Evaluation of plant growth-promoting Rhizobacteria for their efficiency to promote growth and induce systemic resistance in pearl millet against downy mildew disease

Six strains of Plant growth promoting Rhizobacteria (PGPR) were tested for their ability to promote growth and induce resistance in pearl millet against downy mildew disease. All the PGRP strains showed a significant (P < 0.01) increase in growth promotion in laboratory as well as greenhouse conditions. Only two strains of Pseudomonas spp., UOM ISR 17 and UOM ISR 23, were capable of protecting pearl millet against downy mildew significantly. Pseudomonas UOM ISR 17 and UOM ISR 23 were able to offer 56.3 and 47.5%, respectively against downy mildew disease. When tested for the time gap needed to offer maximum protection, it was found that both the strains needed four days to offer maximum protection of 73.3% and 59.7%, respectively. While both the Acetobacter strains UOM Ab9 and Ab11 and Azospirillum strain UOM Az3 were able to promote growth and offered disease protection of 39.2, 22.3 and 17.40% respectively, they were not as efficient as the two Pseudomonas strains in protecting pearl millet against downy mildew. Maximum growth promotion was recorded by Pseudomonas spp. UOM ISR 17 with 33.9 cm height which was 44, 45, 42 and 46.8% more in height, fresh weight, dry weight and leaf area over the control which recorded 27 cm height, 8.1 g fresh weight, 2.1 g dry weight and 29 cm² leaf area, respectively.     

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.     

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.     

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.     

Pearl millet downy mildew as affected by integrating seed dressing,sowing date and cultivar

The effects of seed dressing, sowing date and cultivar on incidence and severity of downy mildew of pearl millet induced by Sclerospora graminicola and yield were studied in a two-year field trial conducted at the Research farm of University of Maiduguri. The millet cultivars, Ex-Borno, SOSAT-C88, GB 8735 and Gwagwa were each dressed with metalaxyl at 0.75 and 1.50 g a.i./kg seed; and a batch of undressed seeds of each cultivar served as control. Both dressed and undressed seeds were used for dry-planting and wet-planting in early and late seasons. The results showed that seed dressing with the fungicides significantly (p ≤ 0.05) reduced the incidence and severity of downy mildew and increased grain yield. Dry-planting also significantly (p ≤ 0.01) increased grain yield irrespective of disease incidence. Delay in sowing led to a significant reduction in incidence and severity of downy mildew. Differences between the cultivars in relation to incidence and severity of downy mildew and grain yield were significant. SOSAT-C88 developed low or no downy mildew in both seasons. Sowing of dressed SOSAT-C88 as soon as rainfall established appeared most beneficial in the control of downy mildew. Dry- or wet-planting Ex-Borno dressed with any of the metalaxyl formulations proved to be effective for downy mildew management and for high yield.     

Comparative evaluation of Pseudomonas fluorescens and their lipopolysaccharides as implicated in induction of resistance against pearl millet downy mildew

Two plant growth promoting Pseudomonas fluorescens isolates namely UOM SAR 14 and UOM SAR 80 most effectively induced resistance against downy mildew disease of pearl millet both under greenhouse and field conditions. Relative assessment of live cultures of P. fluorescens UOM SAR 14 and UOM SAR 80 and their lipopolysaccharides (LPS) extracted from their cell walls were evaluated for their ability to induce resistance against pearl millet downy mildew. Treatment with P. fluorescens and their LPS enhanced the seed germination and seedling vigour considerably. Although both live cultures and their LPS treatment induced resistance in pearl millet against downy mildew disease both under greenhouse and field conditions as evidenced by the significant reduction of the disease, live cultures were more effective than the LPS in level of resistance induced. Live cultures of UOM SAR 14 and UOM SAR 80 induced 66% and 57% protection while their respective LPS extracts offered 59 and 53% protection against downy mildew disease under greenhouse conditions. Similarly, under field conditions with very heavy inoculum pressure live cultures offered 75% and 70%, and their LPS offered 71% and 67% protection, respectively. In either case, the time gap required for the building up of resistance was found to be 3 days and nature of the resistance induced was systemic and durable with both live cultures and their lipopolysaccharides. It was also noticed that the live bacteria significantly varied in the degree of protection offered and so also their respective LPS.     

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     

Influence of nitrogen on the incidence of downy mildew disease of pearl millet (Pennisetum typhoides Stapf & Hubb.)

Summary The influence of different levels of nitrogen on the incidence of downy mildew disease of pearl millet on different varieties was studied under field conditions. Nitrogen nutrition to the host did not have much influence on the disease incidence. Among the three hybrid varieties of pearl millet tested J 1270 exhibited resistance to downy mildew incidence while J 934 and HB₃ were found to be highly susceptible.     

Isolation,Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide‐binding Sites

Sequence analysis of plant disease resistance genes shows similarity among themselves, with the presence of conserved motifs common to the nucleotide‐binding site (NBS). Oligonucleotide degenerate primers designed from the conserved NBS motifs encoded by several plant disease resistance genes were used to amplify resistance gene analogues (RGAs) corresponding to the NBS sequences from the genomic DNA of various plant species. Using specific primers designed from the conserved NBS regions, 22 RGAs were cloned and sequenced from pearl millet (Pennisetum glaucum L. Br.). Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into nine distinct classes. GenBank database searches with the consensus protein sequences of each of the nine classes revealed their conserved NBS domains and similarity to other known R genes of various crop species. One RGA 213 was mapped onto LG1 and LG7 in the pearl millet linkage map. This is the first report of the isolation and characterization of RGAs from pearl millet, which will facilitate the improvement of marker‐assisted breeding strategies.     

Control of pearl millet downy mildew caused by Sclerospora graminicola with systemic fungicides in an artificially-contaminated plot

Among four fungicides, viz. metalaxyl (two formulations), fosetyl-Al, pro-pamocarb and cyomaxanil tested in vitro against sporangial germination inhibition of Sclerospora graminicola, cyomaxanil was found to be most inhibitory. In an artificially contaminated plot, when used as seed treatment or foliar spray for the control of downy mildew of pearl millet, only metalaxyl was effective. Metalaxyl 25 (Ridomil) and metalaxyl 35 (Apron) seed treatments protected the pearl millet plants from downy mildew up to 30 days. As a foliar spray, metalaxyl 25 used once at 20 days or twice after 20 and 38 days of plant growth gave less disease at harvest time. Seed treatment (metalaxyl 25 or 35) followed by one metalaxyl 25 spray was found to be effective in controlling the downy mildew. These treatments improved the growth of plants and yield significantly.     

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     

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.     

DNA fingerprinting detects genetic variability in the pearl millet downy mildew pathogen (Sclerospora graminicola)

Genetic variability in six host genotype-specific pathotypes of pearl millet downy mildew pathogen S. graminicola was studied at the molecular level using mini- and micro-satellites. Our results indicated that microsatellites (GAA)₆, (GACA)₄, and especially (GATA)₄ were quite informative and showed high levels of polymorphism among the pathotypes. The six pathotypes could be classified into five groups based on the cluster analysis of their genetic similarities, thereby confirming the existence of distinct host genotype-specific virulence in S. graminicola pathotypes. We demonstrate, for the first time, the use of DNA fingerprinting to detect genetic variation in downy mildew fungus of pearl millet.     

Synergistic effects of Trichoshield on enhancement of growth and resistance to downy mildew in pearl millet

Trichoshield, a talc formulation consisting of spores of Trichoderma harzianum, Trichoderma lignorum, Gliocladium virens and Bacillus subtilis was tested, following different application methods, for its ability to promote growth of pearl millet plants and to induce resistance to downy mildew of pearl millet. Under laboratory conditions, trichoshield seed treatment enhanced seed germination and seedling vigor of pearl millet significantly over the control; under greenhouse conditions vegetative growth parameters like height, fresh and dry weight, leaf area and number of tillers were significantly enhanced over the control: Trichoshield formulation offered greater protection against downy mildew in comparison with individual strains of T. harzianum, T. lignorum, G. virensand B. subtilis. Among the methods of application, foliar spray was found to be a more efficient delivery method than seed treatment or slurry treatment. Combinations of foliar spray with seed treatment and slurry treatment produced the same effect as foliar spray alone. Under field conditions, trichoshield treatment enhanced reproductive parameters like number of earheads, length and girth of earheads, 1000 seed weight and yield significantly over the control. Days required for 50% flowering was reduced by 4 days compared to the control. Yield enhancement of 28% over the control was highly significant. Trichoshield treatment offered protection ranging from 52 to 71% under field conditions, depending on the application method. However, the chemical fungicide metalaxyl Apron provided the highest protection against downy mildew, both under greenhouse and field conditions.     

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen <Emphasis Type="Italic">Sclerospora graminicola</Emphasis>

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris–SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3′ untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.