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.     

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.     

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.     

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.     

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     

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.     

Changes in total phenolic contents of pearl millet callus resistant and susceptible to downy mildew

Tissue cultures were used as the host-parasite systems to study the total phenolics in relation to downy mildew infection in pearl millet. Total phenolics increased due to infection bySclerospora graminicola. As the age of the callus tissue increased, an increase in total phenolics was observed in both susceptible and resistant cultivars. The possibility of using a dual culture to study the biochemical changes occurring in the host-parasite interactions of an obligate parasite is discussed.     

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.     

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     

Selection of downy mildew resistant somaclones,from a susceptible B line of pearl millet

Plants regenerated from seed-derived callus of a PNMS 6B line of pearl millet (Pennisetum glaucum (L.) R. Br.) were evaluated for their resistance induced by somaclonal variation for downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroter. Among the 201 lines regenerated, only 3 lines consistently proved highly resistant (free from disease incidence) for up to 5 generations; whereas, 17 lines were resistant (disease incidence ranging from 1 to 9%). Resistance was confirmed by testing the plants under both laboratory and field conditions. The plants were evaluated for their agronomic traits. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterisation of populations of Sclerospora graminicola

Nine cultivars of pearl millet were inoculated with 12 collections of Sclerospora graminicola from various locations in Africa and India in a series of experiments which constituted an almost balanced incomplete block design spanning an entire growing season. The character of each pathogen population was defined in terms of incidence of disease on the hosts. Generally the pathogen collections from the sub-Sahel regions of West Africa were more pathogenic than those from Senegal, Zambia or India. All but one host cultivar possessed relative resistance which decreased as the overall pathogenicity of the collections increased. There was one notable exception which exhibited stable resistance to all the pathogen collections tested and may have potential for future resistance breeding programmes.     

Cerebroside as an elicitor for induced resistance against the downy mildew pathogen in pearl millet

Innate defence mechanisms in plants can be triggered and enhanced by certain agents, which are referred to as inducers. Inducing resistance against a broad spectrum of pathogens in otherwise susceptible plants is seen as a potentially safer alternative to other methods of chemical control of plant diseases. Cerebrosides, which are glycosphingolipids extracted from various plant pathogens, have been reported as resistance elicitors in the rice‐pathogen system. In the present study, cerebroside elicited resistance against downy mildew disease (caused by Sclerospora graminicola) of pearl millet (Pennisetum glaucum) that was highly significant. The resistance was of systemic nature and the time required for the resistance to build up was from 2 days onwards. There was a significant yield enhancement due to disease suppression by cerebroside treatment. Promising results were obtained in a preliminary field trial.     

Rust and Downy Mildew Resistance in Pearl Millet (Pennisetum glaucum) Mediated by Heterologous Expression of the afp Gene from Aspergillus giganteus

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T₀ plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.     

Phenol metabolism in downy mildew resistant and susceptible genotypes of pearl millet

Biochemical characterisation of pearl millet genotypes was carried at pre- (45 DAS) and post-infection (57 DAS i.e. 7 days after infection) stages. Total phenol content at pre-infection stage did not show inherent resistance or susceptibility. While the total phenol content was found to be higher in susceptible genotypes at post-infection stage, qualitative analysis of phenol through high-performance thin layer chromatography showed absence of ferulic acid in resistant genotypes at pre-infection stage. Peroxidase (POX) activity was higher in susceptible genotypes at both the stages of analysis. Constitutive activity of phenylalanine ammonia lyase was higher in resistant genotype whereas induced activity was recorded higher in susceptible genotypes. Native poly-acrylamide gel electrophoresis isozyme banding pattern of POX showed some inducible band(s) due to disease infection in resistant and susceptible genotypes.     

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.