K. N. Amruthesh

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.

Nitric oxide is involved in chitosan-induced systemic resistance in pearl millet against downy mildew disease

BACKGROUND The nature and durability of resistance offered by chitosan and the involvement of nitric oxide (NO) in chitosan-induced defence reactions in pearl millet against downy mildew disease were investigated. RESULTS It had previously been reported that chitosan seed priming protected pearl millet plants against downy mildew disease. Further elucidation of the mechanism of resistance showed that chitosan seed priming protects the plants systemically. A minimum 4 day time gap is required between the chitosan treatment and pathogen inoculation for maximum resistance development, and it was found to be durable. Chitosan seed priming elevated NO accumulation in pearl millet seedlings, beginning from 2 h post-inoculation, and it was found to be involved in the activation of early defence reactions such as hypersensitive reaction, callose deposition and PR-1 protein expression. Pretreatment with NO scavenger C-PTIO and nitric oxide synthase (NOS) inhibitor L-NAME before pathogen inoculation reduced the disease-protecting ability of chitosan, and defence reactions were also downregulated, which indicated a possible role for NO in chitosan-induced resistance. CONCLUSION Protection offered by chitosan against pearl millet downy mildew disease is systemic in nature and durable. Chitosan-induced resistance is activated via NO signalling, as defence reactions induced by chitosan were downregulated under NO deficient conditions.

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.

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.

Thiamine seed treatment enhances LOX expression, promotes growth and induces downy mildew disease resistance in pearl millet

Seeds of pearl millet [Pennisetum glaucum (L). R.Br.] susceptible cv. 7042S were treated with thiamine at 5, 10, 15, 20 and 25 mM concentrations and growth promotion and downy mildew resistance were tested. Seed treatment with 20 mM thiamine resulted in 72 and 70 % disease protection under greenhouse and field conditions, respectively, and enhanced vegetative and reproductive growth parameters. Analysis of lipoxygenase (LOX) activity in inoculated pearl millet seedlings at different time intervals indicated that increased LOX activity was initiated at 3 h after inoculation (hai) and maximum activity was observed at 24 hai. Northern analysis showed that LOX mRNA transcript accumulation was higher in the resistant seedlings (cv. IP18292) than in susceptible seedlings. Thiamine seed treatment induces rapid LOX gene expression and results in significant disease protection against downy mildew disease.

Pathogenesis Related Proteins in Plant Defense Response

Proteins encoded by the host plant induced under pathological or related conditions are termed pathogenesis-related proteins. These proteins display high-degree of pathogen specificity and are coordinated at the level of transcription. Induction of PR’s when measured on a time scale is a late event and its effect on the early infection is limited. Application of chemicals or microbially derived metabolites that mimic the effect of pathogen infection induces both PR’s and acquired resistance. Numerous Pathogenesis Related proteins have been detected in rice, wheat, maize, sorghum, barley, tomato, pearl millet, bean, chickpea, soybean, pepper, sunflower, carrot, pepper, grape vine, alfalfa, celery, rubber and many other plants. The localization and distribution of the PR is related directly to the method and nature of the pathogen infection. The PR’s have been classified into various families based on the shared sequence homology. PR’s can also be grouped into different classes based on the migration in the native PAGE, reaction with specific antisera and mRNA probes. PR’s have also been classified based on the biological activity of the induced defense proteins. Seventeen different groups of PR’s have been identified. Several studies have revealed that PR proteins are induced in response more rapidly in resistant interactions. Bacteria, fungi, viruses and nematodes induce PR proteins upon entry into the incompatible host. Several PR genes in the form of cDNAs have been identified and characterized during acquisition of systemic resistance in plants against pathogens. A number of molecules derived from pathogens can serve as elicitors of PR gene expression. In addition to the already existing complexity, some signals are interdependent. Several PR genes encoding PR proteins have been identified in different plants. They are almost silent in healthy plants. Generally most PR protein genes belong to multi gene families. Pathogen induced PR gene expression often occurs at the level of transcription. The occurrence of multi gene families, localization in the apoplast as well as in the vacuolar compartment and differential induction by endogenous signaling compounds indicate an important role in defense not only against pathogen infection but also in eliciting acquired resistance. Several PR proteins like the PR-1, 2, 3, 4 and 5 have been shown to inhibit growth of fungi. Large groups of PR genes which have been well characterized can be put to use to produce plants with better responses to biotic and abiotic stress. Understanding stress signals and transduction mechanisms and identification of additional defense genes will provide opportunities for enhanced resistance engineering in crop plants.

Unsaturated fatty acids from zoospores of Sclerospora graminicola induce resistance in pearl millet

AbstractDowny mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.

Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum

Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.

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.

Crude oligosaccharides mediated resistance and histo-chemical changes in Capsicum annuum against anthracnose disease caused by Colletotrichum capsici

Abstract Enhancing the host resistance using biotic elicitors is one of the eco-friendly approaches developed for plant disease management. The Crude Oligosaccharides (CO) extracted from Colletotrichum capsici (Syd.) Butler & Bisby was evaluated for their efficiency to elicit resistance in chilli against anthracnose disease. Among the different concentrations tested, CO treatment at 2.5 mg/ml concentration for 3 h duration significantly enhanced seed germination (90.5%) and seedling vigor (986.7), compared to control which offered 78% of seed germination and 712.5 of seedling vigor. Application of CO at 2.5 mg/ml concentration also reduced the disease severity with a highest anthracnose disease protection of 68% under greenhouse conditions and enhanced the vegetative growth parameters compared to control. The induction of resistance was evident with higher expression of primary defense responses like hypersensitive response, deposition of lignin, callose, hydrogen peroxide and phenol when compared to control plants. There was a one fold increase in defense enzyme activities phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and lipoxygenase in crude oligosaccharide-treated inoculated seedlings when compared to susceptible inoculated seedlings which were similar to that of resistant inoculated seedlings where a maximum of 1.5-fold increase in enzyme activity was observed.