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.     

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.     

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.     

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.     

Isolation and characterisation of a NBS-LRR resistance gene analogue from pearl millet

Plant resistance (R) proteins belonging to nucleotide-binding site–leucine-rich repeat (NBS–LRR) family are mainly involved in recognition of effectors secreted by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most drought tolerant cereals, staple food crop of the semi-arid tropics but is highly susceptible to the downy mildew disease caused by oomycetous Sclerospora graminicola (Sacc) schroet. Earlier studies have identified several resistance gene analogues (RGAs) in pearl millet which may be involved in resistance against downy mildew. Of these, a clone RGPM213 was shown to have more than 60% identity with R-proteins coding for NBS–LRR-like protein kinase. The exact nature and function of the R-protein encoded by this gene was not known. In the present study, the cDNA of RGPM213 encompassing NBS–LRR region was inserted into an expression vector pRSET-A and transformed into BL21 E.coli cells. The expressed recombinant fusion protein with a His tag was purified using nickel affinity purification and it had a molecular weight of 35 kDa on SDS-PAGE. Immunoaffinity purification using antibodies raised against this recombinant R-protein identified two proteins of molecular weights 55 kDa and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of these proteins followed by homology search in database revealed similarity of the 55 kDa protein with a protein kinase from Brassica oleracia containing serine/ threonine kinase domain.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Control of pearl millet downy mildew by seed treatment with metalaxyl*

Three formulations of the systemic fungicide metalaxyl were tested in various seed treatments for the control of pearl millet downy mildew in three field experiments with downy mildew-susceptible pearl millet hybrid NHB-3. Uniform, high levels of sporangial inoculum of the causal fungus, Sclerospora graminicola, were provided throughout the growth of the test crops from inoculated infector rows of NHB-3, planted earlier between the test plots. Significant reductions in downy mildew were obtained with all fungicide treatments. Best control was obtained when seed was soaked in a 0.5% aqueous solution of a liquid formulation (mean infection index of 9.8% compared with 94.8% in the untreated check). The degree of control with the wettable powder formulations was directly related to fungicide dosage, and there were no significant effects of application method. Simple dusting of seed at 2 g a.i./kg, a rapid and simple operation requiring small quantities of fungicide and no special application equipment, gave a high level of control (infection index of 12.6% compared with 78.9% in the untreated check). In two experiments grain yields from all the treated plots were significantly greater than from the untreated plots (means of 1234 and 1534 kg/ha for treated plots compared with 485 and 743 kg/ha, respectively), and in the third, the treatment with the least downy mildew gave significantly more grain than the untreated check (1228 compared with 727 kg/ha).