Abstract:
Grey leaf spot is an important maize foliar disease caused by the fungal pathogens Cercospora
zeae-maydis and Cercospora zeina. Although methods
exist to detect these Cercospora species in maize,
current techniques do not allow quantification of the
fungi in planta. We developed a real-time SYBR®
Green PCR assay for quantification of grey leaf spot
disease in maize based on the amplification of a fragment
of a cytochrome P450 reductase (cpr1) gene. In
planta fungal DNA content was normalised to a maize
glutathione S-transferase III gene (gst3) to yield values
was specific to the two Cercospora spp., and we
observed no amplification of the cpr1 fragment in
non-target maize leaf pathogens or saprophytes. The
assay was employed to quantify C. zeina in glasshouse
inoculated maize plants and grey leaf spot infected
field plants of resistant and susceptible maize lines.
In both instances, C. zeina DNA content correlated
with symptomatic leaf lesion area, and the susceptible
maize line contained significantly more C. zeina DNA
than the resistant line. Sequence differences between
the C. zeina and C. zeae-maydis cpr1 amplicons enabled
us to perform melt curve analyses to identify the
Cercospora species causing grey leaf spot at a particular
location. This assay has application in the early
detection and quantification of Cercospora spp., both
of which are important tools in grey leaf spot disease
management and maize breeding programmes.
