Alterations in peroxidase activity and peroxidase isozymes in virus-infected plants

Peroxidase activity and isozyme patterns were investigated in two leguminous species infected with viruses which produced either local necrotic or systemic chlorotic symptoms. Highest peroxidase activity was recorded when the hosts reacted to infection with necrotic local lesions. No new virus-specific isozymes were found as a result of infection, but some isozymes, apparently associated with senescence, appeared earlier in extracts from leaves showing necrosis than in extracts from healthy leaves, or from infected leaves showing only very mild chlorosis. Increase in peroxidase activity was accompanied by alteration in isozyme pattern.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Isozyme profiles associated with the hypersensitive response of Chenopodium foetidum to plum pox virus infection

Isozyme profiles of esterases (E.C. 3.1.1.1), glutamate oxaloacetate transaminase (E.C. 2.6.1.1) and peroxidases (E.C. 1.11.1.7) have been determined in healthy tissues of Chenopodium foetidum as well as their modifications during leaf development. The effect of plum pox virus infection on the isozyme profiles has also been studied. Virus-induced necrotic lesions displayed a peroxidase (POX) pattern that has not been found in any other tissue of the plant so far analyzed. The pattern was similar to that of old yellow leaves, except that POX-B, which was detected in the necrotic lesions, has not been detected in any developmental stage of healthy leaves. Changes in the peroxidase profile seem to begin early during infection, even before necrosis is visible. We suggest that senescence is established at necrotic lesions extending from there to the rest of the infected leaf affecting the peroxidase isozyme pattern. However, other changes, which induce POX-B, must also take place at necrotic lesions. These do not extend to the rest of the infected leaves. Plum pox virus infection has less effect on the glutamate oxaloacetate transaminase and esterase isozyme patterns, inducing an almost normal senescence pattern.     

Phenols in Cotton Seedlings Resistant and Susceptible to Alternaria macrospora

In healthy cotton seedlings, stems have a lower phenol content than leaves, but resistant plants have an altogether relatively higher phenol content than susceptible plants. Phenols extracted from infected plants can inhibit the growth of A. macrospora in vitro. In cotton plants infected with A. macrospora, phenols are oxidized by polyphenoloxidase rather than peroxidase and catalase. The main oxidative activity was around the developing necrotic area but activity was detected far from, necrosis as well. Though pre–inoculation mechanical injuries operated the phenol oxidation mechanism in the plant, they neither prevented nor encouraged the increase in disease severity. Isozyme pattern showed that contribution of all participants in the pathological interaction to the oxidative mechanism occurred in the diseased plant. A negative linear correlation was found between polyphenoloxidase activity, phenol accumulation and resistance. This study suggests that the phenol oxidative mechanism, participates in cotton plant resistance to A. macrospora.     

Xanthomonas oryzae pv. oryzae infection triggers accumulation of phenolics,defense-related enzymes and thaumatin-like proteins in rice leaves

Abstract

The effect of Xanthomonas oryzae pv. oryzae infection on induction of phenylalanine ammonia-lyase (PAL), peroxidase (PO), phenolics and thaumatin-like proteins (TLPs) in rice was studied. PAL activity increased significantly one day after inoculation with X. o. pv. oryzae and the maximum enzyme activity was observed two days after inoculation. The phenolic content in rice leaves increased significantly one day after inoculation and the maximum accumulation of phenols was observed two days after inoculation. Significant increase in peroxidase activity was observed in rice leaves one day after inoculation with X. o. pv. oryzae. Isozyme analysis indicated that three peroxidase isozymes (PO-1, PO-2 and PO-3) were induced after inoculation with X. o. pv. oryzae. Immunoblot analysis of protein extracts from control and pathogen inoculated rice plants revealed the induced accumulation of 16 and 24 kDa TLPs in rice leaves in response to X. o. pv. oryzae infection. TLP mRNA accumulation was induced strongly in rice leaves in response to infection by X. o. pv. oryzae.     

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.     

Induction of Resistance in Melon to Didymella bryoniae and Sclerotinia sclerotiorum by Seed Treatments with Acibenzolar-S-methyl and Methyl Jasmonate but not with Salicylic Acid

The plant resistance activator acibenzolar‐S‐methyl (BTH), the signalling molecules salicylic acid (SA) and methyl jasmonate (MeJA) were tested by seed treatment for their ability to protect melon seedlings from gummy stem blight and white mould disease caused by the soil‐borne fungal pathogens Didymella bryoniae and Sclerotinia sclerotiorum, respectively. Didymella bryoniae infection on melon seedlings was completely suppressed by MeJA treatment. Necrotic lesions akin hypersensitive response occurred on all inoculated seedlings and prevented pathogen diffusion into healthy tissues. Didymella bryoniae infection was restricted following BTH seed treatment as well, although the percentage of necrotic lesions in comparison with the water soaked lesions was significantly lower than that from MeJA‐induced seedlings. BTH protected melon seedlings against S. sclerotiorum by the occurrence of a high percentage of necrotic lesions. A lower level of resistance was also achieved by MeJA seed treatment. The augmented level of resistance of tissues from BTH and MeJA‐treated seeds was associated with rapid increases in the activity of the pathogenesis‐related proteins chitinase and peroxidase. MeJA also determined a rapid and transient accumulation of lipoxygenase. Moreover, BTH and MeJA treatments determined the differential induction of particular de novo synthesized isoenzymes of these proteins. Results indicate that BTH and MeJA applied to melon seeds may activate on seedlings diverse metabolic pathways leading to the enhancement of resistance against distinct pathogens.     

Differential in vitro DNA binding activity to a promoter element of the gn1β-1,3-glucanase gene in hypersensitively reacting tobacco plants

In a hypersensitive reaction to pathogen infection, expression of the β-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5′-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the β-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to ?138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from ?250 to ?217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.     

Effect of Tobacco Mosaic Virus on Phospholipid Content and Phospholipase D Activity in Tobacco Leaves

The phospholipid content and phospholipase D activity in the leaves of two tobacco (Nicotiana tabacum L.) cultivars were investigated. These cultivars are characterized by different response to the infection with tobacco mosaic virus (TMV). In the infected leaves of a susceptible cv. Samsun, phospholipid content and phospholipase D activity did not change within seven days after TMV infection. The development of a hypersensitive response in the leaves of a resistant cv. Xanthy necrotic was not accompanied by a change in the total phospholipid content as compared to the noninfected leaves. However, the appearance of necrotic lesions and their subsequent expansion resulted in a steady decrease in the level of phosphatidylglycerol in infected leaves. At the same time, phosphatidic acid and diphosphatidylglycerol contents increased. Leaf zones remote from the regions of necrosis development were also characterized by an increased level of phosphatidic acid. There was a tendency for an increase in phospholipase D activity in both the sites of necrosis development and in the leaf regions remote from these sites. The changes in phosphatidic acid content were of similar nature, and therefore a relative increase in phosphatidic acid could result from the phospholipase D activity. This fact suggests a possible involvement of phospholipase D in the development of the hypersensitive response, and this suggestion is supported by a higher enzyme activity in the leaves of healthy plants of the resistant cultivar as compared to the susceptible one. Causes for the changes in the content of some phospholipids, as well as the physiological role of phospholipase D in the hypersensitive response are discussed.     

Effect of Aliette on AMV Infection of Bean Leaves and on the Resultant Alterations in the Patterns of Proteins and Peroxidases

Increased peroxidase activity as well as changes in the patterns of soluble proteins, and peroxidases were observed following infection of bean leaves with AMV.
Foliar sprays with Aliette® (phosethyl Al) at 2000 ppm a. i. delayed the appearance of necrotic local lesions and reduced their final number on primary bean leaves following inoculation with alfalfa mosaic virus (AMV).
The delay in the appearance of symptoms observed on Aliette-treated inoculated leaves was correlative with a delay in all the above cited alterations, while reduction of final symptoms was correlative with a decrease of these alterations.     

Relationship between peroxidase activity and the amount of fully N-methylated compounds in bean plants infected by Pseudomonas savastanoi pv. phaseolicola

Changes in the level of endogenous formaldehyde (HCHO), some N-methylated compounds (choline and trigonelline) and peroxidase activity were examined in the leaves of bean genotypes (Phaseolus vulgaris L.) with different disease-sensitivity during ontogenesis in the stressfree condition and after natural infection by Pseudomonas savastanoi pv. phaseolicola (until the appearance of lesions). HCHO, as its dimedone adduct, and fully N-methylated compounds were determined by overpressured layer chromatography (OPLC) in different developmental stages and in the infected leaves/leaf discs. Peroxidase activity was measured by a spectrophotometric method. HCHO level decreased with ageing of the primary leaf and accordingly in the leaves at different developmental stages, then increased again in both cases due to the demethylation and methylation processes. Concentration of choline and trigonelline as potential HCHO generators decreased considerably while peroxidase activity increased with ageing of the plants. Comparing the symptomless and the Pseudomonas infected leaf discs (with watersoaked lesions) we found a decrease in the level of HCHO, choline and trigonelline and there was detectable increase in the peroxidase activity in the infected leaf tissues. Our findings are in accordance with previously published results that peroxidases play an important role in oxidative demethylation processes. Our hypothesis is that the high level of HCHO in the old leaves can originate from methylated components as the result of peroxidase activity and this high level may lead to the old leaf being resistant to pathogen. This conclusion is supported by the fact that the leaves of susceptible bean genotypes became resistant to Pseudomonas while growing older.     

Induced Systemic Resistance in Tomato Plants against Phytophthora infestans

Infection of lower leaves of tomato plants with Phytophthora infestans followed by a period unfavourable to disease development increased the general resistance of the plants against the pathogen. Induced resistance to a second infection of upper leaves was expressed in the development of necrotic lesions that were sharply defined and reduced in size. Sporulation of the pathogen was suppressed. Lesions on unprotected plants expanded with a sporulating zone passing gradually to the healthy tissue under the same conditions. Induced resistance delayed and reduced penetration of the pathogen into the epidermis and subsequent colonisation of the mesophyll by formation of papillae in epidermal cells and hypersensitive-like reactions of penetrated, mesophyll cells.     

Effect of thermal shock treatments on symptom expression in test plants inoculated with potato aucuba mosaic virus

Potato aucuba mosaic virus (PAMV) has few reliable local lesion assay hosts. However, lesions formed when PAMV-inoculated leaves were exposed to thermal shock (dipping for 40 s in water at 50 or 2 °C). Leaves of Nicotiana tabacum (cv. Xanthi-nc, Samsun or Samsun NN), Hyoscyamus niger and Datura metel consistently developed necrotic lesions, leaves of Chenopodium amaranticolor developed whitish rings, and leaves of N. glutinosa developed diffuse cream-coloured rings and spots. In PAMV-inoculated leaves of Xanthi-nc tobacco, C. amaranticolor and D. metel, lesions formed only in areas exposed to light. Thermal shocks applied to systemically infected leaves of Xanthi-nc tobacco induced necrotic vein banding patterns. In inoculated Xanthi-nc tobacco leaves, PAMV seemed confined to local lesions. The rate of lesion enlargement was therefore a measure of rate of virus spread. Lesion size increased as the interval between inoculation and shock treatment increased. The mean rates of increase in lesion radius were 17 and 27 Cμm/h at 15 and 25°C respectively. ‘Target’ lesions, composed of concentric necrotic rings, formed when inoculated Xanthi-nc tobacco leaves were given two or more 50°C shocks. The first of two 50°C treatments decreased subsequent rates of lesion enlargement.     

Outdoor infrared imaging for spatial and temporal thermography: A case study of necrotic versus healthy leaf areas on woody plants

The temperature of plants can be measured using infrared (IR) thermography. Despite the extensive use of IR imaging indoors, outdoor IR imaging is uncommon. We used IR imaging to compare leaf temperatures between necrotic spots and healthy areas of oriental cherry (Prunus serrulata var. spontanea), Japanese cornel (Cornus officinalis) and sawtooth oak (Quercus acutissima) in the field. There was a significant difference in the mean leaf temperatures between the necrotic spots (26.4°C) and healthy areas (25.6°C) of oriental cherry (p = .01). The mean temperatures in the necrotic spots of Japanese cornel and sawtooth oak leaves were 22.3°C and 29.6°C, respectively, which were not significantly different from the mean temperatures of the healthy areas. A consecutive, 2‐day temporal leaf analysis in October 2018 revealed that the temperatures in the necrotic spots were generally higher than those in the healthy areas of the three species. The temperature difference between the spots and healthy areas (up to 1.4°C) was more pronounced at 13:00 hr in all three species on both days. These results reveal differences in the spatial and temporal thermal state across the necrotic spotted leaves. There is potential for use of outdoor IR imaging to visualize the response of trees to pathogen infection and abiotic stress.     

Induction of resistance in Phaseolus vulgaris L. cv. Lima against some soil-borne fungal pathogens by pre-inoculation with tobacco necrosis virus (TNV) reacting hypersensitivity

Abstract

Tobacco necrosis virus (TNV) was tested to induce systemic acquired resistance (SAR) in Phaseolus vulgaris cv. Lima against three important soil-borne fungal pathogens viz: Rhizoctonia solani, Macrophomina phaseolina and Fusarium oxysporum. Application of TNV as a local infection of seven-day old primary leaves of Phaseolus vulgaris cv. Lima resulted in reduction of the mean disease rating of root-rot and damping-off caused by the tested fungal pathogens. The pre-inoculated plants with TNV showed a significant enhancement in their content of photosynthetic pigments (chlorophyll a, b and carotenoids) compared to those inoculated with fungal pathogens only. The percentage of cell membrane stability and ion leakage of viral-treated plants were significantly increased confirming the healthy cytological status of the treated plants. Results demonstrated that inoculation of the primary leaves of beans with TNV before infection with the fungal pathogens leads to changes in protein patterns and showed differences compared with control and caused the appearance of at least one new protein band compared with only fungal-infected plants. Also, an increase in peroxidase activity emerged in the thickness of the isozymic pattern in addition to the synthesis of new bands which was observed as a result of TNV application before infection with the three fungal pathogens. Induction of the synthesis of a new protein and increasing peroxidase activity in the inoculated plants enhanced the defense system against the target pathogen. The results greatly supported the successful application of TNV in the induction of systemic acquired resistance in P. vulgaris cv. Lima against the fungal pathogens.     

Leaf scarring by the weevils Neochetina eichhorniae and N. bruchi enhances infection by the fungus Cercospora piaropi on waterhyacinth, Eichhornia crassipes

Additive or synergistic effects among introduced and native insect and plant pathogen agents are necessary to achieve biological control of waterhyacinth (Eichhornia crassipes), a globally damaging aquatic weed. In field plots, plants were infested with waterhyacinth weevils (Neoechetina bruchi and N. eichhorniae) and leaves were scarred by weevil feeding. Subsequent infection by the fungal pathogen Cercospora piaropi caused necrotic lesions to form on leaves. Necrosis development was 7.5- and 10.5-fold greater in plots augmented with both weevils and C. piaropi and weevils alone, respectively, than in plots receiving only C. piaropi. Twenty-four days after weevil infestation, the percentage of laminar area covered by lesions on third-youngest and oldest live leaves was elevated 2.3–2.5-fold in plots augmented with weevils. Scar density and necrosis coverage on young leaf laminae were positively correlated, even though antipathogenic soluble peroxidases were elevated 3-fold in plots augmented with weevils alone or weevils and C. piaropi. Combined weevil and fungal augmentation decreased shoot densities and leaves per plant. In a no-choice bioassay, weevil feeding on oldest but not young leaves was reduced 44 two weeks after C. piaropi inoculation. Protein content and peroxidase activities were elevated 2–6-fold in oldest leaves three weeks after inoculation. Augmentation with both waterhyacinth weevils and C. piaropi led to the development of an additive biological control impact, mediated by one or more direct interactions between these agents, and not plant quality effects.     

Separate and combined effects of excess copper and Fusarium culmorum infection on growth and antioxidative enzymes in wheat (Triticum aestivum L.) plants

The effect of stress combinations on plants cannot be extrapolated from the response to each of the applied stressors. Greenhouse experiments were carried out on soil to which copper ions were introduced at four concentrations (0, 150, 400, and 600 mg kg^?1). Copper treatments without or with Fusarium infection were established. Both stress factors, applied separately or together inhibited growth with the exception of the lowest Cu concentration, which stimulated growth of healthy plants. Depending on concentration, Cu did not change or increased the activity of root peroxidase and leaf catalase, and decreased ascorbate peroxidase (APO) activity in leaves and roots. Infection increased the activities of the enzymes with exception of root APO. The simultaneous presence of these two stress factors modified their individual effects. Generally, the stress combination aggravated the plant status though an opposite trend was observed in some cases.     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

Phenolic Compounds and the Polyphenoloxidase and Peroxidase Activity in Callus Tissue Culture-Pathogen Combination of Red Raspberry and Didymella applanata (Niessl.) Sacc.

The content of phenolic compounds as well as the activity of polyphenoloxidase and peroxidase in the red raspberry callus tissue after D. applanata infection were investigated. Two red raspberry cultivars: Latham — relatively resistant and M. Promise — susceptible were used. In the M. Promise cultivar the amount of phenols, especially o-diphenols, decreased after infection and stayed more or less on the same level in Latham cultivar. The activity of polyphenoloxidase increased in both of the investigated cultivars, however in the tissue of Latham cultivar this rise was more pronounced than in the M. Promise one. The changes in peroxidase activity after infection were inconsiderable. The relation between the changes observed after infection in the phenolic metabolism and the differentiated susceptibility of callus tissue to D. applanata of the investigated cultivars was discussed. The obtained results explain partially the browning of infected callus tissue of one of the investigated cultivar i.e. M. Promise which had already been observed in the earlier investigations.     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.