Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR...

Full description

Saved in:

Bibliographic Details
Published in:	PloS one 2013-06, Vol.8 (6), p.e65990-e65990
Main Authors:	Inderbitzin, Patrik, Davis, R Michael, Bostock, Richard M, Subbarao, Krishna V
Format:	Article
Language:	eng
Subjects:	Actin Assaying Biology Deoxyribonucleic acid Design Diagnostic systems Disease control DNA DNA sequencing DNA, Fungal - genetics Elongation Fungi Genes Glyceraldehyde-3-phosphate dehydrogenase Hybridization Identification Multiplexing Nucleotide sequence Nucleotide sequencing Phosphates Phylogenetics Phylogeny Plant diseases Plant pathology Polymerase chain reaction Polymerase Chain Reaction - methods Primers Sequence Homology, Nucleic Acid Spacer region Species Tryptophan Tryptophan synthase Verticillium - classification Verticillium - genetics Verticillium dahliae
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.
ISSN:	1932-6203 1932-6203