Dataset Information

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Demand for fresh-market sweet basil continues to increase, but in 2009 a new pathogen emerged, threatening commercial field/greenhouse production and leading to high crop losses. This study describes a simple and effective staining method for rapid microscopic detection of basil downy mildew (Peronospora belbahrii) from leaves of basil (Ocimum basilicum). • METHODS AND RESULTS:Fresh leaf sections infected with P. belbahrii were placed on a microscope slide, cleared with Visikol™, and stained with iodine solution followed by one drop of 70% sulfuric acid. Cell walls of the pathogen were stained with a distinct coloration, providing a high-contrast image between the pathogen and plant. • CONCLUSIONS:This new staining method can be used successfully to identify downy mildew in basil, which then can significantly reduce its spread if identified early, coupled with mitigation strategies. This technique can facilitate the control of the disease, without expensive and specialized equipment.

SUBMITTER: Koroch AR

PROVIDER: S-EPMC4103132 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

Koroch Adolfina R AR Villani Thomas S TS Pyne Robert M RM Simon James E JE

Applications in plant sciences 20130705 7

<h4>Unlabelled</h4><h4>Premise of the study</h4>Demand for fresh-market sweet basil continues to increase, but in 2009 a new pathogen emerged, threatening commercial field/greenhouse production and leading to high crop losses. This study describes a simple and effective staining method for rapid microscopic detection of basil downy mildew (Peronospora belbahrii) from leaves of basil (Ocimum basilicum). •<h4>Methods and results</h4>Fresh leaf sections infected with P. belbahrii were placed on a m ...[more]

PMID: 25202569

Similar Datasets

Project description:Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive many disease resistant cultivars have been bred and used. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences can, therefore, be considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed from the metagenome. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.4 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RxLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the predicted secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemi-biotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved in cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.

Project description:Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61-68% compared to the wild-type plants and 69-93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.

Project description:Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.

Dataset Information

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

Publications

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets