Hydrogen peroxide scavenging mechanisms are components of Medicago truncatula partial resistance to Aphanomyces euteiches

The biochemical processes underlying the expression of resistance in the roots of Medicago truncatula against Aphanomyces euteiches infection was investigated, with emphasis on oxidative stress. The levels of H₂O₂, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, soluble phenolics and lignin were measured in the roots of two lines, A17 partially resistant and F83005.5 susceptible to A. euteiches at three infection stages; penetration of the epidermis (1 dpi), colonization of the cortex (3 dpi) and invasion of the root stele (6 dpi). A rapid and large decrease of the H₂O₂ levels in A17 roots occurred. However, in F83005.5 roots, the decrease in H₂O₂ levels was delayed until 3 dpi. In A17 roots, the activities of ascorbate peroxidase, peroxidase and catalase were induced as early as 1 dpi, whereas a general decrease in the activity of the four antioxidant enzymes was observed in F83005.5 roots. The levels of soluble phenolics and lignin were increased in A17 roots at 3 and 6 dpi, respectively. The H₂O₂ levels were negatively correlated to ascorbate peroxidase, catalase and lignin production at 1, 3 and 6 dpi, respectively in A17 roots. Physiological concentrations of H₂O₂ found in M. truncatula infected roots had no detrimental effect on the in vitro growth of this oomycete. Our data suggest that H₂O₂ does not have a direct antimicrobial effect on M. truncatula resistance to A. euteiches, but is involved in cell wall strengthening around the root stele, preventing pathogen invasion of the vascular tissues.     

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.     

Silicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrier

A series of experiments were performed to study the effects of silicon (Si) on rice blast development, H₂O₂ accumulation and lipid peroxidation in a controlled rice—Magnaporthe grisea pathosystem. Rice plants supplied with Si as a single dose immediately after pathogen inoculation (−/+Si) exhibited the same high protection against disease as plants treated continuously with Si for the whole growth period (+/+Si), with disease severity indices of 20.8% and 19.6%, respectively, which were significantly lower than that for the control treatment with no Si supplied (63.7%). A single application of Si to rice plants before inoculation (+/−Si) conferred partial protection (disease severity index of 33.3%) compared with the control treatment. Silicon induced a rapid but transient burst of H₂O₂ at 24 h after inoculation. The addition of Si to rice plants significantly altered the activities of catalase and lipoxygenase and the concentration of malodialdehyde (indicative of lipid peroxidation) in rice plants. We propose that rice plants may respond to Si by increased H₂O₂ accumulation and lipid peroxidation. In turn, these responses are linked to host defence mechanisms such as lignin production, oxidative cross-linking in the cell wall, phytoalexin production, and the hypersensitive reaction. Thus, the mechanisms of Si-stimulated plant disease protection may extend beyond its established role in physically strengthening cell walls.     

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.     

Avirulent strain of Colletotrichum induces a systemic resistance in strawberry

Strawberry plants exposed to an avirulent isolate of Colletotrichum fragariae acquired strong resistance against a virulent strain of C. acutatum. Biochemical, morphological and molecular markers indicated that the strong defence response was associated with an oxidative burst and a transient accumulation of salicylic acid (SA). A maximum accumulation of H₂O₂ and O₂ ^? was observed 8 h after inoculation (hai), callose was detected 48 hai, and a peak of SA was observed 48 hai. Biochemical and phytopathogenic analyses carried out in non-treated tissues revealed that the defence response was systemic and remained fully active 60 days after the first inoculation. Experiments also showed that the resistance acquired by mother plants after the inoculation with the avirulent isolate could be passed to daughter plants through runners. Further characterization of the induced systemic resistance showed that the resistance was not only effective against a virulent strain of C. acutatum but also against Botrytis cinerea.     

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.     

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.     

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.     

Purification of an infection-related acidic peroxidase from pearl millet seedlings

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE–Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS–PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27–60 °C). The K_m for H₂O₂ and V_max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl₂ dependent and both MgCl₂ and ZnCl₂ showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.     

拟南芥转录因子IDD4不同突变体对灰葡萄孢菌的抗性分析

为明确IDD家族IDD4基因在拟南芥Arabidopsis thaliana抵抗灰葡萄孢菌Botrytis cinerea侵染过程中的作用,通过统计病情指数检测拟南芥野生型（wild type,WT）植株、过表达植株IDD4-OE和缺失突变体idd4植株感染灰葡萄孢菌情况,利用组织染色检测叶片细胞死亡和H₂O₂的积累情况,采用实时荧光定量PCR（real-time quantitative-PT-PCR,qRT-PCR）技术分析灰葡萄孢菌肌动蛋白基因Bc. ACTIN在3种植株叶片中的表达情况,并施加0.1 mmol/L外源水杨酸（salicylic acid,SA）后测定IDD4-OE植株的病情指数。结果显示,不同株系对灰葡萄孢菌的抗性由高到低依次为idd4＞WT＞IDD4-OE,IDD4-OE植株中病原菌感染部位的寄主细胞死亡程度比idd4植株严重。染色结果表明,病原菌侵染拟南芥后4 h,接种部位已有H₂O₂积累。qRT-PCR反应结果显示,Bc. ACTIN在IDD4-OE中比在idd4植株中的表达水平更高,表明灰葡萄孢菌在IDD4-OE植株中的繁殖速率更快。对IDD4-OE植株外源施加SA后,其病情指数、Bc. ACTIN表达量与WT植株间均无显著差异,说明SA能将感病植株的抗性提高至WT植株的水平,表明IDD4作为负调控因子参与了拟南芥对灰葡萄孢菌的抗性调控,SA在其中发挥着重要作用。     

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H₂O₂. A small amount of non-specific generation of H₂O₂ was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H₂O₂ at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H₂O₂ at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H₂O₂ generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane. Received 21 September 2001/ Accepted in revised form 25 October 2001     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>- mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defence against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Trichoderma harzianum is an effective biocontrol agent against the devastating plant pathogen Rhizoctonia solani. Despite its wide application in agriculture, the mechanisms of biocontrol are not yet fully understood. Mycoparasitism and antibiosis are suggested, but may not be sole cause of disease reduction. In the present study, we investigated the role of oxidant-antioxidant metabolites in the root apoplast of sunflower challenged by R. solani in the presence/absence of T. harzianum NBRI-1055. Analysis of oxidative stress response revealed a reduction in hydroxyl radical concentration (^•OH; 3.6 times) at 9 days after pathogen inoculation (dapi), superoxide anion radical concentration (O₂^•−; 4.1 times) at 8 dapi and hydrogen peroxide concentration (H₂O₂; 2.7 times) levels at 7 dapi in plants treated with spent maize-cob formulation of T. harzianum NBRI-1055 (MCFT), as compared to pathogen-inoculated plants. The protection afforded by the biocontrol agent was associated with the accumulation of the ROS gene network: the catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and ascorbate peroxidase (APx), maximum activity of CAT (11.0 times) was observed at 8 dapi, SOD (7.0 times) at 7 dapi, GPx (5.4 times) and APx (8.1 times) at 7 dapi in MCFT-treated plants challenged with the pathogen. This was further supported by the inhibition of lipid and protein oxidation in Trichoderma-inoculated plants. MCFT stimulated the accumulation of secondary metabolites of phenolic nature that increased up to five-fold and also exhibited strong antioxidant activity at 8 dapi, eventually leading to the systemic accumulation of phytoalexins. These results suggest that T. harzianum–mediated biocontrol may be related to alleviating R. solani-induced oxidative stress.     

The effect of hydrogen peroxide on the viability of tomato cells and of the fungal pathogen Cladosporium fulvum

An oxidative burst was previously demonstrated to be induced in tomato plants by race specific elicitors of the fungal pathogen Cladosporium fulvum . The in planta levels of H₂O₂estimated to occur during elicitor treatment, were compared with the levels required to show toxicity to host cells and to the fungal pathogen. Injection of Cf-9 tomato leaves with 100 m m H₂O₂caused an insignificant degree of necrosis and 1m H₂O₂was required to cause complete leaf necrosis comparable to that induced by the AVR9 elicitor. Assays with Cf-5 tomato cell suspensions confirmed the low toxicity of H₂O₂to tomato cells but, as expected, the addition of Fe²⁺with H₂O₂(or with intercellular fluids containing AVR5 elicitor) enhanced cell death as determined by the Evans Blue assay. Germination and germ tube growth of conidia of C. fulvum were significantly retarded by 4–5 m m H₂O₂, and at higher concentrations, death of germ tubes was observed (ED₅₀=22 m m), as determined by the fluorescein diacetate assay. The addition of Fe²⁺with H₂O₂had little effect on fungal growth or viability in vitro . These results suggest that the amount of H₂O₂accumulating during an elicitor-induced response in leaves may be sufficient to affect fungal colonization but not to affect viability of host cells unless the Fe²⁺status in the apoplast is in some way altered by the elicitor to facilitate OH^.production via the Fenton reaction.     

Physiological response of Bacillus cereus C1L-induced systemic resistance in lily against Botrytis leaf blight

Bacillus cereus C1L has been demonstrated to induce systemic disease resistance against Botrytis elliptica in lily. The objective of this study was to investigate physiological responses of B. cereus C1L-triggered systemic resistance in lily cv. Star Gazer against B. elliptica. By histological and biochemical analyses, leaves inoculated with B. elliptica displayed cell death, H₂O₂ accumulation and lignin deposition. As plants were elicited with B. cereus C1L, cell death, H₂O₂ accumulation and lignin deposition in leaves caused by B. elliptica infection were suppressed, revealing that suppression of oxidative burst might be associated with B. cereus C1L-induced systemic resistance. In reactive oxygen species inhibitors assays, B. elliptica-caused lesion numbers and H₂O₂ accumulation in lily leaves were significantly reduced as leaves were pretreated with catalase or diphenylene iodonium. Furthermore, the expression of LsGRP1 and LsPsbR in leaves elicited with B. cereus C1L and inoculated with B. elliptica was decreased. The same expression pattern was also observed in leaves pretreated with catalase or diphenylene iodonium and inoculated with B. elliptica. These results suggest that B. cereus C1L-induced systemic resistance may be related to suppression or alleviation of oxidative stress and cell death of lily caused by B. elliptica.     

The effect of salicylic and jasmonic acids on tomato physiology and tolerance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV)

Cucumber mosaic virus (CMV) is one of the most important plant viruses responsible for sharp reductions in the production of many cultivated plants. Activities of antioxidant enzymes, photosynthetic capacity, proline and total soluble carbohydrates (TSC) content were measured in the leaves of tomato (Solanum lycopercicum cv. Falat) plants treated with phytohormones (salicylic and jasmonic acids and their combination) and inoculated with CMV at 0, 1, 2, 4, 6, 8, and 15 days after the treatments. Based on the results, catalase (CAT) activity decreased in the healthy and infected plants, but peroxidase (POD) activity increased in the CMV-infected plants signifying that POD is more active in H₂O₂ scavenging in tomato. Because the hormone treatments inhibited the reduction in the enzyme activity, it may be considered as a controlling method against CMV. Superoxide dismutase (SOD) activity was lower in the control until 6 days post inoculation (dpi), but increased after 8 dpi. The infected plants and the hormone-treated plants showed an increased SOD activity from 0 to 15 dpi. Phenylalanine ammonia lyase (PAL) activity also increased in all the treatments over the time period (0-15 dpi). Net photosynthesis (NP) rate and chlorophyll content decreased under the virus infection and hormone treatment, whereas control plants had the highest NP and chlorophyll content. Proline accumulation occurred in the infected and hormone- treated plants, but TSC content decreased in comparison to the control. Reduction of TSC content was not significant in the hormone and virus- treated plants. Expression of CMV coat protein gene (CMV-CP) was decreased by approximately 34% in SA+JA+CMV treatment in comparison to the CMV-infected plants. In conclusion, CMV had harmful effect on physiological traits of tomato plants, but hormone application induced resistance. This resistance may be accomplished through the combination of both hormone-related signaling pathways which likely established a strong resistance network together.     

Overexpression of StRbohA in Arabidopsis thaliana enhances defence responses against Verticillium dahliae

Plant NADPH oxidases are key regulators of plant–microbe interactions and reactive oxygen species (ROS) are essential to plant defences against pathogens. A significant part in the role played by ROS has been ascribed to plant respiratory burst oxidase homologs (RBOHs). In potato (Solanum tuberosum), where RBOHs were previously shown to be involved in wound-induced oxidative burst, we assessed their expression after inoculation with Verticillium dahliae Kleb. and showed that StRbohA was the only homolog to be differentially induced in potato in response to inoculation. In order to investigate the potential role of this gene in plant protection against wilt diseases, we used Agrobacterium-mediated transformation of Arabidopsis to assess the effects of its overexpression on plant responses to V. dahliae. After inoculation with this pathogen, the transformed Arabidopsis line overexpressing StRbohA showed lower disease severity (percent damaged leaf area and vascular discoloration) as compared to the wild type. It also had higher ROS production and more cell death caused by hydrogen peroxide (H₂O₂), compared to the wild type. Suberization of root cells was also more pronounced in the line overexpressing StRbohA, and supports a possible role for StRBOHA in plant resistance to V. dahliae. Together, these findings indicate that overexpressed StRbohA in Arabidopsis enhances the ROS-mediated defence mechanisms against V. dahliae and can be a potential tool to improve plant resistance to this and other soilborne pathogens that cause wilts in economically important crops.     

Effect of temperature on resistance to Potato virus Y in potato cultivars carrying the resistance gene Rychc

The effect of cultivation temperatures on the resistance reaction to three Potato virus Y strains (PVY^O, PVY^N and PVY^NTN) in potato cultivars carrying Ry_chc was examined. When potato plants carrying Ry_chc were cultivated at 22 °C, a few small necrotic spots developed on inoculated leaves by 5 days after mechanical inoculation (dpi), and systemic infection of a few symptomless plants was confirmed at 28 dpi by IC‐RT‐PCR. At 28 °C, distinct necrotic spots developed on inoculated leaves by 5 dpi, and systemic symptoms occasionally appeared at 28 dpi. Thus, high temperature weakens Ry_chc‐conferred resistance. However, the incidence of systemic infection and the titre of virus in resistant cultivars at 28 °C were lower than in a susceptible cultivar. In graft inoculation under high summer temperatures, some plants developed necrosis on the leaves and stem, but PVY was barely detected by RT‐PCR in leaves on potato carrying Ry_chc. When seedlings from progeny tubers of plants that were inoculated with PVY and grown in a greenhouse at >30 °C in the daytime were examined by ELISA and IC‐RT‐PCR, PVY was not detected in cultivars carrying Ry_chc. These results show that Ry_chc confers an extreme resistance to PVY strains occurring in Japan.