A multiplex TaqMan assay for detection and differentiation of Leptosphaeria maculans and L. biglobosa, causal agents of canola blackleg

• Published in: Phytopathology Vol. 100; no. 6; p. S69
• Main Authors: Leonard, W, Li, X, Smith, D S, Nie, J, Dumonceaux, T
• Format: Journal Article
• Language: English
• Published: 01.06.2010
• Subjects: Leptosphaeria maculans; Oryza sativa
• ISSN: 0031-949X

Blackleg of canola is caused by two related fungal species, Leptosphaeria maculans, a highly virulent species causing severe cankers at the base of stems, and L. biglobosa, a weakly virulent species causing mild stem lesions. For rapid detection and differentiation in canola seed, we designed a multiplex TaqMan assay that targeted two genomic loci of L. maculans, LopB, a pathogenicity gene and LMR1, a repetitive element linked to a virulence region, and the mating protein gene, Mat1-2, of L. biglobosa. The primers specifically amplified 110, 145, and 135 bp products from the three loci, respectively, and TaqMan probes were labelled with Quasar 670, Cal-Fluor-Red 610, or Cal-Fluor-Orange 560 fluorescent dyes and compatible quenchers, respectively. To validate negative results in the mutiplex PCR, a FAM-labelled probe to a 200-bp internal control, designed from a rice sequence bordered by LopB primer sequences, was included in the assay. The final assay involved a 48-hr enrichment of 4-g samples in a minimal medium with antibiotics, sample treatment in a beadbeater, and DNA extraction with magnesil paramagnetic beads prior to assay by multiplex PCR. Under optimized conditions the assay detected single infected seeds in a sample and differentiated between L. maculans and L. biglobosa. Positive detection and diagnosis was based on cycle threshold cutoff values and confirmation of amplicon identity by capillary electrophoresis analyses.