Development of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp. oleifera)

doi:10.1006/pmpp.1999.0207

Physiological and Molecular Plant Pathology

Volume 55, Issue 2, August 1999, Pages 111-119

https://doi.org/10.1006/pmpp.1999.0207 Get rights and content

Abstract

Two sets of PCR primers were designed from DNA sequence which flanked the 3′ end of thePyrenopeziza brassicae mating type idiomorphs (MAT-1 and MAT-2). The first set of primers facilitated the amplification of a 750 bp product from genomic DNA of 50 isolates of P. brassicae , which included both mating types. The 750 bp PCR product was also amplified from DNA extracted from Brassica napus tissue infected with P. brassicae . The 750 bp PCR product was not produced when these diagnostic primers were used in PCR reactions containing DNA from a number of other fungal species, including fungal pathogens of oilseed rape and Tapesia yallundae , which is closely related to P. brassicae . The second set of primers were designed to amplify different sized products from the two mating types of P. brassicae . These mating type specific primers facilitated the amplification of a 440 bp product from all MAT-1 isolates tested and a 535 bp product from 30% of the MAT-2 isolates tested. The value of molecular diagnostic techniques for the identification of P. brassicae in oilseed rape crops, for distinguishing between mating types of the fungus and in disease forecasting is discussed.

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The polymerase chain reaction (PCR) technology has been used to rapidly detect, characterize, and identify a variety of organisms. In a PCR diagnosis study, the development of PCR primers is one of the most important steps. This paper reviews several major approaches that have been used successfully for designing PCR primers specific to various phytopathogenic fungi. These approaches include using species-specific genes or DNA regions, or anonymous unique DNA regions to design PCR primers. Since the problem with PCR inhibitors is prevalent in PCR diagnosis of plant diseases, we also review various techniques that have been used to circumvent PCR inhibitors derived from plant tissues, soil, air, and water samples.
Development of a PCR-based mating-type assay for Clavicipitaceae
2004, FEMS Microbiology Letters
This study developed a PCR-based mating-type assay for Clavicipitaceae. PCR primer sets for the mating-type genes MAT1-1-1 and MAT1-2-1 were designed based on the amino acid sequences of the conserved alpha and HMG boxes, respectively. The PCR-based mating-type assay could be applied for various clavicipitaceous genera (Balansia, Claviceps, Cordyceps and Epichloë). Most of the clavicipitaceous fungi possessed either MAT1-1-1 or MAT1-2-1, and were supposed to be heterothallic. Although the PCR products obtained by the mating-type assay were short, the phylogenetic trees of the mating-type genes gave better resolutions than that of 18S rDNA and agreed well to their econutritional modes.
Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.
2003, Fungal Genetics and Biology
Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny
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A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis
2001, Fungal Genetics and Biology
A multiplex PCR test for determining mating type of the pathogens Tapesia yallundae and Tapesia acuformis is described. The test involves three primers: a “common” primer annealing to DNA sequence conserved in the flanking region of both mating-type idiomorphs and two specific primers annealing to sequence in either the MAT-1 or the MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded PCR products of 812 or 418 bp from MAT-1 and MAT-2 isolates, respectively. The test was used successfully to determine the mating type of 118 isolates of T. yallundae and T. acuformis from Europe, North America, and New Zealand. Isolates of both mating types were found on all continents for both species despite the rarely observed occurrence of sexual reproduction of T. acuformis. The multiplex test design should be applicable to other ascomycete species, of use in studies of MAT distribution and facilitating sexual crossing by identifying compatible isolates.
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2020, Scientia Agricultura Sinica

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^f1: All correspondence should be addressed to: A. M. Ashby, email: [email protected]; Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.

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Physiological and Molecular Plant Pathology

Abstract

References (28)

A molecular view through the looking glass: the Pyrenopeziza brassicae-brassica interaction

Advances in Botanical Research Incorporating Advances in Plant Pathology

Two-allele heterothallism in Tapesia yallundae : the teleomorph of the cereal eyespot pathogenPseudocercosporella herpotrichoides.

Physiological and Molecular Plant Pathology

Heterothallism in Pyrenopeziza brassicae : cause of light leaf spot of brassicas

Transactions of the British Mycological Society

Development of a PCR assay to identify and quantify Microdochium nivale var. nivale andMicrodochium nivale var. majus in wheat

Physiological and Molecular Plant Pathology

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Assessment of benomyl resistance and mating type in field isolates of Pyrenopeziza brassicae : cause of light leaf spot of brassicas

Plant Pathology

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Annals of Applied Biology

Prospects for developing a forecasting scheme to optimise use of fungicides for disease control on winter oilseed rape in the U.K

Aspects of Applied Biology

Diagnosis of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the U.K

Annals of Applied Biology

Benzimidazole resistance in Pseudocercosporella herpotrichoides : the cause of eyespot disease in cereals

International Society of Plant Pathology Chem Contr Newsletter

Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein

Applied and Environmental Microbiology

Survey of benomyl resistance in Fusarium species on winter wheat in England and Wales in 1986

Plant Pathology

Cited by (28)

Approaches for eliminating PCR inhibitors and designing PCR primers for the detection of phytopathogenic fungi

Development of a PCR-based mating-type assay for Clavicipitaceae

Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Detection of airborne fungal spores sampled by rotating-arm and Hirst-type spore traps using polymerase chain reaction assays

A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis

Establishment and application of a multiple PCR method to detect mating types of exserohilum turcicum and bipolaris maydis

Original articleDevelopment of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp. oleifera)

Abstract

Advances in Botanical Research Incorporating Advances in Plant Pathology

Physiological and Molecular Plant Pathology

Transactions of the British Mycological Society

Physiological and Molecular Plant Pathology

Assessment of benomyl resistance and mating type in field isolates of Pyrenopeziza brassicae : cause of light leaf spot of brassicas

Plant Pathology

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Annals of Applied Biology

Prospects for developing a forecasting scheme to optimise use of fungicides for disease control on winter oilseed rape in the U.K

Aspects of Applied Biology

Diagnosis of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the U.K

Annals of Applied Biology

Benzimidazole resistance in Pseudocercosporella herpotrichoides : the cause of eyespot disease in cereals

International Society of Plant Pathology Chem Contr Newsletter

Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein

Applied and Environmental Microbiology

Survey of benomyl resistance in Fusarium species on winter wheat in England and Wales in 1986

Plant Pathology

Original article
Development of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp. oleifera)