Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

Reactive oxygen species (ROS) generation and ROS-induced lipid peroxidation are associated with plasma membrane modifications in host cells in response to AK-toxin I from Alternaria alternata Japanese pear pathotype

AK-toxin I caused plasma membrane modifications with plasma membrane-derived membrane fragments only in sensitive Japanese pear tissues. H₂O₂ generation was abundant in both the membrane fragments and the plasma membranes of the toxin-treated sensitive tissues. Whether lipid peroxidation was induced in plasma membranes of the toxin-treated sensitive tissues was examined biochemically and histochemically. Lipid peroxidation was caused only in the toxin-treated sensitive tissues or the toxin-treated plasma membrane-enriched fractions from sensitive young pear fruits. The results indicated that the peroxidation was probably induced by reactive oxygen species in the modified plasma membranes by action of toxin, suggesting that peroxidation is closely associated with plasma membrane modifications.     

β-1,3-D-Glucan Transported from Golgi Apparatus of Japanese Pear Leaves is a Component of Extracellular Polysaccharides Accumulated after AK-toxin I Treatment

This fluorescence and immunoelectron microscopic study showed that β-1,3-D-glucan accumulated only in leaves of a susceptible cultivar of Japanese pear after treatment with a host-specific toxin, AK-toxin I, from Alternate, alternata Japanese pear pathotype. The positive fluorescent reaction of callose was detected only in aniline blue fluorochrome-stained sections from toxin-treated leaves of the susceptible cultivar: positive sites were observed on cell walls of leaf cells. The sites of callose deposition were probably consistent spatially with modified sites on the plasma membrane that were observed only in the toxin-treated leaves of the susceptible cultivar. The toxin-induced modifications, identified as damage to the plasma membrane, were characterized by invagination of the plasmalemma specifically at plasmodesmata and as the concomitant accumulation of extracellular polysaccharides at the invaginated sites. A positive reaction to anti-β-1,3-D-glucan antibody was detected at the polysaccharides, Golgi vesicles, and trans-Golgi network (TGN) of toxin-treated leaves of the susceptible cultivar, but not at Golgi vesicles and TGN of water-treated ones. The cis-, medial and trans-Golgi stacks of toxin-treated leaves of the susceptible cultivar were negative for the antibody. The results showed that the polysaccharides, Golgi vesicles and TGN contained abundant β-1,3-D-glucan and that the glucan was transported from the Golgi apparatus via Golgi vesicles to the modified sites in cells of toxin-treated leaves of the susceptible cultivar. Received 7 March 2002/ Accepted in revised form 10 June 2002     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

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

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

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

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

BTH处理对甜瓜叶片活性氧代谢的诱导作用

为了明确活性氧（reactive oxygen species,ROS）代谢在甜瓜抗病性诱导中的作用,以抗白粉病甜瓜品种Tam Dew和感病品种卡拉克赛幼苗为材料,通过盆栽试验研究了苯丙噻二唑（BTH）喷雾或白粉菌接种后甜瓜叶片超氧阴离子（O2.-）产生速率、过氧化氢（H2O2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、苯丙氨酸解氨酶（PAL）活性的变化。BTH处理或白粉菌接种均可诱导甜瓜叶片SOD、PAL活性升高,抑制CAT活性,导致叶组织O2.-产生速率和H2O2含量增加,BTH喷雾＋白粉菌接种比二者单独处理效果更好。结果表明,BTH处理后叶片O2.-产生速率提高和H2O2积累是甜瓜抗白粉病能力提高的重要机制,BTH通过诱导ROS代谢酶活性调节H2O2含量,且BTH诱导的甜瓜抗病性与品种的基础抗性有关。     

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.     

Physiological effects of the hydrophilic phytotoxins produced by <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, the causal agent of black sigatoka in banana plants

Although Mycosphaerella fijiensis, the causal agent of black sigatoka disease of banana, has been known to produce numerous lipophilic host-selective (HSTs) and nonhost selective phytotoxins (non-HSTs), only recently we have reported that the pathogen also produces hydrophilic phytotoxins. Here we examined the effect of light on the toxicity of the hydrophilic phytotoxins and estimated the electrolyte leakage and H₂O₂ and superoxide generation in detached banana leaves to study their mode of action at the cellular level. Nonhost plant species were also tested to determine whether the toxins are HSTs or non-HSTs. Our results suggest that the hydrophilic phytotoxins are non-HSTs, that their phytotoxicity is not light dependent, and that they may act at the plasma membrane by altering permeability through oxidative damage, by inducing ROS production as part of their mechanism of action.     

Effect of mal secco toxin on lemon leaf cells

Leaves of Rough lemon (Citrus jambhiri) were inoculated withPhoma tracheiphila or treated with mal secco toxin. The ultrastructural changes in toxin-treated leaves resembled closely those infected by the pathogen, being characterized by separation of the plasmalemma from the cell wall, vesiculation of plasmalemma, disruption of the chloroplast membrane, disorganization of thylakoids, and increase in size and number of osmiophilic droplets in the chloroplasts. The rate of photo-synthetic CO₂ fixation by mesophyll protoplasts was reduced in the presence of the toxin. The toxin induced agglutination of mesophyll protoplasts of lemon only in the presence of an antiserum to the toxin. Agglutination of protoplasts was independent of the biological activity of the toxin and was inhibited by Dmannose or after mild base hydrolysis of the toxin.     

Effect of SS-toxin,a metabolite of <Emphasis Type="Italic">Stemphylium solani</Emphasis>, on H<Superscript>+</Superscript>-ATPase activity and standard redox system in plasma membranes from seedlings leaves of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>)

Leaf blight of garlic (Allium sativum) is a severe disease in garlic-growing regions. SS-toxin is a newly described non-proteinaceous toxin produced by the phytopathogenic fungus, Stemphylium solani, the cause of garlic leaf blight. In this study, the effects of SS-toxin on the H⁺-ATPase activity and standard redox activity in the plasma membranes isolated by aqueous polymer two-phase partitioning from garlic seedling leaves were studied in vitro. The H⁺-ATPase activity, NADH oxidation rate and Fe(CN)₆³⁻ reduction rate of the plasma membranes isolated from susceptible and resistant cultivars were all inhibited in a dose-dependent manner. Our results suggest that, under in vitro conditions, the plasma membrane H⁺-ATPase and standard redox system can be both the cellular targets of SS-toxin.     

向日葵与锈菌互作过程中活性氧的积累

为了探讨向日葵品种与锈菌互作中活性氧的产生和积累与向日葵抗锈病性的关系,采用分光光度计法及联苯胺蓝(DAB)、氮兰四唑(NBT)染色法对过氧化氢(H2O2)及超氧阴离子自由基(O2-)诱导积累的过程进行了检测.结果表明:接种后抗、感病品种均出现H2O2和O2-双峰,侵染早期积累明显,最高峰出现在16 h,在抗病品种中活性氧产生和积累明显高于感病品种;在抗病品种中侵染位点活性氧的产生及积累较明显,接种后16h,侵染位点周围的染色范围较大,染色较深,H2O2及O2-的染色比例均达到最高,分别为65.5%和41%;而在感病品种的侵染位点没有检测到明显的活性氧积累.     

Induced defense dynamics in plant parts is requisite for resistance to <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hubner) infestation in chickpea

Helicoverpa armigera is the most serious insect pest in chickpea that causes significant yield losses due to its feeding on vegetative (leaves) and reproductive (developing pods and seeds) parts of plants. The present aim of study was to explore response dynamics of induced defence mechanism in leaves, podwall and seeds of ten chickpea genotypes (ICC 506, ICCV 10, ICC 10393, 5283, RSG 963, GL 25016, GL 26054, ICCL 86111, ICC 3137, L 550) after insect infestation. Two chickpea genotypes namely ICC 3137 and L 550 were found to be highly susceptible to Helicoverpa armigera infestation due to higher leaf and pod damage in them as compared to rest of eight genotypes which are found to be considerably resistant due to lower damage. Insect infestation induced decreased activities of defensive enzymes such as peroxidase (POD), catalase (CAT), glutatione reductase (GR) and polyphenol oxidase (PPO), decreased free radical scavenging activities in terms of 2,2-diphenyl-1-picryl hydrazyl (DPPH), decreased contents of signaling molecules such as nitric oxide ((NO), hydrogen peroxide (H₂O₂), reduced content of insect feeding behaviour regulating molecules such as total phenols, trypsin inhibitor and accumulation of membrane damage marker such as malondialdehyde (MDA) in leaves of ICC 3137 and L 550; decreased POD activity, nitric oxide content and H₂O₂ in podwall of L550; decreased SOD, GR, nitric oxide content and H₂O₂ in seeds of L550 resulted in aggravation of infestation induced oxidative stress and makes these genotypes more vulnerable to insect damage. The resistance of rest eight chickpea genotypes to insect infestation was due to the integrative effect of up regulated defensive components in leaves, podwall and seeds such as enhanced activities of CAT, POD, GR, PPO and PAL along with accumulation of H₂O₂` and total phenols in leaves, increased SOD, POD, GR and PPO activities along with increased contents of trypsin inhibitor and total phenols in podwall; increased SOD, GR, PPO activities and accumulated total phenols in seeds of resistant chickpea genotypes might be responsible for causing significant shift in oxidative status of these genotypes due to scavenging of free radicals, maintenance of membrane integrity and deterrent to insect feeding. Induced glycine betaine after herbivory was found to be positively correlated with superoxide dismutase and trypsin inhibitors. H₂O₂ content was positively correlated with trypsin inhibitor, DPPH, ferric reducing antioxidant power (FRAP) and total phenols in leaves and with FRAP, DPPH and total phenols in pod wall indicating that H₂O₂ might be stimulating the cascade that will be helping to scavenge free radical species and correlation with phenols and trypsin inhibitor indicated that it act as toxicant to insect feeding.     

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.     

<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.     

Cellular reactions in Arabidopsis following challenge by strains of Pseudomonas syringae: From basal resistance to compatibility

Reactions to strains of the bean halo-blight pathogen Pseudomonas syringae pv. phaseolicola (Pph) strain 1448A and the Arabidopsis and tomato pathogen P.s. pv. tomato (Pst) strain DC3000 were examined by transmission electron microscopy. Wild-type and hrpL mutant strains of Pph failed to multiply in the accession Columbia (Col)-5, but did not cause a hypersensitive reaction (HR). Symptomless non-host resistance to 1448A and the hrpL mutant was associated with the progressive alteration of the plant cell wall adjacent to bacteria, following the accumulation of membrane bound vesicles within the cytoplasm at reaction sites. Large papillae containing callose accumulated within challenged plant cells. Papillae also formed in the pmr4–1 mutant of Col-0 which lacks an inducible callose synthase but immunocytochemical labelling demonstrated that they contained very little β-1, 3 glucan. Some papillae formed in Col-5 in response to the virulent pathogen DC3000, but they dispersed during cell collapse and lesion formation. Transconjugants of Pph expressing the avirulence genes avrPpiA and avrPphB matching the RPM1 and RPS5 resistance genes, caused rapid and slow HR development, respectively. Although corpse morphology was observed our observations suggest that in Arabidopsis, plant cell death during the HR is programmed but represents a variant of necrosis rather than apoptosis. Cerium chloride staining revealed the accumulation of H₂O₂ at reaction sites. The strongest H₂O₂ response was found during the HR activated by avrPpiA but localised generation of peroxide was also found at sites of papilla deposition next to 1448A or the hrpL mutant. Accumulation of H₂O₂ during the HR, but not during wall alterations, was strongly suppressed by inhibition of NADPH oxidase. The differential effect of the inhibitor suggests an alternative source of H₂O₂ to modify the plant wall. Extension of peroxide-driven cross-linking reactions to bacterial cell walls may contribute to the restriction of bacterial multiplication. The lowest level of H₂O₂ occurred during the compatible reaction to DC3000. Characterisation of the cellular co-ordination of basal (non-host) resistance has revealed several potential targets for bacterial effector proteins.     

Mycorrhizal protection of chili plants challenged by <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Hydrogen peroxide (H₂O₂) has been implicated in many stress conditions. Control of H₂O₂ levels is complex and dissection of mechanisms generating and relieving H₂O₂ stress is difficult, particularly in intact plants. Here the role of the mycorrhizal inoculation in chili plants challenged with Phytophthora capsici was investigated to study the effect on hypersensitive response. In the treatment without mycorrhiza (treatment T3) and with mycorrhiza (considered treatment T4) visible disorders were detected two days after inoculation with P. capsici, but in the next days T3 plants rapidly developed 25% more necrotic lesions on the leaves than T4 plants. Leaf necrosis correlated with H₂O₂ accumulation and the greater damage observed in T3 plants coincided with larger accumulation of H₂O₂ after 12 h of inoculation accompanied with an increase in POX (peroxidase) and SOD (superoxide dismutase) activity. T4-infected and mycorrhizal plants exhibited an earlier accumulation of H₂O₂ starting 6 h after inoculation with lower levels compared to T3 plants. Correlated with observed damage, POX and SOD activity measured in T4 plants indirectly suggest a smaller accumulation of ROS (reactive oxygen species) leading to a decrease in the wounds observed and slightly diminishing the advance of the pathogen. According to these findings, we conclude that mycorrhizal colonization contributes significantly in maintaining the redox balance during oxidative stress, but the exact mechanism is still uncertain.