H. S. Shetty

Comparative performance of formulations of plant growth promoting rhizobacteria in growth promotion and suppression of downy mildew in pearl millet

Five plant growth promoting rhizobacterial formulations, each consisting of two Bacilli strains with chitosan as a carrier were tested for their capacity to promote growth and induce resistance against downy mildew in pearl millet under both greenhouse and field conditions. Three modes of applications were tested: seed treatment, soil amendment, and seed treatment+soil amendment. In general, irrespective of application method, most of the formulations, in comparison with the control, increased plant growth and vigor as measured by seed germination, seedling vigor, plant height, fresh and dry weight, leaf area, tillering capacity, number of earheads, length and girth of earhead, 1000 seed weight and yield. The time of flowering was also advanced by 4–5 days over the control. Likewise all the formulations significantly reduced downy mildew incidence relative to the nontreated control. However, the rate of growth enhancement and disease suppression varied considerably with the formulations. Formulations LS256 and LS257 besides being the best growth promoters were also the most efficient resistance inducers. None of the formulations matched the level of the fungicide metalaxyl in offering protection against downy mildew. Among the application methods tested, soil amendment was found to be the most suitable and desirable way of delivering the formulations. Combination of seed treatment and soil amendment produced the same effect that was produced by soil amendment alone. The study demonstrates a potential role for plant growth promoting rhizobacterial formulations in downy mildew management.

Seed bio-priming with Pseudomonas fluorescens isolates enhances growth of pearl millet plants and induces resistance against downy mildew

Biopriming pearl millet seeds with Pseudomonas fluorescens isolates resulted in improved growth of the plants and also induction of resistance against downy mildew disease caused by the fungus Sclerospora graminicola. Although all of the test isolates reduced disease severity and promoted growth both under greenhouse and field conditions, high variability was observed in their efficiency. Treatment due to Pseudomonas fluorescens resulted in enhancement of: germination, seedling vigour, plant height, leaf area, tillering capacity, seed weight (measured for 1000 seeds) and yield. The time required for flowering was advanced by 5 days. Isolate UOM SAR 14 registered higher levels of vegetative and reproductive growth; most importantly there was a 22% increase in grain yield. The isolates also effectively induced resistances against downy mildew disease both under greenhouse and field conditions. The isolates offered protection ranging from 20 to 75%. However, the level of disease control was less than that achieved by the systemic fungicide Apron. UOM SAR 14 offered 71 and 75% and remained consistent under all test conditions. Further studies showed that the resistance induced was systemic, required a minimum of 3 days to build up, and was sustained throughout the plants life.

Induction and accumulation of polyphenol oxidase activities as implicated in development of resistance against pearl millet downy mildew disease

Polyphenol oxidase (PPO) activity was analysed in seedlings of resistant and susceptible pearl millet [Pennisetum glaucum (L.) R.Br] cultivars with or without inoculation of the downy mildew pathogen Sclerospora graminicola (Sacc.) Schroet. Seedlings of resistant varieties had greater PPO activity than susceptible seedlings, and inoculated seedlings had significantly higher PPO levels than uninoculated seedlings. Temporal accumulation of PPO showed a maximum activity at 24 h post-inoculation in resistant seedlings, whereas in susceptible seedlings it peaked at 48 h. PPO activity was positively correlated with levels of downy mildew resistance in different pearl millet cultivars under field conditions. Native PAGE staining showed four isoforms of PPO, which were differentially induced in relation to the time of appearance and intensities in the uninoculated seedlings, whereas a fifth PPO isoform appeared after inoculation with S. graminicola. PPO activity was significantly higher in the shoot and leaves of pearl millet than in the root. Tissue printing analysis of the enzyme expression showed that the enzyme is predominantly expressed after pathogen inoculation and is localised in the epidermal and vascular regions. Temporal analysis of transcript accumulation showed that in resistant seedlings PPO mRNAs was expressed earlier and more abundantly than in susceptible seedlings. Our studies demonstrate, for the first time, that PPO is actively involved in plant defence and can be used as a marker of resistance to downy mildew infection in pearl millet.

Induction of Growth Promotion and Resistance Against Downy Mildew on Pearl Millet (Pennisetum glaucum) by Rhizobacteria

A series of laboratory, greenhouse, and field experiments were conducted to evaluate seven strains of plant growth-promoting rhizobacteria (PGPR). The PGPR were tested as suspensions of fresh cultures and talc-based powder formulations. Evaluations were conducted on pearl millet (Pennisetum glaucum) for growth promotion and management of downy mildew caused by Sclerospora graminicola. All treatments with fresh suspensions and powdered formulations showed enhancement in germination and vigor index over the respective untreated controls. With fresh suspensions, maximum vigor index resulted from treatments by Bacillus pumilus strain INR7 followed by B. subtilis strain IN937b (64 and 38% higher than the untreated control, respectively). With powdered formulation, treatment with strain INR7 also resulted in the highest germination and vigor indexes, which were 10 and 63%, respectively, over the untreated control. Under experimental plot conditions, prominent enhancement in growth also was observed in the disease tests. Yield was enhanced 40 and 37% over the untreated control by seed treatment with powdered formulations of strains INR7 and SE34, respectively. The same strains also increased yield by 36 and 33%, respectively, when applied as fresh suspensions. Studies on downy mildew management resulted in varied degrees of protection by the PGPR both under greenhouse and field conditions. With fresh suspensions, treatment with INR7 resulted in the highest protection (57%), followed by B. pumilus strain SE34 and B. subtilis strain GBO3, which resulted in 50 and 43% protection, respectively, compared with the untreated control. With powdered formulation, PGPR strain INR7 suppressed downy mildew effectively, resulting in 67% protection, while SE34 resulted in 58% protection, followed by GBO3 with 56% protection. Treatment with Apron (Metalaxyl) resulted in the highest protection against downy mildew under both greenhouse and field conditions. Thus, the present study suggests that the tested PGPR, both as powdered formulations and fresh suspensions, can be used within pearl millet downy mildew management strategies and for plant growth promotion.

Peroxidase activity and isozyme analysis of pearl millet seedlings and their implications in downy mildew disease resistance

Investigation has been carried out on differential expression of peroxidase isozymes in pearl millet seedlings inoculated with downy mildew disease-causing pathogen Sclerospora graminicola. Two-day-old seedlings of eight different pearl millet cultivars were assessed for downy mildew disease reaction under greenhouse conditions. Based on the results, pearl millet cultivars, IP 18292 and IP 18294, were categorized as highly resistant, P 310-17 and MBH 110 as resistant, 5141 B and 81 B as susceptible and 23 B and HB 3 as highly susceptible. Time-course study of peroxidase analysed spectrophotometrically showed increased activity at 8 and 16 h after inoculation in highly resistant cultivar (IP 18292) and highly susceptible cultivar (HB 3), respectively. Time-dependent expression as analysed by isoelectric focusing (IEF) recorded 22 isozymes with pI ranging from 3.6 to 9.8 in highly resistant and induced-resistant seedlings, whereas highly susceptible seedlings showed only 12 isozymes. Cultivar-specific studies showed 55.2 and 54.9, 40.9 and 44.8, 40.8 and 41.9, and 33.6 and 32.9 units of peroxidase activity in highly resistant, resistant, susceptible and highly susceptible seedlings at constitutive level, respectively, and there was a clear-cut difference between them. On inoculation highly resistant, resistant and induced-resistant seedlings recorded increase in enzyme activity, whereas susceptible and highly susceptible seedlings recorded decrease in enzyme activity. The same observation was also recorded in native-PAGE analysis.

RESISTANCE TO DOWNY MILDEW IN PEARL MILLET IS ASSOCIATED WITH INCREASED PHENYLALANINE AMMONIA LYASE ACTIVITY

Phenylalanine ammonia lyase (PAL) activity was studied in pearl millet cultivars with different levels of resistance to the downy mildew disease caused by Sclerospora graminicola, an important oomycete pathogen. PAL activity was elevated in resistant host cultivar and decreased in susceptible cultivars following downy mildew pathogen infection. The enzyme activation varied between cultivars and was correlated with the degree of resistance to downy mildew disease. The induction of PAL as a response to pathogen inoculation was further corroborated by a time-course study in seedlings and cultured cells of pearl millet. The level of PAL activity was highest at 1.5 h in cultured cells and 4 h in seedlings of resistant host cultivar after inoculation with Sclerospora graminicola. Further studies on PAL activity in different tissues of seedlings showed highest enzyme activity in the young growing region of the root of the resistant host cultivars. The accumulation of wall-bound phenolics and lignin was higher in the resistant cultivar seedlings as evidenced by phloroglucinol-HCl staining and p-coumaric acid assay. The temporal changes in lignin concentration and the concentration of soluble phenolics were greater in root tissues of resistant cultivars than in those of susceptible cultivars. Treatment of resistant seedlings with a PAL inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the enhancement of the enzyme activity, whereas in the presence of 1 mm trans-cinnamic acid the pathogen-induced PAL was completely inhibited. Treatment of pearl millet seedlings with exogenously applied PAL inhibitors induced downy mildew disease susceptibility in the resistant pearl millet cultivar, consistent with direct involvement of PAL in downy mildew resistance. Results are discussed with respect to the presumed importance of host phenolic compounds and lignin accumulation and its relation to PAL activation as a response to the pathogen infection.

Plant Growth Promoting Rhizobacteria: Potential Green Alternative for Plant Productivity

Use of plant growth promoting rhizobacteria (PGPR) for the benefits of agriculture is gaining worldwide importance and acceptance and appears to be the trend for the future. PGPR are bioresources which may be viewed as a novel and potential tool for providing substantial benefits to the agriculture. These beneficial, free-living bacteria enhance emergence, colonize roots, stimulate growth and enhance yield. PGPR are known to induce resistance against various plant pathogens in different crops ranging from cereals, pulses, ornamentals, vegetables, plantation crops, spices and some trees. Most studies have emphasized exploration and potential benefits of PGPR in agriculture, horticulture and forestry. The plausible mechanisms adopted by these rhizobacteria in growth promotion and resistance, though abundantly documented but still remains to be fully explored. Integrated use of PGPR allows the combination of various mechanisms thereby enhancing their beneficial abilities. However, their use has not been to the full potential due to inconsistency in their performance and their commercialization limited to few developed countries. Use of PGPR as bioinoculants, biofertilizers and biocontrol agents, advantages and disadvantages, practical potential in improved agriculture and future prospects are also discussed.

Purification and properties of lipoxygenase induced in downy mildew resistant pearl millet seedlings due to infection with Sclerospora graminicola

Induction of lipoxygenase (LOX) was studied in pearl millet seedlings upon inoculation with Sclerospora graminicola. Resistant pearl millet seedlings exhibited a 2.4-fold increase in LOX activity after inoculation with the downy mildew pathogen S. graminicola. This increase was mainly due to the synthesis of a new LOX isozyme designated as LOX-6. Three of the six isozymes of lipoxygenase designated LOX-1, -3 and -6, were purified from inoculated downy mildew resistant pearl millet seedlings as electrophoretically homogeneous proteins. The isozymes were purified stepwise by ammonium sulfate fractionation, DEAE-sephadex A-50 and sephadex G-200 gel-filtration. Purification factor for LOX-1, -3 and -6 were 46.5, 73.6 and 115.7, respectively. The purified LOX-1, -3 and -6 had molecular weight of 83, 77 and 73 kDa with a pI of 5.5. 5.8 and 6.2, respectively. The results indicate that the LOX isozymes were dimers composed of two unequal subunits of 43 and 40 for LOX-1. 40 and 37 for LOX-3, 38 and 35 for LOX-6. The optimum pH for LOX 6 was 6.5 being stable from pH 4.5 to 8.0. The LOX-1 and -3 had a similar optimum pH of 9.0. LOX isozymes had a different thermal stability ranging from 0 to 30degreesC. Esculetin, NDGA, SHAM, N-propylgallate and copper sulfate were found to be the potent inhibitors of all three isozymes of LOX. All the isozymes of LOX revealed similar reaction with the metal ions like Ca2+, Ba2+, Mn2+, Cu2+, Fe2+ and Mg2+ with very few exceptions. Butyl hydroxyanisole caused the strongest inhibition of the pearl millet LOX isozymes among the various antioxidants tested. The LOX isozymes showed preferential activity towards linoleic acid followed by linolenic acid as substrate

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

Trichoshield, a talc formulation consisting of spores of Trichodermaharzianum, Trichodermalignorum, Gliocladiumvirens and Bacillussubtilis 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.

Rhizosphere fungus Penicillium chrysogenum promotes growth and induces defence-related genes and downy mildew disease resistance in pearl millet

Susceptible pearl millet seeds (cv 7042S) were treated with the plant growth promoting fungus Penicillium chrysogenum (PenC-JSB9) at 1 × 10(8) spores·ml(-1) to examine mRNA expression profiles of five defence responsive genes and test its ability to induce resistance to downy mildew caused by Sclerospora graminicola. PenC-JSB9 treatment at 1 × 10(8) CFU·ml(-1) for 6 h significantly enhanced seed germination (9.8- 89%), root length (4.08% to 5.1 cm), shoot length (18.9% to 7.77 cm) and reduced disease incidence (28%) in comparison with untreated controls. In planta colonisation of PenC-JSB9 showed that all three root segments (0-6 cm) and soil dilutions incubated on PDA produced extensive mycelial growth, however colonisation frequency of PenC-JSB9 was significantly higher in soil than in root segments. Spatiotemporal studies revealed that induction of resistance was triggered as early as 24 h and a minimum 2-3 days was optimal for total resistance to build up between inducer treatment and challenge inoculation in both experiments. In Northern blot analysis, transcript accumulation of resistant and PenC-JSB9 induced susceptible cultivars showed higher basal levels of defence gene expression than non-pretreated susceptible controls. Transcript accumulation in resistant seedlings challenge-inoculated with the pathogen showed maximum expression of CHS (3.5-fold increase) and Pr-1a (threefold increase) at 24 and 12 h, respectively. While PenC-JSB9 pretreated susceptible seedlings challenge-inoculated showed rapid and enhanced expression of LOX and POX at 48 h and for CHT at 24 h, whereas non-pretreated susceptible seedlings after pathogen inoculation showed weak expression of hybridised defence genes. Enhanced activation of defence genes by PenC-JSB9 suggests its role in elevated resistance against S. graminicola.