Genome-wide characterization of the NBLRR gene family provides evolutionary and functional insights into blast resistance in pearl millet (Cenchrus americanus (L.) Morrone).

MAIN CONCLUSION: The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Plants have evolved multiple integral mechanisms to counteract the pathogens' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host-pathogen interactions.

Elicitation of Novel Trichogenic-Lipid Nanoemulsion Signaling Resistance Against Pearl Millet Downy Mildew Disease.

Nanoemulsion was formulated from membrane lipids of Trichoderma spp. with the non-ionic surfactant Tween 80 by the ultrasonic emulsification method. Nanoemulsion with a droplet diameter of 5 to 51 nm was obtained. The possible effects of membrane lipid nanoemulsion on pearl millet (PM) seed growth parameters and elicitation of downy mildew (DM) disease resistance in PM was analyzed to develop an eco-friendly disease management strategy. Seed priming with nanoemulsion illustrates significant protection and elevated levels of early defense gene expression. Lipid profiling of Trichoderma spp. reveals the presence of oleic acid as a major fatty acid molecule. The prominent molecule in the purified lipid fraction of T. brevicompactum (UP-91) responsible for the elicitation of induction of systemic resistance in PM host against DM pathogen was predicted as (E)-N-(1, 3-dihydroxyoctadec-4-en-2yl) acetamide. The results suggest that protection offered by the novel nanoemulsion formulation is systemic in nature and durable and offers a newer sustainable approach to manage biotrophic oomycetous pathogen.

Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation.

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.

Elicitation of resistance and associated defense responses in Trichoderma hamatum induced protection against pearl millet downy mildew pathogen.

Endophytic Trichoderma hamatum UoM 13 isolated from pearl millet roots was evaluated for its efficiency to suppress downy mildew disease. Under laboratory conditions, T. hamatum seed treatment significantly enhanced pearl millet seed germination and seedling vigor. T. hamatum seed treatment resulted in systemic and durable immunity against pearl millet downy mildew disease under greenhouse and field conditions. T. hamatum treated seedlings responded to downy mildew infection with high lignification and callose deposition. Analysis of defense enzymes showed that T. hamatum treatment significantly enhanced the activities of glucanase, peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase in comparison to untreated control. RT-PCR analysis revealed differentially expressed transcripts of the defense enzymes and PR-proteins in treated, untreated, and checks, wherein PR-1, PR-5, and cell wall defense HRGPs were significantly over expressed in treated seedlings as against their lower expression in controls. T. hamatum treatment significantly stimulated endogenous salicylic acid (SA) levels and significantly upregulated important SA biosynthesis gene isochorismate synthase. The results indicated that T. hamatum UoM13 treatment induces resistance corresponding to significant over expression of endogenous SA, important defense enzymes, PR-proteins, and HRGPs, suggesting that SA biosynthetic pathway is involved in pearl millet for mounting systemic immunity against downy mildew pathogen.

Enhancement of downy mildew disease resistance in pearl millet by the G_app7 bioactive compound produced by Ganoderma applanatum.

Pearl millet (Pennisetum glaucum) stands sixth among the most important cereal crops grown in the semi-arid and arid regions of the world. The downy mildew disease caused by Sclerospora graminicola, an oomycete pathogen, has been recognized as a major biotic constraint in pearl millet production. On the other hand, basidiomycetes are known to produce a large number of antimicrobial metabolites, providing a good source of anti-oomycete agrochemicals. Here, we report the discovery and efficacy of a compound, named G_app7, purified from Ganoderma applanatum on inhibition of growth and development of S. graminicola, as well as the effects of seed treatment with G_app7 on protection of pearl millet from downy mildew. G_app7 consistently demonstrated remarkable effects against S. graminicola by recording significant inhibition of sporangium formation (41.4%), zoospore release (77.5%) and zoospore motility (91%). Analyses of G_app7 compound using two-dimensional nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry revealed its close resemblance to metominostrobin, a derivative of strobilurin group of fungicides. Furthermore, the G_app7 was shown to stably maintain the inhibitory effects at different temperatures between 25 and 80 °C. In addition, the anti-oomycete activity of G_app7 was fairly stable for a period of at least 12 months at 4 °C and was only completely lost after being autoclaved. Seed treatment with G_app7 resulted in a significant increase in disease protection (63%) under greenhouse conditions compared with water control. The identification and isolation of this novel and functional anti-oomycete compound from G. applanatum provide a considerable agrochemical importance for plant protection against downy mildew in an environmentally safe and economical manner.

Association between accumulation of allene oxide synthase activity and development of resistance against downy mildew disease of pearl millet.

The present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars.

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.

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.

Chitosan enhances disease resistance in pearl millet against downy mildew caused by Sclerospora graminicola and defence-related enzyme activation.

BACKGROUND: The present study investigated the effect of chitosan seed priming on the induction of disease resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroet. RESULTS: Pearl millet seeds were primed with chitosan at different concentrations: 0.5, 1.5, 2.5 and 3 g kg(-1) seed. Of the different concentrations, 2.5 g kg(-1) was found to be optimum, with enhanced seed germination of 99% and seedling vigour of 1782, whereas the untreated control recorded values of 87% and 1465 respectively. At optimum concentration, chitosan did not inhibit sporulation and release of zoospores from sporangia. Furthermore, pearl millet seedlings raised after seed treatment with chitosan showed an increased level of the defence-related enzymes chitosanase and peroxidase as compared with the untreated pearl millet seedlings on downy mildew pathogen inoculation. The effect of chitosan in reducing downy mildew incidence was evaluated in both greenhouse and field conditions, in which respectively 79.08 and 75.8% disease protection was obtained. CONCLUSION: Chitosan was effective in protecting pearl millet plants against downy mildew under both greenhouse and field conditions by inducing resistance against the pathogen. Thus, chitosan formulation can be recommended for seed treatment in the management of downy mildew disease.