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.     

Arachidonic acid-induced hypersensitive cell death as an assay of downy mildew resistance in pearl millet

Arachidonic acid (AA) induces hypersensitive response (HR) on coleoptile/root regions of two-day-old pearl millet seedlings. The response is comparable to the HR induced by the downy mildew pathogen, Sclerospora graminicola. A time gap in the appearance of cell necrosis among genotypes of pearl millet was related to the degree of resistance to downy mildew. Based on the time required for the development of necrotic spots induced by AA, the pearl millet genotypes were categorised as highly resistant/resistant (HR in 3–6 h), susceptible (HR in 7–12 h) and highly susceptible (HR in 13 h and above). The percentage disease incidence in each genotype was compared with the time required for the development of AA-induced HR. The appearance of hypersensitive cell necrosis was rapid in genotypes having high resistance to downy mildew and was slow in genotypes with high susceptibility. This simple method of screening various pearl millet genotypes in the absence of the pathogen aids in identifying the downy mildew resistant/susceptible host cultivars without the risk of introducing the virulent race of the pathogen.     

The Possible Involvement of Lipoxygenase in Downy Mildew Resistance in Pearl Millet

The downy mildew disease, incited by Sclerospora graminicola,is a major biotic constraint for pearl millet production inthe semi-arid tropics. Sources of resistance to this diseasehave been identified. However, the mechanism of host resistancestill remains obscure. The enzyme lipoxygenase (LOX) is knownto play a role in disease resistance in many host-pathosystems.In the present study, LOX activity was tested in seeds of differentgenotypes of pearl millet with different susceptibility to downymildew. The LOX assay of the seeds indicated a good correlationbetween enzyme activity and their downy mildew reaction in thefield. Maximum activity was recorded in seeds of highly resistantgenotypes and minimum activity was found in the highly susceptiblegenotypes. Seeds obtained from plants recovered from the downymildew disease had more LOX activity than that of the originalparent seeds. Thus, in seeds, the LOX activity can be used asa biochemical marker for screening different genotypes of pearlmillet for downy mildew. The study, carried out in the susceptiblegenotype of pearl millet seedlings, showed that LOX activitydecreased after inoculating with S. graminicola zoospores whencompared with uninoculated controls. However, a significantincrease in the enzyme activity was observed on the second andthird days after inoculation in resistant seedlings. The possiblerole of LOX in conferring resistance to downy mildew infectionof pearl millet is discussed. Key words: Lipoxygenase, pearl millet, downy mildew     

Comparative analysis of activities of vital defence enzymes during induction of resistance in pearl millet against downy mildew

Pearl millet [Pennisetum glaucum (L.) R. Br.] has the seventh largest annual production in the world giving it significant economic importance. Although generally well adapted to the growing conditions in arid and semi-arid regions, major constraints to yields are susceptibility to downy mildew disease caused by the oomycete Sclerospora graminicola (Sacc.) Schroet. Induction of resistance against downy mildew disease of pearl millet has been well established using various biotic and abiotic inducers. The present study demonstrated the comparative analysis of the involvement of the important defence enzymes like β-1,3-Glucanase, chitinase, phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and lipoxygenase (LOX) during induced systemic resistance (ISR) mediated by inducers like Benzo(1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (BTH), Beta amino butyric acid (BABA), Chitosan and Cerebroside against pearl millet downy mildew disease. Native-PAGE showed six POX isozymes in all categories of uninoculated pearl millet seedlings and maximum intensity of bands was noticed in resistant seedlings. After inoculation in Cerebroside-treated seedlings, there were seven isoforms, POX-4 was not present in any other seedlings. Native-PAGE analysis showed the presence of five PPO isozymes in all categories of uninoculated pearl millet seedlings and after inoculation seven isoforms of PPO-7 were noticed, and the intensity of banding was more in resistant and Cerebroside-treated seedlings. The isoforms PPO-3 were present as an extra band after inoculation in all seedlings. Isoform PPO-7, though found in all seedlings, was very prominent in Chitosan- and Cerebroside-treated seedlings. β-1,3-Glucanase Native-PAGE analysis showed the presence of only one isozyme in all categories of uninoculated/inoculated pearl millet seedlings. Glu-1 isozyme was very prominent in all seedlings including resistant and susceptible seedlings. Among the induced resistant seedlings, highest intensity was observed in Cerebroside-treated seedlings. Native-PAGE analysis showed the presence of three LOX isozymes in all categories of uninoculated pearl millet seedlings, and the intensity of banding pattern was very low in BTH-treated seedlings. LOX-1 and LOX-2 were very prominent in resistant, Chitosan- and Cerebroside-treated seedlings. Upon inoculation, one extra band, LOX-3, was exclusively noticed in Cerebroside-treated seedlings. In inoculated seedlings, LOX-1, LOX-2 and LOX-4 were very prominent in Chitosan Cerebroside-treated seedlings compared to other seedlings.     

Sclerospora graminicola- and arachidonic acid-induced autofluorescence in downy mildew resistant and susceptible genotypes of pearl millet

Autofluorescence of downy mildew resistant and susceptible cells of pearl millet seedlings undergoing hypersensitive reaction (HR) upon Sclerospora graminicola-inoculation and arachidonic acid (AA)-treatment was studied. Two-day-old seedlings of a highly resistant (IP 18296) and a highly susceptible (23D₂B) genotype of pearl millet were either inoculated with zoospore suspension of S. graminicola or treated with AA for 24 h. The coleoptiles with hypersensitive necrotic spots were processed by the standard procedure, and the tissues were subjected to fluorescence microscopy. A differential accumulation of autofluor-escent compounds in resistant and susceptible pearl millet genotypes was observed with most accumulation occurring in resistant cells treated with AA. The variation in the degree of fluorescence and the spatial accumulation of autofluorescent compounds among the two inoculated/treated genotypes is discussed.     

Biochemical events involved in downy mildew disease resistance in pearl millet in relation to H+-ATPase

Pearl millet (Pennisetum glaucum L. Br.) is the most important crop in India and Africa. Downy mildew disease of pearl millet caused by the oomycetous fungus Sclerospora graminicola (Sacc.) Schroet., is the major biological constraint in the production of pearl millet. Plasma membrane H⁺-ATPase is induced in resistant pearl millet against downy mildew pathogen. Sodium orthovanadate, an inhibitor of H⁺-ATPase, was used in this study to understand its effect on other known defence responses in pearl millet including H⁺-ATPase. Results suggest that vanadate down-regulates all defence responses tested, such as H⁺-ATPase (53 ± 5.0%), peroxidase (36 ± 5.6%), phenylalanine ammonia lyase (43 ± 4.5%), β-1,3 glucanase (25 ± 4.2%), lytic activity (32 ± 3.0%), hypersensitive response (57 ± 4.3%) and pathogen colonisation. These data indicate that the plasma membrane H⁺-ATPase may be a key step in the signaling pathway leading to defence activation in pearl millet against downy mildew disease.     

Detection of Genetic Variability in Pearl Millet Downy Mildew (Sclerospora graminicola) by AFLP

Downy mildew, caused by Sclerospora graminicola, is an economically important disease of pearl millet in the semiarid regions of Asia and Africa. Amplified restriction fragment length polymorphism (AFLP) was used to detect the extent of genomic variation among 19 fungal isolates from different cultivars of pearl millet grown in various regions of India. Fourteen AFLP primer combinations produced 184 polymorphic bands. An unweighted pair-group method of averages cluster analysis represented by dendrogram and principal coordinate analysis separated the mildew collections into four distinct groups. Isolates having characteristic opposite mating abilities, geographic relatedness, virulence, common host cultivars, and changes through asexual generations reflected heterogeneity of the pathogen. The use of AFLP to detect genetic variation is particularly important in selecting mildew isolates to screen breeding material for identification of resistant millet and monitoring changes in S. graminicola in relation to changes in host for effective disease management.     

Histo-chemical changes induced by PGPR during induction of resistance in pearl millet against downy mildew disease

Plant growth-promoting rhizobacteria Bacillus pumilus strain INR-7 effectively induced downy mildew resistance in pearl millet. The histo-chemical analysis of B. pumilus INR-7 mediated systemic resistance showed that induced resistance is associated with the expression of hypersensitive response (HR), enhanced lignification, callose deposition, and hydrogen peroxide in addition to the increased expression of the defense enzymes β-1,3-glucanase, chitinase, phenylalanine ammonia lyase (PAL), peroxidase (POX), and polyphenol oxidase (PPO). There was rapid expression of HR in the resistant pearl millet as well as the susceptible seedlings induced by treatment with INR-7 after pathogen infection when compared to the susceptible seedlings, which expressed HR at later hours. Examination of inoculated pearl millet tissues by microscopy showed that lignin, callose, and hydrogen peroxide accumulated earlier and to higher levels in resistant and induced resistant seedlings. Accumulation of various defense enzymes was an immediate response to Sclerospora graminicola infection and preceded the development of induced resistance elicited by strain INR-7. Tissue print analysis showed that defense enzymes were found to be localized in the vascular bundles and revealed the visual difference in the expression pattern of β-1,3-glucanase, chitinase, PAL, POX, and PPO whose intensity varied among resistant, INR-7 treated, and susceptible pearl millet seedlings. This study clearly demonstrated that the differences between the responses, susceptible, INR-7 treated or resistant pearl millet seedlings recorded differences in the speed, intensity, and pattern of different histo-chemical responses to S. graminicola infection.     

Inheritance of downy mildew resistance, β-1,3-glucanases and peroxidases in pearl millet [Pennisetum glaucum (L.) R. Br.] crosses

The inheritance of resistance to downy mildew disease and the defense-related enzymes β-1,3-glucanase and peroxidase was studied in crosses of pearl millet using a generation-mean analysis. The study material comprised six generations (susceptible and resistant parents, F₁, F₂, BC₁ and BC₂) in three crosses. Seedlings from these generations were inoculated with the downy mildew pathogen Sclerospora graminicola and disease incidence was recorded. Analysis of constitutive levels of β-1,3-glucanase and peroxidase in the seedlings of different generations indicated that the resistant populations showed higher enzyme activities, while lower activities of the enzymes were recorded in the susceptible populations. In the generation-mean analysis, the significance of scaling tests revealed the existence of non-allelic interactions in the inheritance of resistance to downy mildew as well as with the enzymes. Among the gene effects, both additive and dominant effects were significant. All the non-allelic interaction effects were significant in the crosses. Studies on the isozyme patterns of the enzymes substantiated the results of the disease-incidence experiments in most of the generations. The results indicated that the inheritance of downy mildew disease resistance and the expression of β-1,3-glucanase and peroxidase in pearl millet is not only under the control of additive and dominant genes but are also governed by complex non-allelic interactions. Received: 30 April 2000 / Accepted: 17 October 2000     

Seed treatment with aqueous extract of Viscum album induces resistance to pearl millet downy mildew pathogen

Abstract

Downy mildew (Sclerospora graminicola [Sacc.] Schroet.) is a serious agricultural problem for pearl millet (Pennisetum glaucum [L.] R. Br.) grain production under field conditions. Six medicinally important plant species Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and Viscum album were evaluated for their efficacy against pearl millet downy mildew. Seeds of pearl millet were treated with different concentrations of aqueous extract of the plants to examine their efficacy in controlling downy mildew. Among the plant extracts tested, V. album treatment was found to be more effective in enhancing seed quality parameters and also in inducing resistance against downy mildew disease. Germination and seedling vigor was improved in seeds treated with V. album extracts over control. Seeds treated with 10% concentration of V. album showed maximum protection against downy mildew disease under greenhouse and field conditions. The downy mildew disease protection varied from 44–70% with different concentrations. Leaf extract of V. album did not inhibit sporulation and zoospore release from sporangia of Sclerospora graminicola, indicating that the disease-controlling effect was attributed to induced resistance. Seed treatment with V. album extract increased pearl millet grain yield considerably. In V. album, treated pearl millet seedlings increased activities of peroxidase, and phenylalanine ammonia-lyase enzyme was detected. FTIR analysis of V. album extracts showed the presence of amides and other aromatic compounds which are antimicrobial compounds involved in plant defense.     

Plant Growth‐promoting Fungus Penicillium oxalicum Enhances Plant Growth and Induces Resistance in Pearl Millet Against Downy Mildew Disease

Plant Growth‐promoting Fungus (PGPF) Penicillium oxalicum was isolated from rhizosphere soil of pearl millet and was tested for its ability to promote growth and induce systemic resistance in pearl millet against downy mildew disease. The fungal isolate P. oxalicum UOM PGPF 16 was identified as P. oxalicum using ITS sequencing and morphological analysis and sequence was deposited at NCBI with accession number KF150220. Pearl millet susceptible seeds were treated with three different inducers (CS, CF and LCF) of PGPF P. oxalicum and all the inducers significantly reduced the downy mildew disease and enhanced plant growth. Among the inducers tested, CS treatment recorded highest seed germination of 91% and 1427 seedling vigour followed by LCF and CF treatments. The vegetative growth parameter and NPK uptake studies under greenhouse conditions revealed that the CS treatment of P. oxalicum remarkably enhanced the parameters tested when compared to control plants. A significant disease protection of 62% and 58% against downy mildew disease was observed in plants pretreated with CS of P. oxalicum under greenhouse and field conditions, respectively. The spatio‐temporal studies revealed that inducers P. oxalicum required a minimum of 3 days for developing maximum disease resistance which was maintained thereafter. The maximum Peroxidase (POX) activity (62.7 U) was observed at 24 h in seedlings treated with CS of PGPF P. oxalicum and the activity gradually reduced at later time points after pathogen inoculation. Chitinase (CHT) activity was significantly higher in inducer treated seedlings when compared to control seedlings inoculated with pathogen after 48 h and remained constant at all time points.     

Phenylalanine Ammonia Lyase Activity in Pearl Millet Seedlings and its Relation to Downy Mildew Disease Resistance

Phenylalanine ammonia lyase (PAL) activity was studied in differentgenotypes of pearl millet with varying degrees of susceptibilityto downy mildew disease, after inoculating with Pathotype 1of Sclerospora graminicola. In resistant genotypes, the enzymeactivity significantly increased 24 h after fungal inoculationwhile in the susceptible genotypes, the activity decreased.The increase or decrease in enzyme activity was well-correlatedwith the degree of host resistance to the pathogen. A time-courseof change in activity of PAL after inoculation showed a considerabledifference between resistant and susceptible genotypes. Studieson the activity of PAL in different parts of pearl millet seedlingsrevealed that in the resistant genotype, enzyme activity significantlyincreased at 24 h post-inoculation only in the shoot portion,whereas in mesocotyl and root the activity decreased. In susceptibleseedlings, enzyme activity decreased at 24 h post-inoculationin shoot, mesocotyl and root. The activity of PAL was also foundto be pathotype-specific. Histochemical tests for lignin werepositive in infected cells in the resistant genotypes. The roleof PAL in imparting resistance to pearl millet against downymildew disease is discussed. Key words: Sclerospora graminicola, resistance screening, enzyme activity     

Enzyme Changes Associated with Host-Parasite Interactions between Pearl Millet and Downy Mildew Fungus

During the pathogenesis of pearl millet by the downy mildew (Sclerospora graminicola [Sacc.] Schroet.), the activities of peroxidase (PO) and indoleacetic acid oxidase (IAAO) and their isozyme pattern determined by isoelectric focusing on polyacrylamide gels, were observed in extracts of leaves and ears at different stages of development. The PO activity in extracts from infected plants of a susceptible cultivar was found to be higher than in healthy plants. The higher activity was most probably due to the acceleration of host senescence by the pathogen. Quantitative differences in the isozyme patterns of PO and IAAO were found. In inoculated plants of the resistant cultivar, no symptoms developed under the conditions used for infection of the susceptible cultivar and the changes in enzyme activities after inoculation were not significant. The results indicated that different proteins are synthesized in the two cultivars.