A Diagnostic Guide for Basil Downy Mildew

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is one of the most important diseases affecting sweet basil worldwide. Field- and greenhouse-grown basil may be affected, and crop losses are observed as the reduction of marketable leaves during both the production and postharvest handling stages. As an obligate biotroph, P. belbahrii cannot be cultured and maintained without live plant tissue, which may complicate efforts to diagnose and identify the causal agent. Thus, the goal of this diagnostic guide is to outline the appropriate methods required to identify basil downy mildew based on the symptoms of the disease and signs of the pathogen. Additionally, methods for pathogen identification, pathogen isolation, storage of single-sporangium cultures on live plants, and pathogenicity testing are described in detail.

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Morphological Characteristics and Susceptibility of Basil Species and Cultivars to Peronospora belbahrii

HortScience ◽

10.21273/hortsci09778-16 ◽

2016 ◽

Vol 51 (11) ◽

pp. 1389-1396 ◽

Cited By ~ 5

Author(s):

Kathryn Homa ◽

William P. Barney ◽

Daniel L. Ward ◽

Christian A. Wyenandt ◽

James E. Simon

Keyword(s):

United States ◽

Downy Mildew ◽

Stomatal Density ◽

Morphological Characteristics ◽

The United States ◽

Ocimum Sanctum ◽

Phenotypic Traits ◽

Sweet Basil ◽

Oomycete Pathogen ◽

Stomatal Length

Sweet basil (Ocimum basilicum) is the most economically important culinary herb in the United States. In 2007, a new disease, basil downy mildew (BDM), caused by the oomycete pathogen Peronospora belbahrii, was introduced into the United States and has since caused significant losses in commercial basil production. Although no commercial sweet basils available are resistant to P. belbahrii, other species of Ocimum have exhibited potential tolerance, resistance, or both. The objectives of this work were to determine if leaf morphological characteristics including stomata density and leaf curvature correlated with infection of plants by P. belbahrii, and thus could be used as selected characters in plant breeding. In 2011, 20 Ocimum cultivars including sweet (O. basilicum), cinnamon (O. basilicum), clove (O. basilicum), citrus (Ocimum ×africanum syn. Ocimum citriodorum), spice (Ocimum americanum syn. Ocimum canum), and holy basils (Ocimum tenuiflorum syn. Ocimum sanctum) were evaluated for susceptibility to downy mildew. Sweet basils were determined to be the most susceptible; cinnamon, clove, and Thai types were moderately susceptible; and citrus, spice, and holy types were least susceptible to downy mildew. Using those same 20 Ocimum species and cultivars, stomata length and density and leaf curvature were measured and correlated with downy mildew incidence and severity. In general, basil species with higher stomatal densities had higher downy mildew incidence and severity. High stomatal densities were mainly found in the sweet, cinnamon, and clove basils. Citrus and spice species with longer stomatal lengths generally exhibited lower downy mildew incidence. Holy basil, the least susceptible of all Ocimum sp. to P. belbahrii evaluated in this study, had the greatest stomatal density and shortest stomatal length. Some sweet basil cultivars with the highest downy mildew incidence also had the greatest downward leaf curvature, whereas other sweet basil cultivars with moderate downy mildew incidence had leaves that were nearly flat or curved upward. Holy, citrus, and spice basils with low downy mildew incidence had leaves that were nearly flat or curved upward. This study suggests that leaf curvature and stomatal density and length affect downy mildew development and sporulation. Considerations of these leaf morphological characteristics may be useful phenotypic traits in breeding for downy mildew resistance in Ocimum.

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CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance

PLoS ONE ◽

10.1371/journal.pone.0253245 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0253245

Author(s):

Jeremieh Abram R. Hasley ◽

Natasha Navet ◽

Miaoying Tian

Keyword(s):

Downy Mildew ◽

Susceptibility Gene ◽

Downy Mildew Resistance ◽

Mildew Resistance ◽

Sweet Basil ◽

Guide Rnas ◽

Oomycete Pathogen ◽

Hyaloperonospora Arabidopsidis ◽

S Genes ◽

Downy Mildew Disease

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.

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Co-Occurrence of Two Phylogenetic Clades of Pseudoperonospora cubensis, the Causal Agent of Downy Mildew Disease, on Oriental Pickling Melon

Mycobiology ◽

10.1080/12298093.2021.1880310 ◽

2021 ◽

pp. 1-8

Author(s):

Dong Jae Lee ◽

Jae Sung Lee ◽

Young-Joon Choi

Keyword(s):

Downy Mildew ◽

Causal Agent ◽

Pseudoperonospora Cubensis ◽

Downy Mildew Disease

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The zoospore of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

Nordic Journal of Botany ◽

10.1111/j.1756-1051.1989.tb00529.x ◽

1989 ◽

Vol 8 (5) ◽

pp. 511-516 ◽

Cited By ~ 8

Author(s):

Lene Lange ◽

Ulla Edén ◽

Lauritz W. Olson

Keyword(s):

Downy Mildew ◽

Causal Agent ◽

Pseudoperonospora Cubensis ◽

Cucurbit Downy Mildew

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The First Report of Downy Mildew Caused by Peronospora belbahrii on Sweet Basil in Greenhouses in the Czech Republic

Plant Disease ◽

10.1094/pdis-07-13-0747-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1579-1579 ◽

Cited By ~ 3

Author(s):

I. Šafránková ◽

L. Holková

Keyword(s):

Czech Republic ◽

Downy Mildew ◽

Width Ratio ◽

Specific Sequence ◽

The Czech Republic ◽

First Report ◽

Sweet Basil ◽

Potted Plants ◽

Length Width ◽

Necrotic Spots

Sweet basil (Ocimum basilicum L.) is an aromatic plant that is cultivated as a pot plant in greenhouses or in fields in the Czech Republic. The plants are intended for direct consumption or for drying. In April of 2012, the first large chlorotic from the middle necrotic spots occurred gradually on leaves of pot plants O. basilicum cv. Genovese in greenhouses in Central Bohemia. The characteristic gray to brown furry growth of downy mildew appeared on abaxial surfaces of leaves in the place of chlorotic spots within 3 to 4 days. The infested leaves fell off in the late stages of pathogenesis. The infestation gradually manifested itself in ever-younger plants and in July, cotyledons and possibly the first true leaves were already heavily infected and damaged and these plants rapidly died. The plant damage reached 80 to 100%, so it was necessary to stop growing the plants in the greenhouse at the end of July. The causal agent was isolated and identified as Peronospora belbahrii Thines by means of morphological and molecular characters (2,3). Conidiophores were hyaline, straight, monopodial, 280 to 460 μm, branched three to five times, ended with two slightly curved branchlets with a single conidia on each branchled tip. The longer branchlets measured 13 to 24 μm (average 18.2 μm), the shorter one 4 to 15 μm (average 9.7 μm). Conidia were rounded or slightly ovoid, from brownish to dark brownish, measured 22 to 31 × 20 to 28 μm (length/width ratio 1.2). A pathogen-specific sequence was detected with the help of the pathogen ITS rDNA specific primers in symptomatic leaves (1). DNA from plant tissues was isolated using the DNeasy plant Mini Kit (Qiagen, Germany) following the standard protocol. PCR was performed using KAPA2G Robust HotStar kit (Kapa Biosystems, United States) according to the conditions recommended in Belbahri et al. (1). The specific products were visualized by electrophoresis through 1.5% agarose gels. Leaves of 20-day-old potted plants O. basilicum ‘Genovese’ were inoculated by spraying with 5 × 105 conidia/ml of the pathogen. Each pot contained 10 plants. Sterilized distilled water was applied to control plants. Plants were covered with polyethylene bags during the entire incubation period to maintain high humidity, and kept at a temperature of 22 to 24°C. Typical disease symptoms appeared on leaves 5 to 9 days after inoculation. Control plants were symptomless. P. belbahrii was re-isolated from the lesions of inoculated plants, thus fulfilling Koch's postulates. Downy mildew on sweet basil was reported in countries in Africa, Europe, and South and North America (4). To our knowledge, this is the first report of downy mildew on sweet basil in the Czech Republic. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) Y.-J. Choi et al. Mycol. Res. 113:1340, 2009. (3) M. Thines et al. Mycol. Res. 113:532, 2009. (4) C. A. Wyenandt et al. HortScience 45:1416, 2010.

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