visualisation methods (benchtop- and synchrotron-based micro-XRF [µXRF]), as well as the quantitative inductively coupled plasma optical emission spectroscopy (ICP-OES) method, to investigate the changes that occur in the ionome of compatible and incompatible plant–pathogen interactions. We utilised the agronomically important and comprehensively studied interaction between potato (Solanum tuberosum) and the late blight oomycete pathogen Phytophthora infestans as an example. We used one late blight-susceptible potato cultivar and two resistant transgenic plant lines (only differing from the susceptible cultivar in one or three resistance genes) both in control and P. infestans-inoculated conditions. In the lesions from the compatible interaction, we observed rearrangements of several elements, including a decrease of the mobile macronutrient potassium (K) and an increase in iron (Fe) and manganese (Mn), compared with the tissue outside the lesion. Interestingly, we observed distinctly different distribution patterns of accumulation at the site of inoculation in the resistant lines for calcium (Ca), magnesium (Mg), Mn and silicon (Si) compared to the susceptible cultivar. The results reveal different ionomes in diseased plants compared to resistant plants. Our results demonstrate a technical advance and pave the way for deeper studies of the plant–pathogen ionome in the future.     

Peroxidase involvement in the defense response of red raspberry to Didymella applanata (Niessl/Sacc.)

Peroxidase activity of red raspberry canes was dependent on the cultivar and influenced the subsequent lignification. After inoculation with Didymella applanata, responsible for the spur blight cane disease, the activity of soluble cytoplasmic enzyme increased in the moderately resistant ‘Latham’ and susceptible ‘Malling Promise’, similarly for syringaldazine and guaiacol as hydrogen donors. Systemic induction found in ‘Latham’ was recognized as a symptom of defence mechanism responsible for fungal restriction. Locally enhanced peroxidase activity in the ‘M.Promise’ tissues was related to the local lignification and/or may be associated with the loss of cell integrity caused by pathogen penetration. Pathogen-induced changes of cell wall peroxidases were similar in both cultivars mentioned above. No influence of the infection was found in the high susceptible Zeva cultivar. Using native-PAGE analysis and horizontal starch electrophoresis of soluble fraction five constitutive acidic isoperoxidases were detected in ‘Latham’ and three in ‘M. Promise’. The infection process was accompanied by the appearance of two new anodic isoforms.     

The Role of Polyphenol Oxidase and Peroxidase in Potato Tuber Resistance to Soft Rot Caused by Erwinia carotovora

Tubers from somatic hybrids produced by protoplast fusion between Solanum brevidens, a diploid, non-tuber-bearing wild species, and a tetraploid S. tuberosum showed resistance to decay caused by soft rot Erwinia. Tubers of the S. tuberosum fusion parent and potato cultivar Russet Burbank are susceptible to bacterial soft rot. Tubers of somatic hybrids indicated higher levels of activities of polyphenol oxidase and peroxidase than tubers of the parental line of S. tuberosum and cultivar Russet Burbank. This is true for intact tubers and also for injured or inoculated tubers. Polish commercial potato cultivars indicated a higher susceptibility to soft rot than somatic hybrids. However, there were some differences in susceptibility to soft rot between Polish commercial potato cultivars, only slight differences were observed in the activities of the polyphenol oxidase and peroxidase between Polish cultivars. A relation between soft rot resistance and the activity of each enzyme was not found for intact, injured or inoculated tissue of commercial cultivars. On the contrary, the activities of both enzymes were significantly higher in the periderm than in the medullary tissue of somatic hybrids, the parental line and the commercial cultivars.     

Leakage of electrolytes and phenols and altered activity profile of peroxidase and its isoenzymes from wheat leaves inoculated withPuccinia recondita

Second and third leaves of 25-day old wheat (Triticum aestivum L.) planta, cultivar WL-711, were inoculated with three races of the brown rust pathogen,Puccinia recondita f. sp.tritici. The host reflected a differential interaction response towards these races, viz., susceptible reaction to race 77 (reaction type 4) intermediate reaction to race 104 (reaction type 1 - 3) and resistant reaction to race 63 (reaction type 0). Post-inoculation mutual interaction brought about observable changes in peroxidase activity along with leakage of electrolytes and phenolic constituents indicating altered permeability of host tissue. Differential increase in leakage of electrolytes and phenols occurred with increase of peroxidase activity during 12 h and 24 h post-inoculation, however, during 36 h, 48 h and 72 h post-inoculation, electrolyte leakage increased continuously with corresponding increases in peroxidase activity in all three interactions, while leakage of phenols showed a differential pattern. The differential changes in isoenzymic pattern of peroxidase activity were also noted during the progressive infection process which may also be an indication of altered permeability of the host issue at the very onset of brown rust infection.     

Reaction of Melon (Cucumis melo L.) Cultivars to Monosporascus cannonballus (Pollack & Uecker) and their Effect on Total Phenol,Total Protein and Peroxidase Activities

Eighteen melon cultivars were screened for resistance to Monosporascus cannonballus under greenhouse conditions. The melon cultivars were grown in pasteurized sand, which had been inoculated with a high level (60 CFUs/g of soil) of M. cannonballus mycelium from culture. Cultivars Nabijani, Sfidak khatdar, Sfidak bekhat, Ghandak, Mollamosai, Chappat, Hajmashallahi and Shadgan were moderately resistant to M. cannonballus but all other melon cultivars were moderately to highly susceptible (HS) to this pathogen. A second screening was performed for resistance to M. cannonballus under greenhouse conditions. In the second screening, cultivars Nabijani, Sfidak khatdar, Sfidak bekhat, Ghandak, Mollamosai, Chappat, Hajmashallahi and Shadgan were moderately resistant to M. cannonballus. To examine the melon resistance mechanism against M. cannonballus, the activities of total phenol, total protein and peroxidase in two melon cultivars Nabijani (as resistant) and Khaghani (as susceptible) were determined at 0, 24, 48 and 72 h after inoculation. Inoculated resistant cultivar roots had always higher content of total phenol, total protein and peroxidase than the corresponding inoculated susceptible cultivar roots. The results indicated that there was a relationship between resistance in Nabijani and accumulation of total phenol, total protein and peroxidase.