Polygalacturonase-inhibitor proteins in pearl millet: possible involvement in resistance against downy mildew.

Polygalacturonase-inhibitor protein (PGIP) is a defense protein found in plant cell walls. It prevents the degradation of pectin by modulating the endo-polygalacturonase activity. The present study has used heterologous anti-bean PGIP probes to investigate the role of PGIP in pearl millet [Pennisetum glaucum (L) R. Br.] resistance against downy mildew caused by oomycete pathogen Sclerospora graminicola (Sacc.) Schroet. Northern blot analysis using bean pgip2 DNA fragment as probe showed an early and marked induction of transcripts (∼1.2 kb) upon pathogen-inoculation in pearl millet cultivar resistant to downy mildew, with the maximum level observed at 24 and 48 h post-inoculation (h.p.i.). Western blot analysis of pearl millet total cell wall proteins using antibodies against bean PGIP showed the presence of a major band of ∼43 kDa, and several minor ones. The protein accumulation was higher in resistant seedlings than in susceptible seedlings with a differential expression observed only in the case of incompatible interaction. Immunocytochemical localization in epidermal peelings of coleoptiles and tissue-printing showed a similar trend in the PGIP accumulation. PGIP was found to localize in the epidermal as well as in the vascular regions of tissues. Higher accumulation was observed in the stomatal guard cells of resistant cultivar inoculated with the pathogen. PGIP activity of pearl millet total protein extracts when assayed against Aspergillus niger PG displayed differential PG inhibitory activities between the resistant and suceptible cultivars with resistant sample showing the highest inhibition of 16%, post-pathogen treatment. Thus, PGIP appeared to be an important player in pearl millet-S. graminicola interaction leading to host resistance.

Infection biology and defence responses in sorghum against Colletotrichum sublineolum.

AIMS: To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes. METHODS AND RESULTS: Infection biology and defence responses were studied quantitatively by light microscopy, H(2)O(2) accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H(2)O(2), HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H(2)O(2) accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration. CONCLUSIONS: The early accumulation of H(2)O(2) and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that there is a significant correlation between induced accumulation of H(2)O(2), papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.

Transposition of great arteries with natural partial Senning: A rare case report.

The association of transposition of the great arteries (TGA) and anomalous pulmonary venous connection is extremely rare. Children with transposition of the great arteries improved dramatically with the advent of the atrial repair. In this report, we describe a 40-day old male infant with TGA and associated anomalous pulmonary venous connection who presented with the history of cyanosis and hurried breathing. This patient underwent successful balloon atrial septostomy and discharged with uneventful recovery.

Mycotoxin contamination of maize grains grown in Karnataka (India).

One hundred and ninety seven maize samples representing different cultivars, collected from different agroclimatic regions of Karnataka (India) were analysed for moisture content, mould incidence, ergosterol and extent of mycotoxin contamination. Moisture content determination by the hot-air oven method revealed significantly high levels of moisture content (15-18%) in 34 (17%) samples, which exceeded the permissible limit for safe storage. Ergosterol quantification by HPLC revealed the presence of ergosterol in many samples collected from rural areas of Karnataka irrespective of the moisture content. Mould enumeration based on blotter and agar plating methods revealed the association of 24 diverse species of both field and storage moulds belonging to 14 genera. Mycotoxins analyses using monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) revealed mycotoxin contamination in 69 (34.8%) samples. Maize samples with a high incidence of diverse species of moulds and alarmingly high levels of mycotoxins in many samples indicate the need for proper surveillance and monitoring exclusively for the prevention of moulds and mycotoxins in maize produce in Karnataka before it reaches the consumer.

Seed treatment with beta-aminobutyric acid protects Pennisetum glaucum systemically from Sclerospora graminicola.

beta-Aminobutyric acid (BABA) treatment of pearl millet [Pennisetum glaucum (L) R Br] seeds influenced seedling vigour and protected the seedlings from downy mildew disease caused by the oomycetous biotropic fungus Sclerospora graminicola (Sacc) Schroet. Of the different concentrations of BABA tested, viz 25, 50, 75 and 100 mM, seeds treated with 50 mM for 6 h resulted in the maximum of 1428 seedling vigour and showed 23% disease incidence in comparison with the control which recorded a seedling vigour of 1260 and 98% disease incidence i.e. 75% protection from disease. Seeds treated with BABA when challenged for downy mildew disease using zoospores of S graminicola required 48 h after inducer treatment to develop maximum resistance. Durability of induced resistance was also tested in plants raised from seeds treated with the inducer and identified as resistant, by second challenge inoculation with the downy mildew pathogen at tillers and inflorescence axes. Reduced disease incidence of only 10 and 12% in these plants, compared with 71 and 76% disease in control plants inoculated at the tillers and inflorescence axes, respectively, suggested that resistance induced in seeds with BABA remained operative through vegetative and reproductive growth of pearl millet plants. Induction of resistance by seed treatment with BABA enhanced vegetative growth, viz height, fresh weight, leaf area and tillering, and reproductive growth, viz early flowering, number of productive ear heads and 1000 seed weight. Studies on induction of resistance in different cultivars of pearl millet with varying resistance reaction to downy mildew indicated that the protection offered by BABA is independent of the nature of cultivars used and not dependent on their constitutive resistance.

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.

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.

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.

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.

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-ß-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.

An approach to obtain specific polyclonal antisera to Xanthomonas campestris pv. cyamopsidis and its potential application in indexing of infected seeds of guar.

Clusterbean seed health testing is warranted since the pathogen (Xanthomonas campestris pv. cyamopsidis (Xccy)) is seed-borne and seed-transmitted. A polyclonal antibody was developed in rabbit via subcutaneous and intramuscular injections and characterized for sensitivity, specificity and its applicability to ELISA which: (i) was sensitive in detecting as few as 102 cells ml - 1 at a titre of 1: 4000; (ii) was specific, since it reacted only with Xccy and not with other xanthomonads; (iii) reacted both with Xccy cells and culture filtrate, indicating that the antigenic determinant is a secretory component; (iv) was applicable and reliable in seed health testing since it reacted only with infected seeds and plant materials and not with healthy seeds and (v) a purified fraction of antibody was virulent-specific since heat-denatured and avirulent isolates were not detected. The ELISA thus developed is highly reproducible and therefore suitable for the evaluation of the potential disease status of seeds and plant health, which is appropriate for routine seed health testing.