Changes in ascorbate–glutathione pathway enzymes in response to Mycosphaerella fragariae infection in selected strawberry genotypes

Ten strawberry genotypes, resistant and moderately resistant (Joliette, Seascape, Aromas, FIN005-55 and FIN005-50) and susceptible ones (FIN00132-8, FIN00134-11, FIN00132-14, FIN005-7 and Kent) were used to assess the role of the antioxidative defence system against Mycosphaerella fragariae infection. The pathogen-induced changes of hydrogen peroxide (H₂O₂) and antioxidant enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the ascorbate–glutathione (ASC–GSH) cycle were examined in leaves of the selected genotypes. A significant different response was observed among the genotypes. A marked increase in H₂O₂ content, APX, MDHAR, DHAR and GR activities were observed in resistant and moderately resistant genotypes after inoculation by M. fragariae. In contrast, weak changes were observed in susceptible genotypes for the aforementioned enzymes and compounds. It seems that resistant genotypes capable of overproducing H₂O₂ have a higher capacity to scavenge and reduce the injury to strawberry leaves by regulating the ASC–GSH cycle. The results may be useful in future breeding programmes to select those individuals with high scavenging properties to breed new resistant lines.     

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.     

Cloning of the spoT gene of "Candidatus Phlomobacter fragariae" and development of a PCR-restriction fragment length polymorphism assay for detection of the bacterium in insects

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium "Candidatus Phlomobacter fragariae" is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between "P. fragariae" and other insect-associated proteobacteria, isolation of "P. fragariae" genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from "P. fragariae"-infected strawberry plants. It encodes part of a "P. fragariae" open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish "P. fragariae" from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry "P. fragariae." Interestingly however, the "P. fragariae" spoT sequence could be easily detected in whiteflies proliferating on "P. fragariae"-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.     

Regulation of superoxide dismutase isoforms in resistant and susceptible strawberry cultivars subjected to leaf spot disease

Abstract

Macroscopic symptoms were observed in two strawberry cultivars, with the degree of symptom intensity varying depending on the susceptibility of the cultivars, i.e. resistant or susceptible. The symptoms presented as red spots and were observed 30 d following leaf tissue inoculation with the Mycosphaerella fragariae pathogen. A comparison of the superoxide dismutase isoform profiles obtained by gel electrophoresis in all samples extracted from both resistant and susceptible cultivars indicated one constant sharp band, identified as Mn[sbnd]SOD with a molecular mass of 19 kDa. The intensity of this band was higher in all samples derived from the resistant cultivar than in those from the susceptible cultivar. Another superoxide dismutase (SOD) isoform, identified as CuZn[sbnd]SOD with a molecular mass of 16 kDa, was detected in all soluble proteins derived from the resistant cultivar. This isoform was not observed in the susceptible cultivar; however, following an incremental increase in the amount of loaded protein, it was illuminated as a faint band in a sample collected 3 d after inoculation, indicating insufficient production of the CuZn[sbnd]SOD isoform in the susceptible cultivar during an oxidative burst induced by the M. fragaria pathogen. Several bands were also characterized in both cultivars containing Fe and Mn as their co-factors (Fe, Mn[sbnd]SOD). Unlike in the resistant cultivar, where the activity of Fe, Mn[sbnd]SOD isoforms gradually and regularly increased and reached its highest level on the third day after inoculation, the activity of the isoforms changed irregularly over 20 days of study in the susceptible cultivar.     

Induction of new isoforms of superoxide dismutase and catalase enzymes in the flag leaf of wheat during monocarpic senescence.

Leaf senescence is a programmed cell death phenomenon and involves oxidative stress. Superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT EC 1.11.1.6) activities were studied in the flag leaf of Triticum aestivum cv. Kundan at different stages of grain development. Both SOD and CAT activities showed a decline during monocarpic senescence. Three SOD isozymes were observed in the cytosol, of which one isozyme was observed in the chloroplasts as well. Mitochondria showed the presence of three low abundant SOD isoforms. Inhibitor studies revealed the cytosolic and chloroplastic isoforms to be Cu/Zn SODs. In mitochondria however, two isozymes were MnSOD and one of them appeared to be FeSOD. These isoforms present in the mitochondria increased in activity as senescence progressed. Three isoforms of CAT were observed in peroxisomes which responded differentially during monocarpic senescence. The changes in the kind and pattern of the antioxidant enzymes supported the ordered sequence of events during leaf senescence. This is the first report showing an increase in mitochondrial FeSOD activity during leaf senescence.     

Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in <Emphasis Type="Italic">Phytophthora</Emphasis> blight disease resistance

Assessment of the differential expression of antioxidative enzymes and their isozymes, was done in 30 day-old ex vitro raised plants of three highly resistant (DP-25, Jhankri and Duradim) and one highly susceptible (N-118) genotypes of taro [Colocasia esculenta (L.) Schott]. Antioxidative enzymes were assayed in the ex vitro plants, 7 days after inoculation with the spores (15,000 spores ml⁻¹ water) of Phytophthora colocasiae Raciborski to induce taro leaf blight disease. Uninoculated ex vitro plants in each genotype were used as control. The activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased under induced blight condition when compared with control. Increase in antioxidative enzymes was more (67–92%) in the resistant genotypes than that (21–29%) of the susceptible genotype. The zymograms of SOD and GPX in the resistant genotypes, with pathogenic infection, showed increased activity for anodal isoform of SOD and increased expression and/or induction of either POX 1 or POX 2 isoforms of GPX. In susceptible genotype, expression of the above isoforms was faint for SOD and nearly absent for GPX under both blight free and induced blight conditions. Induction and/or increased activity of particular isoform of SOD and GPX against infection of Phytophthora colocasiae in the resistant genotypes studied led to the apparent conclusion of linkage of isozyme expression with blight resistance in taro. This might be an important criterion in breeding of taro for Phytophthora leaf blight resistance.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae

Background and aims

Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars.

Methods

Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4?h to 7?d post inoculation (pi).

Results

Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues.

Conclusions

This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder.     

Changes in Activities of Antioxidant Enzymes in Sunflower Leaves of Different Ages

Role of superoxide dismutase isozymes and other antioxidant enzymes was studied in relation to leaf age in sunflower (Helianthus annuus L. cv. ACC 1508) at pre-flowering and grain filling stages. Relative water content (RWC) did not change much in leaves of different age and at the two stages. Protein content declined continuously from the youngest to the oldest leaf, while chlorophyll (Chl) and carotenoids (Car) contents increased down to 7^th/9^th leaf and declined in subsequent older leaves. Protein, Chl and Car contents were higher at pre-flowering than at seed filling stage. Superoxide dismutase (SOD), its isozymes, and ascorbate peroxidase (APO) and catalase (CAT) activities were highest in the 9^th leaf and declined in subsequent older leaves. SOD and APO activities were higher at seed filling, except in oldest senescent (13^th, 15^th) leaves. Among SOD isozymes, Cu/Zn-SOD and Mn-SOD activities accounted for most of the total SOD, and only marginal activity was observed for Fe-SOD. Peroxidase activity increased from youngest to the oldest leaf at pre-flowering stage and down to 13^th leaf at seed filling stage. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Pratylenchus penetrans on development of strawberry wilt caused by Verticillium dahliae

The nematode Pratylenchus penetrans or the closely related P. fallax occurred in three out of four strawberry plantations infested with Verticillium dahliae surveyed in Kent. When plants of cv. Cambridge Vigour were inoculated with P. penetrans and V. dahliae together in field ‘micro-plots’ the nematode increased the rate of wilt development except when the concentration of V. dahliae microsclerotia in the soil was very low. In a pot experiment, under conditions not conducive to symptom expression, the incidence of infection was increased by nematodes in Cambridge Favourite, Cambridge Vigour and Redgauntlet; the incidence was least in Red-gauntlet, the most resistant cultivar. The nematode appeared to cause local changes in the root cortex which aided hyphal penetration of the adjacent tissues. Growth of strawberry plants in the absence of V. dahliae was not significantly affected even by large populations of the nematode, but growth was reduced by V. dahliae at all rates of inoculation in the field. Infection by Verticillium did not affect the total numbers of nematode per plant at the end of the experiments, although the number per g fresh weight was greater on the small amount of white root on severely diseased plants than on the larger amount on healthy or mildly diseased plants.     

Impact of reflective mulch on yield of strawberry plants and incidence of damage by tarnished plant bug (Heteroptera: Miridae).

The current study investigated the impact of reflective mulch on yield of strawberry plants and incidence of damage by tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), for three strawberry cultivars: 'Honeoye', 'Earliglow', and two sibling Dayneutrals ('Tribute' and 'Tristar', herein considered as one cultivar). Of all cultivars tested, Honeoye was the most productive and least susceptible to tarnished plant bug. For Earliglow and Honeoye, reflective mulch enhanced productivity of strawberry plants and suppressed density of nymphs per flower cluster and proportion of damaged fruits, but did not significantly impact numbers of nymphs or damaged fruits per hectare, Results with Dayneutrals were not consistently significant. Both in the presence or absence of reflective mulch, proportion of damaged fruits increased with increasing density of nymphs per flower cluster and with decreasing number of fruits harvested per row section, suggesting that planting productive strawberry cultivars or maintaining cultural practices that promote high yield may provide an effective line of defense against tarnished plant bug. These results also suggest that reflective mulch may suppress incidence of damage by tarnished plant bug both directly, by reducing number of nymphs per flower cluster, and indirectly, by enhancing productivity of strawberry plants. Economic analyses evaluating costs and benefits of using reflective mulch, as well as studies investigating mechanisms that underlie the impact of reflective mulch on yield and incidence of damage by tarnished plant bug, are still needed before reflective mulch can be implemented as a management strategy in commercial strawberry fields.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Histological study of the responses of two Vitis vinifera cultivars (resistant and susceptible) to Plasmopara viticola infections

Leaves of Vitis vinifera L. cvs. Chasselas (susceptible) and Solaris (resistant) were inoculated with Plasmopara viticola. Samples were then examined by scanning electron microscopy, light and epifluorescence microscopy. On the resistant cv. Solaris, stomatal deposits, identified as callose, were visible around the germinating zoospores 7 h after inoculation. Twenty-four hours after inoculation, infected stomata exhibited secretions that enveloped the zoospores. At this time, infected stomata were surrounded by necrotic tissues. At 120 h after inoculation, undefined material was deposited on the cuticle in the necrotic areas. Stomata in the vicinity of the infection sites contained callose deposits in and around the stomatal openings, but no necrotic zones were observed. On the sensitive cv. Chasselas neither secretion nor callose deposits were observed. Sporangiophores emerged about 96 h after inoculation and were fully developed 24 h later. Sporulation through small stomata-like apertures present all along the primary vein was also observed on the upper leaf surface. The role of stomatal callose deposits in the defense reactions of the Solaris grapevine cultivar against P. viticola is discussed.     

Superoxide Dismutase Activity and Lipid Peroxidation in Relation to Senescence of Rica Leaves

Superoxide dismutase (SOD), lipid peroxidation, ribulose 1, 5-bisphosphate earboxylasc and chlorophyll content in the leaves of rice were investigated during bloomJug to ripening stages. The results indicated that leaf senescence was associated with the decreascs of SOD and RuBP earboxylase activities and chlorophyll content. A marked increase of malondialdehyde(MDA)contcnt, a produet of lipid pcroxidation was observed during leaf senescence, However, the relative high activities of SOD and RuBP earboxylase and chlorophyll content, lower MDA content in hybrid rice senescenee leaves, compared with their three lines, suggest the presence of physiological hybrid vigor. The analysis of polyacrylamide gel eleetrophoresis showed that three SOD isozymes in leaf extract, which were Cu-Zn SOD. Changes of SOD isozymes were observed during leaf agling and at different time of storage of its extract. The destroying effect of oxygen free radical on enzymes and membrane in the course of leaf senescence was discussed.     

水稻叶片的衰老与超氧物歧化酶活性及脂质过氧化作用的关系

研究了从抽穗开花到籽粒成熟过程中,水稻植株顶部三片叶子的超氧物歧化酶(SOD),脂质过氧化产物丙二醛(MDA)含量及二磷酸核酮糖羧化酶活性的变化。实验结果表明:叶片的衰老伴随着 SOD 活性、RuBP 羧化酶活性及叶绿素含量的降低、丙二醛含量显著增高。分离了三个 SOD 的同工酶,证明为 Cu—Zn SOD。观察了 SOD 同工酶在叶片老化及酶液存放不同时间中的变化。讨论了叶片衰老过程中氧自由基对酶及质膜的损伤影响。     

Susceptibility of strawberry cultivars to the vine weevil Otiorhynchus sulcatus (Coleoptera: Curculionidae)

Abstract 1 The feeding preference of vine weevil, Otiorhynchus sulcatus (Fabricius), adults for foliage from 21 commercial cultivars of strawberries is investigated using binary choice tests with leaf disks, using ‘Honeoye’ as a standard against which all other cultivars are compared. ‘Delmarvel’, ‘Idea’, ‘Lester’, ‘Primetime’ and ‘Seneca’ are not preferred. 2 Variation in leaf nitrogen content is correlated with feeding preference, but does not explain all the variation, because outliers exist for both preferred and nonpreferred cultivars. 3 Removal of leaf hairs with adhesive tape permits their role in deterrence to be evaluated. Eight of the 21 cultivars have deterrent leaf hairs. Paradoxically, some highly preferred cultivars (e.g. ‘Latestar’ and ‘Tristar’) have deterrent leaf hairs, and four of the five nonpreferred cultivars lack significantly deterrent leaf hairs. 4 The 21 tested cultivars do not differ in their suitability for vine weevil larval development. 5 Differences in cultivar susceptibility to vine weevil in the field may involve interactions between the palatability and nutritional value of foliage, which influence fecundity and egg placement, and the tolerance of strawberry plants to larval feeding, determined by root biomass. The nonpreference traits found in the foliage of commercial cultivars of strawberry are present by chance. Accordingly, further selective breeding to enhance strawberry varietal tolerance to vine weevils may be possible.     

Transmission Electron Microscopy of Susceptible and Resistant Tomato Leaves Following Infection with Pseudomonas syringae pv. tomato

Ultrastructural changes in tomato leaves of susceptible cv. Peto 95 and resistant cv. Ontario 7710 infected with Pseudomonas syringae pv. tomato were followed by transmission electron microscopy. Up to 48 hours from the inoculation host cells of both cultivars looked quite normal and no bacteria were visible in the intercellular spaces; bacterial cells were found only in the substomatal chambers. Afterwards, the leaf cells of cv. Peto 95 began to degenerate and bacteria invaded the intercellular spaces which seemed enlarged. After 15 days the disorganization was complete: tomato cells were plasmolyzed and the intercellular spaces were filled with bacteria. In the leaves of resistant cv. Ontario 7710 no bacteria were observed later than 48 hours and no visible modifications occurred up to 15 days after the inoculation.     

Triterpene phytoalexins from strawberry fruit

Strawberry cv. Houkouwase is resistant to infection by Colletotrichum fragariae. The formation of antifungal compounds was observed in unripe fruit which had been wounded and inoculated with conidia of C. musae. Three antifungal compounds were isolated and identified as euscaphic acid, tormentic acid and myrianthic acid. Myrianthic acid inhibited the growth of C. musae at 3 microg, and euscaphic and tormentic acids showed inhibitory effects at 100 microg. A quantitative analysis of their contents showed that the triterpenes increased in wounded fruit, and in wounded and inoculated fruit, but not in non-treated fruit. These findings indicate that unripe fruit of Houkouwase produced the triterpenes as phytoalexins. The triterpene phytoalexins seem to be involved in the resistance of strawberry to the fungus.     

Flower Volatiles,Crop Varieties and Bee Responses

Pollination contributes to an estimated one third of global food production, through both the improvement of the yield and the quality of crops. Volatile compounds emitted by crop flowers mediate plant-pollinator interactions, but differences between crop varieties are still little explored. We investigated whether the visitation of crop flowers is determined by variety-specific flower volatiles using strawberry varieties (Fragaria x ananassa Duchesne) and how this affects the pollination services of the wild bee Osmia bicornis L. Flower volatile compounds of three strawberry varieties were measured via headspace collection. Gas chromatography showed that the three strawberry varieties produced the same volatile compounds but with quantitative differences of the total amount of volatiles and between distinct compounds. Electroantennographic recordings showed that inexperienced females of Osmia bicornis had higher antennal responses to all volatile compounds than to controls of air and paraffin oil, however responses differed between compounds. The variety Sonata was found to emit a total higher level of volatiles and also higher levels of most of the compounds that evoked antennal responses compared with the other varieties Honeoye and Darselect. Sonata also received more flower visits from Osmia bicornis females under field conditions, compared with Honeoye. Our results suggest that differences in the emission of flower volatile compounds among strawberry varieties mediate their attractiveness to females of Osmia bicornis. Since quality and quantity of marketable fruits depend on optimal pollination, a better understanding of the role of flower volatiles in crop production is required and should be considered more closely in crop-variety breeding.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.