Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Salicylic acid alleviates growth inhibition and oxidative stress caused by zucchini yellow mosaic virus infection in Cucurbita pepo leaves

The effects of zucchini yellow mosaic virus (ZYMV) infection and pretreatments with salicylic acid (SA) on biomass accumulation of pumpkin (Cucurbita pepo cv. Eskandarani) were investigated. The response of photosynthesis, transpiration and the activities of antioxidant enzymes in leaves was also considered. Significant reductions in growth parameters (i.e. leaf area, biomass and shoot height), photosynthesis and chlorophyll a and b content were detected in ZYMV-infected leaves in comparison to healthy controls. Antioxidant enzyme activities were increased up to 3-fold for peroxidase (POD), 2-fold for ascorbate peroxidase (APX) and catalase (CAT) activities and 1.3-fold for SOD activity by virus infection. ZYMV infection also caused increases in H₂O₂ and malondialdehyde (MDA) contents. These results suggest that ZYMV infection causes oxidative stress in pumpkin leaves leading to the development of epidemiological symptoms. Interestingly, spraying pumpkin leaves with SA led to recovery from the undesirable effects of ZYMV infection. Leaves treated with 100 μM SA three days before inoculation had the appearance of healthy leaves. No distinct disease symptoms were observed on the leaves treated with 100 μM SA followed by inoculation with ZYMV. In non-infected plants, SA application increased activities of POD and superoxide dismutase (SOD) and inhibited APX and CAT activities.In contrast, SA treatment followed by ZYMV inoculation stimulated SOD activity and inhibited activities of POD, APX and CAT. In addition, MDA displayed an inverse relation, indicating inhibition of lipid peroxidation in cells under SA treatment. It is suggested that the role of SA in inducing plant defense mechanisms against ZYMV infection might have occurred through the SA-antioxidant system. Such interference might occur through inhibition or activation of some antioxidant enzymes, reduction of lipid peroxidation and induction of H₂O₂ accumulation following SA application.     

A study on the susceptibility of the model legume plant <Emphasis Type="Italic">Medicago truncatula</Emphasis> to the soil-borne pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Fusarium wilt is a soil-borne disease caused by formae specialis of Fusarium oxysporum on a large number of cultivated and wild plants. The susceptibility of the model legume plant Medicago truncatula to Fusarium oxysporum was studied by root-inoculating young plants in a miniaturised hydroponic culture. Among eight tested M. truncatula lines, all were susceptible to F. oxysporum f.sp. medicaginis, the causal agent of Fusarium wilt in alfalfa. However, a tolerant line, F83005.5, and a susceptible line, A17, could be distinguished by scoring the disease index. The fungus was transformed with the GFP marker gene and colonisation of the plant roots was analysed by epifluorescence and confocal microscopy. A slightly atypical pattern of root colonisation was observed, with massive fungal growth in the cortex. Although colonisation was not significantly different between susceptible and tolerant plants, the expression of some defence-related genes showed discrimination between both lines. A study with 10 strains from various host-plants indicated that M. truncatula was a permissive host to F. oxysporum.     

硅对盐胁迫下葡萄幼苗生理效应的影响

以酿酒葡萄"赤霞殊"(Cabernet Sauvignon,CS)1年生扦插苗为试材,采用营养液水培系统,研究了根施外源硅(K2SiO3·9H2O,Si)对盐(50 mmol/L NaCl)胁迫下赤霞珠葡萄幼苗叶片生理生化特性的影响.结果表明:盐胁迫处理下,葡萄幼苗叶片超氧阴离子(O2-')产生速率加快,过氧化氢(H2O2)、丙二醛(MDA)含量和相对膜透性增加;超氧化物歧化酶(SOD),过氧化物酶(POD)和过氧化氢酶(CAT)活性增强;组织含水量降低;施用2.0mmol/L Si明显抑制了O2-、H2O2和MDA的积累,增加了组织含水量,并进一步提高了抗氧化酶SOD和POD的活性.说明外源Si可通过提高盐胁迫下葡萄幼苗体内的抗氧化酶活性,从而降低活性氧(ROS)水平,一定程度上减轻盐胁迫对植株所造成的细胞膜脂过氧化伤害,增强其耐盐性.     

Nitric Oxide Treatment and Induced Genes Role Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Effects of sodium nitroprusside (SNP; nitric oxide donor) treatments on enhancement of secondary metabolites production, oxidative stress mediators (\(\mathrm{O}_{2}^{-}\)) accumulation and antioxidant defense enzymes of Potato Spunta Sp. suspension culture cells elicited by a fungal extract from phytophthora infestans mycelium. The obtained data confirmed the significant increase in various oxidative burst (super oxide anion, hydrogen peroxide and total glutathione) contents. The administration of various NO concentrations strongly decreased hydrogen peroxide concentration and superoxide anion levels. Moreover, the SNP treatments regulate elicitor-induced activation of phenylalanine ammonium-lyase and total soluble phenols accumulation. The highest concentrations of NO donor sodium nitroprusside potentiated elicitor-induced H₂O₂ production. On the other hand, the lowest H₂O₂ contents coincided with elicitation regulated various activities of enzymes superoxide-dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and Phenyl alanine ammonia lyase (PAL) activity, the decrease in H₂O₂ concentration was probably due to a direct reduction interaction of NO-H₂O₂. On the other hand, the addition of these previous NO treatments affects mRNA peroxidase gene expression using RT-PCR techniques. In general, the addition of lower concentrations of nitric oxide reduce the mRNA peroxidase activity on contrary, the higher concentrations induced the mRNA peroxidase activity, which induce the hypersensitive reactions against fungus infection.     

<Emphasis Type="Italic">Sunflower Chlorotic Mottle Virus</Emphasis> in Compatible Interactions with Sunflower: ROS Generation and Antioxidant Response

Sunflower chlorotic mottle virus (SuCMoV) is a recently described potyvirus that causes systemic infections in sunflower plants leading to chlorotic mottling and important growth reductions and yield losses. Oxidative damage is expressed after symptom development in this host-pathogen combination. The involvement of antioxidant enzyme activities in disease susceptibility was studied in two sunflower lines differing in the intensity and rate of development of SuCMoV infections: L2 is more susceptible than L33. A transient superoxide production peak was detected in leaves of both lines before symptom development. H₂O₂ accumulation increased before symptom expression in infected plants of L33 but in L2 such increase was registered only after symptoms became evident. In healthy plants of both lines, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) showed similar activity levels. In inoculated plants of line L2, but not in L33, SOD and CAT activities increased significantly before the appearance of symptoms, and APX increases were detected later. A 1 mM SA treatment effectively decreased SuCMoV accumulation in plants of L2 but it did not affect it in L33. This treatment increased H₂O₂ accumulation and prevented the increase in antioxidant enzyme activities in infected plants of L2. It is suggested that increases in antioxidant enzyme activities interrupted the signals generated by the increase in ROS, which may have otherwise triggered defence reactions in the host and thus, resulted in a compatible interaction.     

Bentazone and bromoxynil induce H and H2O2 accumulation, and inhibit photosynthetic O2 evolution in Synechococcouselongatus PCC7942

Using a unicellular cyanobacterium, Synechococcouselongatus PCC7942, we have shown that cytosolic acidification, O₂⁻; H₂O₂ production and photosystem II-inactivation are the causes of cell death from bentazone/bromoxynil incubations. Butyric acid evoked solely pH lowering response and yet inhibited PS II activity indicating that herbicide-caused acidification is sufficient to kill the cyanobacterial cells, but other factors like excess H₂O₂ production due to an imbalance in the peroxide sequestration machinery might be contributory. While the activities of superoxide dismutase and pyrogallol peroxidase increased consequent to herbicide incubations and displayed oligomeric states with mobility shift, catalase and glutathione peroxidase though present remained insensitive.     

Spatiotemporal patterns of oxidative burst and micronecrosis in resistance of wheat to brown rust infection

The accumulation of H₂O₂ (oxidative burst) and the progress of pathogen development were studied in compatible and incompatible wheat‐brown rust interactions. The accumulation of H₂O₂ was detected in 98·7% of guard cells with appressoria 8 h post inoculation (hpi). The reaction in both susceptible and resistant plants declined 2–3 days post inoculation (dpi). The second phase of the oxidative burst was observed in the mesophyll and/or epidermis. In susceptible plants it began 4–5 dpi and was detected only in the epidermis. In resistant plants the response was observed in the mesophyll. In moderately resistant plants it was induced 1–3 dpi, and the percentage of infection units reached 80–90% 8 dpi. This corresponded with severe necrotic symptoms. In highly resistant plants, the oxidative burst was short and transient. The percentage of infection units with H₂O₂ accumulation reached its highest level (60–70%) 2 dpi, and decreased thereafter. Four days later, the low percentage and weak DAB staining indicated very low H₂O₂ accumulation. The localization and the time‐course changes of the oxidative burst correlated with the profiles of the micronecrotic response, haustorium mother cell formation and pathogen development termination. An early and localized induction of oxidative burst followed by its rapid quenching correlated with high resistance and almost no disease symptoms. The possible correlation of the oxidative burst and pathogen development patterns with the level and durability of resistance conferred by Lr genes are discussed.     

Oxygen metabolism in plant cell culture/bacteria interactions: role of bacterial concentration and H2O2-scavenging in survival under biological and artificial oxidative stress

Preliminary studies with plant cell suspensions indicated that high concentrations of bacterial inoculum can significantly reduce the H₂O₂levels detected immediately following inoculation. Further investigation suggested that bacterial concentration is an important factor in H₂O₂-scavenging, and is often overlooked in both plant/pathogen interactions as well asin vitrosurvival studies.In vitrostudies withPseudomonasspp. characterized the relationship between H₂O₂-scavenging and culturability of bacteria. Because H₂O₂permeates membranes rapidly, the intracellular scavenging activity of the bacteria could be detected as a decrease in the external H₂O₂concentration. Therefore, the effective scavenging activity of a given suspension of bacteria was proportional to the bacterial concentration. The culturability of populations of bacteria exposed to H₂O₂concentrations which are intolerable to individual cells (≥100 μm) was also dependent on bacterial concentration. Studies with a catalase-deficient mutant ofP. putidaindicated that catalase, due to its high K_m, had little effect on scavenging H₂O₂at the lower concentrations of H₂O₂(<100 μm) that are likely to be encountered in suspension cells and other plant tissues. Using high concentrations of bacteria (1×10⁹cfu ml^-1), there was little difference between the catalase-deficient mutant and various isolates ofPseudomonasspp. in the reduction of H₂O₂levels in suspension cells. The studies indicated that an additional scavenging system other than catalase exists in bacteria and this may play an important role in scavenging H₂O₂during plant/bacteria interactions.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

The Oxidants and Antioxidant Enzymes in Tomato Leaves Treated With O-Hydroxyethylorutin and Infected with Botrytis cinerea

Application of o-hydroxyethylorutin restricted the development of Botrytis cinerea in tomato leaves. Superoxide anion and hydrogen peroxide generation rates and changes in superoxide dismutase, peroxidase and catalase activities were studied in uninfected tomato plants, in plants infected with B. cinerea, and in plants treated with o-hydroxyethylorutin and infected with pathogen. About two times higher hydrogen peroxide concentration were found in plants treated with o-hydroxyethylorutin and infected with the pathogen at the early infection stages compared with untreated infected plants. In vitro tests showed that germination of B. cinerea conidia was significantly inhibited by H₂O₂. Higher H₂O₂ concentrations were needed to inhibit mycelial growth. The results indicate that o-hydroxyethylorutin triggers hydrogen peroxide production in tomato plants and suggest that enhanced levels of H₂O₂ are involved in restricted B. cinerea infection development.     

Tomato spotted wilt virus triggers specific and shared defense mechanisms in hypersensitive and susceptible Solanaceae hosts

In situ and in vitro techniques were employed to investigate the metabolic changes caused by Tomato spotted wilt virus in hypersensitive and susceptible hosts; Petunia hybrida and Nicotiana tabacum, respectively. In petunia, H₂O₂ accumulation preceded increased peroxidase and shikimate dehydrogenase activity at local lesion sites. In systemic tobacco plants, peroxidase activity was induced prior to symptom onset and the activity of shikimate dehydrogenase was disrupted upon viral infection. Taken together, our data suggest that reactive oxygen species-based mechanisms of defense are shared by hypersensitive and susceptible hosts, although downstream components and regulatory mechanisms are distinct.     

Application of energy-filtering electron microscopy (EFEM) for analysis of hydrogen peroxide and lignin in epidermal walls of cucumber leaves triggered by acibenzolar-S-methyl treatment prior to inoculation with Colletotrichum orbiculare

Hydrogen peroxide (H₂O₂) and lignin induced by acibenzolar-S-methyl (ASM) during the expression of systemic acquired resistance (SAR) of cucumber plants against the fungus Colletotrichum orbiculare was analysed with electron microscopy and energy-filtering electron microscopy (EFEM). ASM pretreatment consistently led to the blockage of appressorial penetration pegs into leaves at early stages after inoculation, whereas the fungus was able to grow into the vascular tissue after distilled water pretreatment. The blocked pegs, seen frequently at epidermal pectin layers in leaves of ASM-treated plants, were surrounded by abundant electron-dense amorphous material and dots. These were identified as lignin, based on their appearance and high reactivity with KMnO₄. Lignin first appeared as an amorphous material at pectin layers, and then as dots in some areas of the material. The results showed that ASM caused SAR in leaves via faster formation of lignin within 1 day after inoculation. After CeCl₃ treatment to detect H₂O₂, H₂O₂-reactive products (cerium perhydroxides) were seen near ASM-induced lignin formation sites in pectin layers. EFEM analysis showed that Ce and O were located at the same sites as the lignin dots, suggesting that lignin was associated with sources of H₂O₂ generation. The results indicate that the faster H₂O₂ generation and lignification induced by ASM were the potential causes of SAR.     

混合盐碱处理下蚕豆叶片生理指标的变化

为了研究蚕豆在混合盐碱胁迫下的生理变化,试验采用3种盐碱胁迫方式KCl/NaCl、KCl/Na₂CO₃、KCl/Na₂SO₄处理2周,测定蚕豆鲜重、丙二醛和脯氨酸含量、抗氧化酶活性等生理指标。结果表明,随着盐碱胁迫时间的延长,蚕豆鲜重表现为先升高后下降趋势,丙二醛和脯氨酸含量呈上升趋势,其中脯氨酸含量在处理11d时急剧增加;随胁迫时间的延长,4种抗氧化酶SOD、POD、CAT、APX活性增加幅度呈先升高后降低趋势,其中处理11d时,酶活性增加幅度顺序是SOD＞ CAT＞ APX或POD。结论:蚕豆盐碱胁迫中,SOD酶活性发挥首要作用,脯氨酸是一种重要的渗透物质。     

Post infection application of DL-3-amino-butyric acid (BABA) induces multiple forms of resistance against <Emphasis Type="Italic">Bremia lactucae</Emphasis> in lettuce

DL-3-amino-butyric acid (BABA) induces local and systemic resistance against disease in numerous plant species. In a recent study we showed that preventive application of BABA to lettuce (Lactuca sativa) plants induced resistance against downy mildew caused by the oomycete Bremia lactucae by callose encasement of the primary infection structures of the pathogen. Now we show that post-infection application of BABA to the foliage or the roots, even at progressive stages of disease development, is highly protective against B. lactucae. Resistance induced by BABA is manifested in multiple microscopic forms, depending on the time of its application. When applied at 1 day post inoculation (dpi) BABA induced HR in penetrated epidermal cells; at 2 dpi it caused massive encasement with callose of the primary haustoria; and, at 3 or 4 dpi it enhanced the accumulation of H₂O₂ in the developing mycelia runners and altered their colour to red. The pronounced change in the colour of the mycelium was visually apparent to the naked eye. In all cases the pathogen failed to sporulate on the treated plants. This is the first indication that an immunizing compound may be protective at advanced stages of disease development.     

Population structure,races, and host range of <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> from alfalfa production fields in the central USA

Aphanomyces euteiches (races 1 and 2) causes root rot of alfalfa; however, its population biology and distribution are poorly understood where alfalfa is a major crop. The objectives of this study were to (1) characterise the distribution and frequency of races of A. euteiches in Illinois alfalfa fields, (2) determine host range of A. euteiches on cultivated and native legumes, and (iii) to describe genetic diversity and population genetic structure of A. euteiches in alfalfa fields. To accomplish this, soil samples (n = 103) were collected from 30 alfalfa fields in 18 Illinois counties. Using the susceptible cv. ‘Saranac’, 148 isolates of A. euteiches were baited from the soil. The virulence phenotype of isolates representing all 18 counties was tested, and 54% were R1 and 46% were R2. Both races were detected in 61% of the counties, whereas only R1 was detected in 22% and R2 in 17%. Thirteen legume hosts for isolates from alfalfa fields were identified based on symptoms and/or production of oospores in roots. In addition to six previously known hosts, seven species were susceptible to infection: kura clover, purple prairie clover, white prairie clover, ladino clover, hairy vetch, Canadian milk vetch, and Illinois tick trefoil. AFLP analysis revealed high levels of genetic diversity among the isolates from different fields and counties and a lack of genetic structuring of populations based on race or geographical origin. The results suggest that populations of A. euteiches in alfalfa fields are diverse, often composed of races 1 and 2, and create risk for alfalfa and to multiple cultivated and native legume species.     

Tomato partial resistance to Rhizoctonia solani involves antioxidative defense mechanisms

The resistance interactions of four tomato cultivars to five Rhizoctonia solani isolates, causing foot rot, were investigated. Priming H₂O₂ accumulation, peroxidase and ascorbate peroxidase activity was observed in CH Falat, as a partially resistant cultivar. Maximum peroxidase activity in CH Falat was observed at 24 h post inoculation (hpi) which was earlier than maximum phenolics accumulation at 72 hpi. In addition, application of peroxidase inhibitor reduced phenolics level. Therefore, peroxidase might be involved in phenolics production, as an effective resistance mechanism in our pathosystem.     

Natural variation of Medicago truncatula resistance to Aphanomyces euteiches

We analysed the resistance variation in 14 natural populations of Medicago truncatula from Tunisia to Aphanomyces euteiches infection. The reaction of M. truncatula lines to A. euteiches infection varied from susceptibility to full resistance. Of the overall level of phenotypic variation, 65.4 % was found to occur within populations. Principal component analysis showed a high spread of lines belonging to the same population, indicating no clear structure in the Tunisian M. truncatula populations and supporting the hypothesis of gene flow among populations. Likewise, there was no association between local resistance composition and the geographical distances between populations, ruling out isolation by distance as an explanation. Furthermore, significant correlations were observed between quantitative traits and ecological factors consistent with the local adaptation hypothesis. A cluster analysis of the populations showed the presence of three groups. The first group comprised the populations originating from the centre of the country, containing the main resistant lines. The second group included the populations collected in the south and the mountain region of Thala and contained the main partially resistant lines. The third group comprised the populations sampled from the north regions and saline soils and included the main susceptible lines. Overall, we found that the natural M. truncatula lines were more likely to be susceptible (71.3 %) than resistant (28.7 %) to A. euteiches attack. Nevertheless, many resistant lines exhibiting new reaction patterns to A. euteiches attack were identified in the natural populations and these can be used for the identification of potentially new resistance genes.