Establishing inoculum threshold levels for Bean common mosaic virus strain blackeye cowpea mosaic infection in cowpea seed

Bean common mosaic virus strain blackeye cowpea mosaic (BCMV-BlCM) is an important seed-borne virus infecting cowpea and is transmitted both by seeds and aphids. Infected cowpea seeds can act as primary source of inoculum for disease epidemics. Four field experiments were conducted during 2003 - 2006 to assess the role of different amounts of seed-borne inoculum in the dissemination of BCMVBlCM virus in cowpea under field conditions. The identity of BCMV-BlCM was confirmed by ELISA and IC-RT-PCR. Plants infected at an early growth stage appeared to serve as the primary source for subsequent virus spread by aphids. The mean disease incidence during four field experiments reached88-93% in plots sown with 10% infected seed. The disease incidence in plots sown with 5% infected seed recorded 46-63% while for plants raised from 3 and 2% BCMV-BlCM seed infection, disease incidence reached 32-49% and 17-23%, respectively. Mean yield losses in terms of seed yield per plant from four field experiments were 74 and 54% for initial seed infection of 10 and 5%, respectively. Seed infection of 2% BCMV-BlCM incidence resulted in an average of 24% mean seed yield loss/plant-1. The infection appeared to decrease the seed yield in terms of number and size. The BCMV incidence in harvested seed ranged from 0.3 - 19% for the different levels of initial seed infection. The field experiments demonstrated that sowing > 1% BCMV-BlCM infected seed can lead to significant losses in grain yield, while the spread of BCMV-BlCM infection resulting from sowing 1% infected seed did not significantly decrease seed yield. The role of establishing damage or inoculum thresholds from BCMVBlCM seed-borne infections is discussed in the present study.Keywords: Cowpea, potyvirus, seed-borne virus, thresholds, yield los

Context Matters: Intertextuality and Voice in the Early Modern English Controversy about Women

This dissertation examines three clusters of works from the early modern English controversy about women--the debate about the merits and flaws of womankind--in order to argue that authors in the controversy took advantage of the malleability of women's voices to address issues beyond the worth of women. I depart from standard treatments of the controversy by giving priority to the intertextual contexts among works that engage with one another. Attending to the intertextual elements of this genre reveals the metapoetic concerns of the authors and the way such authors fashion their feminine apologists as discursive agents in order to express those concerns. Chapter 1 examines Edward Gosynhyll's sixteenth-century works in tandem with Geoffrey Chaucer's The Legend of Good Women and "The Wife of Bath's Prologue and Tale," arguing that Gosynhyll's revisions of Chaucer--revisions embodied by the feminine apologists in the texts--are integral to his project of establishing the controversy genre as multivalent and dialectical. The resulting metacommentary examines in a new light the age-old rhetorical tradition of exemplarity, a persuasive tool used in diverse literary genres. Chapter 2 considers the way the anonymous play Swetnam the Woman-Hater uses cross-voicing and cross-dressing to establish the performative nature of controversy conventions. In doing so, the play argues for the social benefits of abandoning essentialist logic in favor of gender performance, as such performance makes the role of apologist available to men and women alike. This cluster reconsiders the very processes by which a person--male or female--can be known to others. Finally, I trace John Taylor's use of the marginal woman in his controversy works in order to demonstrate the extent to which Taylor makes these women instrumental in establishing his own poetic and social identity. This project contributes to studies on the English controversy as well as to the field of early modern women and women's writing by arguing that authors found the genre generally and the woman's voice specifically to be fit vehicles for articulating poetic agendas beyond the immediate task of debating the nature of womankind

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen:Genome sequence of pearl millet downy mildew pathogen

Sclerospora graminicola pathogen is one of the most important biotic production constraints of pearl millet worldwide. We report a de novo whole genome assembly and analysis of pathotype 1. The draft genome assembly contained 299,901,251 bp with 65,404 genes. Pearl millet [Pennisetum glaucum (L.) R. Br.], is an important crop of the semi-arid and arid regions of the world. It is capable of growing in harsh and marginal environments with highest degree of tolerance to drought and heat among cereals (1). Downy mildew is the most devastating disease of pearl millet caused by Sclerospora graminicola (sacc. Schroet), particularly on genetically uniform hybrids. Estimated annual grain yield loss due to downy mildew is approximately 10?80 % (2-7). Pathotype 1 has been reported to be the highly virulent pathotype of Sclerospora graminicola in India (8). We report a de novo whole genome assembly and analysis of Sclerospora graminicola pathotype 1 from India. A susceptible pearl millet genotype Tift 23D2B1P1-P5 was used for obtaining single-zoospore isolates from the original oosporic sample. The library for whole genome sequencing was prepared according to the instructions by NEB ultra DNA library kit for Illumina (New England Biolabs, USA). The libraries were normalised, pooled and sequenced on Illumina HiSeq 2500 (Illumina Inc., San Diego, CA, USA) platform at 2 x100 bp length. Mate pair (MP) libraries were prepared using the Nextera mate pair library preparation kit (Illumina Inc., USA). 1 ?g of Genomic DNA was subject to tagmentation and was followed by strand displacement. Size selection tagmented/strand displaced DNA was carried out using AmpureXP beads. The libraries were validated using an Agilent Bioanalyser using DNA HS chip. The libraries were normalised, pooled and sequenced on Illumina MiSeq (Illumina Inc., USA) platform at 2 x300 bp length. The whole genome sequencing was performed by sequencing of 7.38 Gb with 73,889,924 paired end reads from paired end library, and 1.15 Gb with 3,851,788 reads from mate pair library generated from Illumina HiSeq2500 and Illumina MiSeq, respectively. The sequences were assembled using various assemblers like ABySS, MaSuRCA, Velvet, SOAPdenovo2, and ALLPATHS-LG. The assembly generated by MaSuRCA (9) algorithm was observed superior over other algorithms and hence used for scaffolding using SSPACE. Assembled draft genome sequence of S. graminicola pathotype 1 was 299,901,251 bp long, with a 47.2 % GC content consisting of 26,786 scaffolds with N50 of 17,909 bp with longest scaffold size of 238,843 bp. The overall coverage was 40X. The draft genome sequence was used for gene prediction using AUGUSTUS. The completeness of the assembly was investigated using CEGMA and revealed 92.74% proteins completely present and 95.56% proteins partially present, while BUSCO fungal dataset indicated 64.9% complete, 12.4% fragmented, 22.7% missing out of 290 BUSCO groups. A total of 52,285 predicted genes were annotated using BLASTX and 38,120 genes were observed with significant BLASTX match. Repetitive element analysis in the assembly revealed 8,196 simple repeats, 1,058 low complexity repeats and 5,562 dinucleotide to hexanucleotide microsatellite repeats.publishersversionPeer reviewe

Selection of downy mildew resistant somaclones, from a susceptible B line of pearl millet

Plants regenerated from seed-derived callus of a PNMS 6B line of pearl millet (Pennisetum glaucum (L.) R. Br.) were evaluated for their resistance induced by somaclonal variation for downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroter. Among the 201 lines regenerated, only 3 lines consistently proved highly resistant (free from disease incidence) for up to 5 generations; whereas, 17 lines were resistant (disease incidence ranging from 1 to 9%). Resistance was confirmed by testing the plants under both laboratory and field conditions. The plants were evaluated for their agronomic traits

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 similar to 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 SCAR primer 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

Infection processes of Sclerospora graminicola on Pennisetum glaucum lines resistant and susceptible to downy mildew

The host-pathogen interaction between pearl millet seedlings and Sclerospora graminicola, causing downy mildew disease, was studied. Two-day-old seedlings of pearl millet, with high resistance (0% of the plants systemically infected), resistance (1-10%), susceptibility (11-25%) and high susceptibility (> 25%) to downy mildew disease were inoculated with zoospores of the pathogen. Infected tissues were macerated in NaOH and stained with cotton blue in lactophenol to observe the differential behaviour in germination and colonization of zoospores in tissues of the coleoptile, mesocotyl and root regions of different lines of pearl millet. The formation of the infection structures by the pathogen such as appressoria, vesicles, infection hyphae, haustorial mother cells and haustoria, as well as the formation of papillae in the host cell walls was observed in the inoculated tissues. The percentage of host cells colonized by the pathogen, and the number of haustoria formed per 100 infected host cells were highest in the coleoptile, followed by the mesocotyl and root. The percentage of infected cells and the number of haustoria formed were least in highly resistant and highest in highly susceptible lines. The number of papillae, however, was maximum in highly resistant lines, the number reducing with increasing susceptibility of the host to the disease. These responses may be helpful in developing a rapid screening method to determine the resistance of pearl millet lines to downy mildew

Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease

Nitric oxide (NO) donors Nitroso-R-Salt, 2-Nitroso-1-Naphthol and Sodium Nitro Prusside (SNP) were evaluated for their effectiveness in protecting pearl millet (Pennisetum glaucum L.) R. Br. plants against downy mildew disease caused by Sclerospora graminicola (Sacc). Schroet. Optimization experiments with NO donors showed no adverse effect either on the host or pathogen. Aqueous SNP seed treatment with or without polyethylene glycol (PEG) priming was the most effective in inducing the host resistance against downy mildew both under greenhouse and field conditions. Potassium Ferrocyanide, a structural analog of NO donor lacking NO moiety failed to protect the pearl millet plants from downy mildew indicating a role for NO in induced host resistance. Spatio-temporal studies corroborated that the protection offered by NO donor treatment was systemic in nature and a minimum of 3-day time gap between the inducer treatment and subsequent pathogen inoculation was necessary for maximum resistance development. Disease protection ability of NO donors was also validated as durable in nature. Conversely, prior-treatment with NO scavenger 2-4-carboxyphenyl-4,4,5,5 tetrazoline-1-oxyl-3-oxide potassium salt (C-PTIO) rendered the pearl millet plants relatively susceptible for pathogen infection. Expression of primary defense responses like hypersensitive response, lignin deposition and defense related enzyme phenylalanine ammonialyase −EC 4.3.1.5 (PAL) were enhanced by NO donor treatments

Seed priming with plant gum biopolymers enhances efficacy of metalaxyl 35 SD against pearl millet downy mildew

`Priming' the plant and seed induces a physiological state in which plants are able to activate defense responses. Plant-based exudates are excellent gum biopolymers which contain plant growth-regulating hormones with priming potential without any side effects. In this study, gum exudates of Acacia arabica, Moringa oleifera, Carica papaya and Azadirachta indica were evaluated for synergistic effects of seed priming with exuded gum biopolymer combined with metalaxyl (Apron 35 SD) on pearl millet seed quality, growth parameters, and resistance to Sclerospora graminicola. Seeds of 7042S were primed with gum biopolymers and metalaxyl 35 SD and evaluated under laboratory and greenhouse conditions. Seed germination and vigor were synergistically enhanced using gum biopolymers solution (1:2 w/v) with 3 g kg(-1) metalaxyl 35 SD. A. arabica and A. indica gum biopolymers alone or with 3 g kg(-1) of metalaxyl 35 SD resulted in seed germination of > 91%. Seed priming with 6 g kg(-1) of metalaxyl 35 SD gave 89% seed germination and was not significantly different from control. A similar trend in vigor was observed among treatments. Seed priming with gum biopolymers alone provided varied disease protection levels when compared with the control. A. arabica or A. indica gum with 3 g kg(-1) of metalaxyl 35 SD was the superior treatment, offering significant 86% disease reduction while exhibiting a growth-promoting effect. Synergistic use of gum biopolymers and metalaxyl 35 SD by seed priming is highly effective in growth promotion and management of pearl millet downy mildew disease

Purification and partial characterization of manganese superoxide dismutase from downy mildew resistant pearl millet seedlings inoculated with Sclerospora graminicola

The Mn-SOD was purified from pearl millet by ammonium sulfate precipitation followed by column chromatography using DEAE-cellulose and Sephadex G-100. The isozyme has a molecular weight of 35 kDa. Electrophoresis revealed a single band of SOD activity corresponding to the purified enzyme. The purified enzyme was stable over a pH range of 7.0–9.0 for 24 h, and a temperature range of 20–35°C. The purified pearl millet SOD exhibited insensitivity to hydrogen peroxide and cyanide, which is typical of Mn-containing SOD. SOD was purified 73-fold from pearl millet