Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

90
Mating Type-Specific PCR Primers for Stagonospora
nodorum Field Studies
Bennett, R.S., 1 S.-H. Yun, 1 T.Y. Lee, 1 B.G. Turgeon, 1 B. Cunfer, 2
E. Arseniuk, 3 and G.C. Bergstrom 1 * (Poster)
1 Department of Plant Pathology, Cornell University, Ithaca, NY, USA
2 Department of Plant Pathology, University of Georgia, Griffin, GA, USA
3 Plant Breeding and Acclimatization Institute, Radzików, Poland
* corresponding author
Abstract
Conserved regions of the mating type genes (MAT) in Cochliobolus heterostrophus, Mycosphaerella zeaemaydis,
and Alternaria alternata were used to design primers to identify the mating type genes and thus assign
mating types to Stagonospora nodorum. These primers successfully distinguished between mating types of S.
nodorum isolates that had been identified through traditional crosses. The primers were used to screen a small sample of
S. nodorum isolates from New York, thus revealing the presence of both mating types. This efficient method of
identifying mating types may help determine the role of sexual recombination in the epidemiology of S. nodorum, as
well as elucidate phylogenetic relationships among related species.
Stagonospora nodorum (Berk.)
Castellani & E.G. Germano
(teleomorph = Phaeosphaeria
nodorum (E. Müller) Hedjaroude) is
the causal agent of stagonospora
nodorum blotch, a major disease of
wheat around the world.
Phaeosphaeria nodorum is
heterothallic and thus requires
strains of the two different mating
types to produce ascospores
(Müller, 1989). The potential
significance of sexual reproduction
in the epidemiology of
stagonospora nodorum blotch has
been heightened by the high RFLP
variability found among field
isolates (McDonald et al., 1994).
Fungi that regularly reproduce
sexually have more opportunities
to develop fungicide resistance and
to overcome cultivar resistance.
Furthermore, airborne ascospores
would permit dissemination over
longer distances than splashdispersed
conidia.
MAT genes have been identified
for several species of
Loculoascomycetes including
Setosphaeria turcica, Mycosphaerella
zeae-maydis, Alternaria alternata, and
several Cochliobolus spp. (Arie et al.,
1997). Both genes, MAT-1 and MAT-
2, contain conserved regions that
allow the relatively rapid
identification of these genes from an
increasing group of ascomycetes. A
high mobility group (HMG) and abox
motif are the conserved regions
in MAT-2 and MAT-1, respectively
(Turgeon, 1998). These sequences,
besides elucidating sexual
mechanisms and phylogeny, may
also help address epidemiological
questions.
We have developed primers to
identify the mating types of P.
nodorum from the conserved MAT
regions of related fungi. Preliminary
data on the mating type prevalence
in a small sample of New York field
isolates is also presented.
Materials and Methods
Fungal isolates and media
Three S. nodorum isolates of
different mating types (two (-)
(SN435PL-98, SN437GA-98) and
one (+) (SN436GA-98)) (Arseniuk
et al., 1997a,b) were grown on V-8
juice agar (200 ml V8 juice, 3 g
CaCO3 , 15 g agar per 800 ml of
distilled water). After
approximately seven days, mycelial
plugs were taken and placed into
yeast-malt-sucrose broth (5 g yeast
extract, 5 g malt extract, 20 g
sucrose per 1 liter of distilled
water), and placed on a shaker (150
rpm) at room temperature. Mycelia
were harvested after 5-7 days by
filtering through 3-ply sterile
cheesecloth lining a Buchner funnel
and were rinsed with distilled
water. The samples were then
frozen and lyophilized overnight.
MAT-1 and MAT-2 C. heterostrophus
laboratory strains (C5 and C4,
respectively) were used as controls.