J. Sudisha

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.

Cloning and development of pathotype-specific SCAR marker associated with Sclerospora graminicola isolates from pearl millet

Downy mildew pathogen of pearl millet in India is associated with the spread of the highly virulent Sclerospora graminicola pathotype-1. Twenty-seven S. graminicola isolates were screened using 20 inter simple sequence repeats (ISSR). Dinucleotide repeat primer [17898A-(CA)6 AC] amplified a ∼600 bp fragment specific to five isolates of pathotype-1 (Sg 048, Sg 153, Sg 212, DM-11 and DM-90). The ISSR fragment linked with pathotype-1 was cloned successfully and sequenced. To convert ISSR fragments into pathotype-specific sequence characterised amplified region (SCAR) markers, PCR primers were designed using a sequence of the cloned DNA fragment. PCR amplification using SCAR primer pair (UOM3-Sg-Path1-F/R) amplified a single 284 bp band only in isolates of S. graminicola pathotype-1. This SCARprimer pair did not amplify the 284 bp product from the other five S. graminicola pathotypes or a negative control, which demonstrates primer specificity for pathotype-1. The SCAR primer pair (UOM3-Sg-Path1-F/R) obtained in this study will provide a valuable tool for rapid identification and specific detection of S. graminicola pathotype-1.

Anti-oomycete compounds from Ganoderma appalantum, a wood rot basidiomycete

Solvent extracts of 17 different basidiomycetes were tested for their ability to inhibit Sclerospora graminicola. Among those tested, only three basidiomycete crude extracts exhibited significant inhibition on pathogen sporulation, zoospore release and zoospore motility. In vitro, the chloroform extract of Ganoderma appalantum (3 mg mL−1) recorded a maximum sporangial inhibition of 52.5 and 82.0% zoospore release and 92.5% motility. Crude extracts of three different basidiomycete fungi were treated to pearl millet seeds and assessed for seed germination, seedling vigour and effectiveness against downy mildew disease under greenhouse conditions. None of the solvent extracts were found to be phytotoxic. Chloroform and petroleum ether extracts of G. appalantum (3 mg mL−1) proved to be the best by offering disease protection of 55.6 and 43.7%, respectively, followed by chloroform and petroleum ether extracts of Polyphorus spp., with 42.5 and 39.1% disease protection. The thin layer chromatography (TLC) spots that developed were eluted and tested for inhibition of S. graminicola zoosporangia. The partially purified compound from TLC chloroform extract of G. appalantum consistently showed good inhibitory effect against S. graminicola, which exhibited inhibition of sporangia (42.3%), zoospore release (76.7%) and zoospore motility (86.7%), compared to the chloroform control which offered only 1.9, 2.9 and 1.7% inhibition of sporangia, zoospore release and zoospore motility, respectively. The partially purified compound from petroleum ether extract of G. appalantum resulted in 38.0, 63.0 and 81.6% inhibition of sporangia, zoospore release and motility, respectively, compared to petroleum ether control. However, azoxystrobin 250 SC (2 µg mL−1) and apron 35 SD (0.015 mg mL−1) treated on sporangial suspension showed the highest inhibition of S. graminicola pathogen compared to chloroform and petroleum ether TLC fractions.

Osmopriming enhances pearl millet growth and induces downy mildew disease resistance.

Abstract The osmoconditioning effect on pearl millet seeds was tested with different concentrations among which 1% showed an optimum effect. Seeds osmoprimed with mannitol for 3 h followed by overnight drying offered maximum germination of 99% and seedling vigour of 1465 followed by PEG and glycerol. All the three osmopriming agents recorded a significant increase on growth parameters like height of the plant, leaf area, number of tillers per plant. The results indicated that mannitol offered a maximum positive effect followed by PEG and glycerol. Notably, osmopriming has advanced five days of flowering and also has a positive effect on number and length of the earheads. The osmoconditioning agent increased the 1000 seed weight significantly over the untreated control. When the osmoprimed seeds were germinated and inoculated with downy mildew pathogen, the maximum protection of 61.76% was observed in mannitol treatment followed by PEG and glycerol under greenhouse conditions. Osmopriming has promoted seed germination percentage, seedling vigour index and growth parameters in pearl millet and also reduced downy mildew incidence.

First report of the seed-borne nature of root and collar rot disease caused by Rhizoctonia solani in sunflower from India

This is the first report of the seed-borne nature of root and collar rot disease caused by Rhizoctonia solani in sunflower from India. The disease incidence has increased from 17% in 2006 to 21% in 2008. Consequently, disease monitoring and management measures need to be taken.