Publications by authors named "Roshan Sharma Poudel"

10 Publications

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Expansion of internal hyphal growth in Fusarium Head Blight infected grains contribute to the elevated mycotoxin production during the malting process.

Mol Plant Microbe Interact 2021 Mar 15. Epub 2021 Mar 15.

North Dakota State University, 3323, Department of Plant Sciences, Fargo, North Dakota, United States;

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Worldwide surveys of commercial beers have reported that the trichothecene mycotoxin deoxynivalenol (DON) is the most frequent contaminant in beer. Although the DON content of grain generally declines during steeping due to its solubilization, can continue to grow and produce DON from steeping through the early kilning stage of malting. DON present on malt is largely extracted into beer. The objective of the current study was to localize the growth of within FHB infected kernels by developing an improved method and to associate fungal growth with the production of DON during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in the amount of DNA and trichothecene mycotoxins following malting were screened for hyphal localization. The growth of fungal hyphae associated with grain and malt was imaged by scanning electron microscope and confocal laser scanning microscope assisted with WGA-Alexa Fluor 488 staining, respectively. In barley, hyphae were present on or within the husk, vascular bundle, and pericarp cavities. Following malting, vast hyphal growth was observed not only in these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. However, these grains already had an extensive internal presence of hyphae in the unmalted grain, thus representing an enhanced chance of fungal expansion during the malting.
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http://dx.doi.org/10.1094/MPMI-01-21-0024-RDOI Listing
March 2021

Identification of fungal species associated with crown and root rots of wheat and evaluation of plant reactions to the pathogens in North Dakota.

Plant Dis 2021 Mar 1. Epub 2021 Mar 1.

North Dakota State University, Plant Pathology, Walster Hall 306, NDSU Dept. 7660, Fargo, North Dakota, United States, 58108-6050;

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on sub-crown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared to the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. Bipolaris sorokiniana and Fusarium species including F. acuminatum, F. avenaceum, F. culmorum, F. graminearum, F. equiseti, F. pseudograminearum, F. oxysporum, F. redolens, F. sporotrichioides, and F. solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were non-pathogenic and considered as secondary invaders associated with the root and crown rot diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with root and crown rots of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated.
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http://dx.doi.org/10.1094/PDIS-11-20-2412-REDOI Listing
March 2021

The Barley Transcription Factor Is Required for Resistance Against f. .

Front Genet 2020 15;11:601500. Epub 2021 Jan 15.

Department of Plant Pathology, North Dakota State University, Fargo, ND, United States.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen f. () has emerged as a global threat to barley production and diverse populations of have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised resistance were identified from an M population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of in barley line CI5791 resulted in susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that functions as a conserved signaling component of defense mechanisms that restricts growth in barley.
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http://dx.doi.org/10.3389/fgene.2020.601500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844392PMC
January 2021

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

Viruses 2020 06 10;12(6). Epub 2020 Jun 10.

United States Department of Agriculture, Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58102, USA.

"Rhizomania" of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, . Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus (genus ; family ). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of . Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.
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http://dx.doi.org/10.3390/v12060626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354460PMC
June 2020

Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance.

BMC Genomics 2019 Dec 16;20(1):985. Epub 2019 Dec 16.

Department of Crop and Soil Sciences, Washington State University, Pullman, WA, USA.

Background: Stem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidate Puccinia graminis f. sp. tritici (Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifying Pgt elicitors that interact with the rpg4-mediated resistance locus (RMRL), the only effective source of Pgt race TTKSK resistance in barley.

Results: Thirty-seven Pgt isolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process. In planta RNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to the Pgt race SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent -vs- the 15 RMRL avirulent Pgt isolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses.

Conclusions: This transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses.
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http://dx.doi.org/10.1186/s12864-019-6369-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915985PMC
December 2019

Microscopic, Biochemical, and Molecular Comparisons of Moderately Resistant and Susceptible Genotypes Inoculated with .

Phytopathology 2019 Dec 5;109(12):2074-2086. Epub 2019 Nov 5.

Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331.

, the causal agent of Septoria leaf spot and stem canker, is responsible for mortality and yield loss in plantations. However, little is known about the mode of infection and the mechanisms of resistance in this pathosystem. To characterize these phenomena, microscopic, biochemical, and transcriptome comparisons were performed between leaves of moderately resistant and susceptible genotypes of inoculated with conidia. Using scanning electron, cryofracture, and laser-scanning confocal microscopy, the infection and colonization of leaves by were examined across five time points (48 h, 96 h, 1 week, 2 weeks, and 3 weeks). The infection process was similar regardless of the host genotype. Differences in host colonization between susceptible and moderately resistant genotypes were apparent by 1 week postinoculation. However, the germination of conidia was greater on the susceptible than on the moderately resistant genotype ( < 0.008). Diaminobenzidine staining, a measure of hydrogen peroxide accumulation, was different ( < 0.001) between the host genotypes by 2 weeks postinoculation. Transcriptome differences between genotypes indicated that the speed and amplitude of the defense response were faster and more extensive in the moderately resistant genotype. Changes in gene expression support the microscopic and biochemical observations.
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http://dx.doi.org/10.1094/PHYTO-03-19-0105-RDOI Listing
December 2019

Mapping of barley susceptibility/resistance QTL against spot form net blotch caused by Pyrenophora teres f. maculata using RIL populations.

Theor Appl Genet 2019 Jul 20;132(7):1953-1963. Epub 2019 Mar 20.

Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108-6050, USA.

Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an important disease of barley worldwide including the major barley production regions of North America. To characterize SFNB resistance/susceptibility quantitative trait loci (QTL), three recombinant inbred line (RIL) populations were developed from crosses between the malting barley cultivars, Tradition (six row) and Pinnacle (two row), and the two world barley core collection lines, PI67381 and PI84314. Tradition and Pinnacle were susceptible to many North American Ptm isolates, while PI67381 and PI84314 carry resistances to diverse Ptm isolates from across the globe. The RIL populations, Tradition/PI67381, Pinnacle/PI67381, and Pinnacle/PI84314 were genotyped using polymerase chain reaction-mediated genotype-by-sequencing single nucleotide polymorphism marker panels and phenotyped at the seedling stage with six geographically distinct Ptm isolates: FGOB10Ptm-1 (North Dakota, USA), Pin-A14 (Montana, USA), Cel-A17 (Montana, USA), SG1 (Australia), NZKF2 (New Zealand) and DEN2.6 (Denmark). The goal was to determine if the susceptible elite lines contained common susceptibility genes/QTL or if the resistant lines had common resistant genes/QTL effective against diverse Ptm isolates. The QTL analyses identified a total of 12 resistance and/or susceptibility loci on chromosomes 2H, 3H, 4H, 6H, and 7H of which three had not been previously reported. Common major QTL were detected on chromosome 2H (R = 14-40%) and 7H (R = 24-80%) in all three RIL populations, suggesting underlying genes with broad resistance specificity. The major 7H QTL was shown to be a dominant susceptibility gene in both susceptible malting barley varieties.
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http://dx.doi.org/10.1007/s00122-019-03328-xDOI Listing
July 2019

Pyramiding - and -Mediated Stem Rust Resistance in Barley Requires the Gene for Both to Function.

Front Plant Sci 2018 5;9:1789. Epub 2018 Dec 5.

Department of Plant Pathology, North Dakota State University, Fargo, ND, United States.

Stem rust, caused by f. sp. () is an economically important disease of wheat and barley. is the only resistance gene deployed in Midwestern US barley varieties and provides remarkable resistance to most North American races, except race QCCJB. is also ineffective against race TTKSK and its lineage that originated in Africa. The barley -mediated resistance locus (RMRL) conferring resistance to races QCCJB and TTKSK was isolated from line Q21861, which is resistant to all known races due to and RMRL. To develop elite barley varieties RMRL was pyramided into the varieties, Pinnacle and Conlon (both contain ), producing the near isogenic lines (NILs), Pinnacle RMRL-NIL (PRN) and Conlon RMRL-NIL (CRN). The CRN was resistant to races QCCJB (RMRL specific) and HKHJC ( specific) at the seedling stage and race TTKSK (RMRL specific) at the adult stage. In contrast, PRN was susceptible to QCCJB and HKHJC at the seedling stage and TTKSK at the adult stage. Interestingly, PRN's susceptibility to QCCJB and HKHJC showed that RMRL was non-functional in the Pinnacle background but its presence also suppressed -mediated resistance. Thus, in the absence of a gene/s found in the Q21861 background, becomes non-functional if RMRL is present, suggesting that another polymorphic gene, that we designated (required for -mediated resistance 1), is required for RMRL resistance and -mediated resistance in the presence of RMRL. Utilizing a PRN/Q21861 derived recombinant inbred line (RIL) population, was delimited to a ∼0.5 MB physical region, slightly proximal (∼1.8 MB) of RMRL on barley chromosome 5H. A second gene, designated required for -mediated resistance 2 (), with duplicate gene action to in -mediated resistance function, was genetically delimited to a physical region of ∼0.7 MB, slightly distal (∼3.1 MB) to on the short arm of barley chromosome 7H. Thus, is required for RMRL resistance and or is required for functional -mediated resistance in the presence of the RMRL introgression. Candidate and genes were identified that need to be considered when pyramiding and RMRL in barley.
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http://dx.doi.org/10.3389/fpls.2018.01789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290389PMC
December 2018

Identification and Validation of a New Source of Low Grain Cadmium Accumulation in Durum Wheat.

G3 (Bethesda) 2018 03 2;8(3):923-932. Epub 2018 Mar 2.

Department of Plant Sciences, North Dakota State University, Fargo, North Dakota 58108

Cadmium (Cd) is a heavy metal that has no known biological function and is toxic for many living organisms. The maximum level of Cd concentration allowed in the international market for wheat grain is 0.2 mg kg Because phenotyping for Cd uptake is expensive and time consuming, molecular markers associated with genes conferring low Cd uptake would expedite selection and lead to the development of durum cultivars with reduced Cd concentrations. Here, we identified single nucleotide polymorphisms (SNPs) associated with a novel low Cd uptake locus in the durum experimental line D041735, which has hexaploid common wheat in its pedigree. Genetic analysis revealed a single major QTL for Cd uptake on chromosome arm 5BL within a 0.3 cM interval flanked by SNP markers. Analysis of the intervening sequence revealed a gene with homology to an aluminum-induced protein as a candidate gene. Validation and allelism tests revealed that the low Cd uptake gene identified in this study is different from the closely linked gene, which also resides on 5BL. This study therefore showed that the durum experimental line D041735 contains a novel low Cd uptake gene that was likely acquired from hexaploid wheat.
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http://dx.doi.org/10.1534/g3.117.300370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844312PMC
March 2018

Characterization of recombinants of the Aegilops peregrina-derived Lr59 translocation of common wheat.

Theor Appl Genet 2015 Dec 4;128(12):2403-14. Epub 2015 Aug 4.

Department of Plant Sciences, North Dakota State University, Fargo, ND, 58108, USA.

Key Message: A compensating, recombined Lr59 translocation with greatly reduced alien chromatin was identified. Microsatellite locus Xdupw217 occurs within the remaining segment and can be used as a co-dominant marker for Lr59. In earlier studies, leaf rust (caused by Puccinia triticina Eriks.) resistance gene Lr59 was transferred from Aegilops peregrina (Hackel) Maire et Weiler to chromosome arm 1AL of common wheat (Triticum aestivum L.). The resistance gene was then genetically mapped on the translocated chromosome segment following homoeologous pairing induction. Eight recombinants that retained the least alien chromatin apparently resulted from crossover within a terminal region of the translocation that was structurally different from 1AL. These recombinants could not be differentiated by size, and it was not clear whether they were compensating in nature. The present study determined that the distal part of the original translocation has group 6 chromosome homoeology and a 6BS telomere (with the constitution of the full translocation chromosome being 1AS·1L(P)·6S(P) ·6BS). During the allosyndetic pairing induction experiment to map and shorten the full size translocation, a low frequency of quadrivalents involving 1A, the 1A translocation, and two 6B chromosomes was likely formed. Crossover within such quadrivalents apparently produced comparatively small compensating alien chromatin inserts within the 6BS satellite region on chromosome 6B of seven of the eight recombinants. It appears that the Gli-B2 storage protein locus on 6BS has not been affected by the recombination events, and the translocations are therefore not expected to affect baking quality. Simple sequence repeat marker results showed that Lr59-151 is the shortest recombinant, and it will therefore be used in breeding. Marker DUPW217 detects a homoeo-allele within the remaining alien chromatin that can be used for marker-assisted selection of Lr59.
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http://dx.doi.org/10.1007/s00122-015-2594-xDOI Listing
December 2015