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Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster

1
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy
2
Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, EA 3882, IBSAM, ESIAB, Université de Brest, Technopôle Brest-Iroise, 29280 Plouzané, France
3
CABI Europe-UK, Bakeham Lane, Egham, Surrey TW20 9TY, UK
4
Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), University of Salamanca, Calle del Duero 12, 37185 Villamayor (Salamanca), Spain
*
Authors to whom correspondence should be addressed.
Plants 2019, 8(7), 222; https://doi.org/10.3390/plants8070222
Received: 20 June 2019 / Revised: 7 July 2019 / Accepted: 12 July 2019 / Published: 14 July 2019
(This article belongs to the Special Issue Interactions between Colletotrichum Species and Plants)
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Abstract

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01–0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed. View Full-Text
Keywords: IGS; ribosomal intergenic spacer; Colletotrichum acutatum; Lupinus; lupins; legumes; fungal pathogens IGS; ribosomal intergenic spacer; Colletotrichum acutatum; Lupinus; lupins; legumes; fungal pathogens
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Pecchia, S.; Caggiano, B.; Da Lio, D.; Cafà, G.; Le Floch, G.; Baroncelli, R. Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster. Plants 2019, 8, 222.

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