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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: The authors declare no conflict of interest pertaining to this research work.

Downy mildew (DM) caused by Sclerospora graminicola is the most calamitous disease of pearl millet. Therefore, for introgression of DM resistance (DMR) in HHB 197 (MH-1302), an elite pearl millet hybrid, a marker-assisted breeding was undertaken by targeting three DMR loci on linkage groups (LGs) 1, 2 and 4. Breeding programme was initiated by crossing HBL 11 (DM susceptible), male parent of HHB 197 hybrid with ICMP 451 (DM-resistant) to produce true F 1 plants. By conducting three rounds of backcrossing and selection, BC 3 F 1 lines were generated. Foreground selection was employed using six polymorphic simple sequence repeat (SSR) markers of the 18 total selected markers. Four of these markers were linked to LG 1, five to LG 2 and nine to LG 4. Background selection was performed in BC 3 F 1 generation using 33 polymorphic SSR markers of a total of 56 evenly spread SSR markers in the pearl millet genome to check recovery of recurrent parent genome. On the basis of genotypic selection (foreground as well as background) using selected SSR markers, agronomic performance in field and DM screening in greenhouse; 10 improved HBL 11 lines were selected and crossed with ICMA 97111 to produce DM-resistant HHB 197 hybrid versions. Six putatively improved HHB 197 hybrids were successfully tested in first year trials at Hisar and Bawal locations of Haryana and two selected versions with higher yield and zero DM incidence will be further tested in multilocation trials.

Pearl millet downy mildew (DM), caused by Sclerospora graminicola, is of serious economic concern to pearl millet farmers in the major crop-growing areas of the world. To study the inheritance and allelic relationship among genes governing resistance to this disease, three DM-resistant pearl millet lines (834B, IP 18294-P1, and IP 18298-P1) and one susceptible line (81B) were selected on the basis of disease reaction under greenhouse conditions against two isolates of S. graminicola (Sg 526-1 and Sg 542-1). Three resistant parents were crossed with the susceptible parent to generate F 1 , F 2 , and backcross BC 1 P 1 (susceptible parent × F 1 ) and BC 1 P 2 (resistant parent × F 1 ) generations for inheritance study. To carry out a test for allelism, the three resistant parents were crossed with each other to generate F 1 and F 2 generations. The different generations of these crosses were screened for disease reaction against two isolates (Sg 526-1 and Sg 542-1) by artificial inoculation under greenhouse conditions. The segregation pattern of resistance in the F 2 and corresponding backcross generations revealed that resistance to DM is controlled by a single dominant gene in 834B and IP 18294-P1 and by two dominant genes in IP 18298-P1. A test for allelism inferred that a single dominant gene for resistance in 834B is nonallelic to that which governs resistance in IP 18294-1, whereas one of the two dominant genes for DM resistance in IP 18298-P1 against the test isolates is allelic to the gene for DM resistance in 834B and a second gene is allelic to the resistance gene present in IP 18294-P1.

Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction.

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.

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., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

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.

Studies were undertaken to assess the induction of defence response pathways in pearl millet (Pennisetum glaucum) in response to infection with the leaf rust fungus Puccinia substriata. Pretreatment of pearl millet with salicylic acid (SA) conferred resistance to a virulent isolate of the rust fungus, whereas methyl jasmonate (MeJA) did not significantly reduce infection levels. These results suggest that the SA defence pathway is involved in rust resistance. In order to identify pearl millet genes that are specifically regulated in response to SA and not MeJA, and thus could play a role in resistance to P. substriata, gene expression profiling was performed. Substantial overlap in gene expression responses between the treatments was observed, with MeJA and SA treatments exhibiting 17% co-regulated transcripts. However, 34% of transcripts were differentially expressed in response to SA treatment, but not in response to MeJA treatment. SA-responsive transcripts represented genes involved in SA metabolism, defence response, signal transduction, protection from oxidative stress and photosynthesis. The expression profiles of pearl millet plants after treatment with SA or MeJA were more similar to one another than to the response during a compatible infection with P. substriata. However, some SA-responsive genes were repressed during P. substriata infection, indicating possible manipulation of host responses by the pathogen.

Few crop breeding programs today are breeding crops in their areas of diversity and origin. This study reports on a Malian breeding program in an area of genetic diversity. It has the objective to accumulate resistance to major populations of Sclerospora graminicola (= Sg) with modern breeding and selection methods. This study is part of the development of pearl millet top cross hybrids, with a reduced plant height, Sg-resistance (= resistance to pearl millet downy mildew) and 'stay green' at physiological maturity. The parent entries, among other relevant characteristics, were selected for a high level of resistance (good sources of resistance) making use of a combination of artificial young plant screening methods and single location field testing, in 1998. Pedigree selection in F1 to F4 was from 1999 to 2002. Its synthetics and composites were selected for low S. graminicola-levels, in 2003 to 2005 and in 2003 and 2006 tested for S. graminicola-resistance together with 5 checks at two Locations differing in S. graminicola-virulence responses. The 2006 test seemingly indicated the expected quadratic checks, whereby entry 1 is resistant at location 1 and susceptible at location 2 and entry 2 is susceptible at location 1 but resistant at location 2. This quadratic check is indicating differences in virulence between the two S. grominicola-populations and also an adaptation of the pathogen populations on the newly accumulated genes for resistance in the host. It is also indicating that one or more genes for resistance against each of the two populations were accumulated. A good number of synthetics and composites combined low S. graminicola-incidences with relatively high yields and some had 'stay green' at physiological maturity. One too late entry seemingly had immunity. The 2006 results indicate presence of several S. graminicola-resistance genes in the parent entries and accumulation of one or more genes in certain derived entries, and were obtained in combination with reduced plant height and for the first time in pearl millet also with 'stay green' at physiological maturity. The accumulation of S. graminicola-resistance is expected to increase the chance for regional or global 'stay green' hybrids for grain (medium tall) and fodder (tall).

Transgenic pearl millet lines expressing pin gene--exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola--were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T(0), 2T(0), and 3T(0) showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T(1) progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T(1) plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T(1) plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T(2) progenies of 1T(0) conformed to dihybrid segregation of 15 resistant:1 susceptible plants.

Induction of resistance to downy mildew in pearl millet was studied after treatment with L-methionine. The disease severity was drastically reduced in the seedlings after treatment with L-methionine. Northern hybridization was used to study the transcript (mRNA) accumulation of defense response genes upon treatment with L-methionine in pearl millet seedlings and the mechanism during the induction of resistance. The gene activation patterns in the highly resistant cultivar IP18294 were compared with those exhibited by the susceptible cultivar 7042S at various time intervals to determine if differences in timing or levels of transcript accumulation could be correlated with the differences in the susceptibility of pearl millet to the downy mildew pathogen, Sclerospora graminicola. The mRNA levels of genes for Pr-1a, beta-1,3-glucanase, chitinase, peroxidase, lipoxygenases and chalcone synthase showed an increase after inoculation with S. graminicola. Significant gene activation was observed in the susceptible cultivar 7042S after treatment with L-methionine followed by challenge inoculation with the downy mildew pathogen which was comparable to the resistant cultivar.