Journal of Plant Pathology - Sipav.org

S1.8 Fungicide sensitivity of Septoria tritici Journal of Plant Pathology (2011), 93 (1, Supplement), S1.7-S1.13
sensitivity of fungi towards DMIs (Leroux et al., 2007).
Furthermore, experimental evidence suggests that ABC
(ATP-Binding Casette)-transporters located in the outer
membrane of fungal cells are involved in fungicide resistance
of S. tritici by exporting the toxic compounds
(Stergiopoulos et al., 2003). DMIs were first introduced
in the 1970s (prochloraz 1977, propiconzole 1979, tebuconazole
1986, epoxiconazole 1990, and prothioconazole
2002; Russell, 2005).
In contrast to DMIs and QoIs, folpet and
chlorothalonil are non-systemic fungicides that are not
taken up by the plant in significant amounts (Tomlin,
2000). They belong to the group of multi-site inhibitors,
which interfere with various steps of fungal metabolism
(Tomlin, 2000). Folpet and chlorothalonil were introduced
in 1952 and 1964, respectively (Russell, 2005).
Northern Germany is a region with high yield/cropping
potential with an average yield of about 9 tons
wheat per ha (Stratmann et al., 2004). For comparison,
wheat yield was reported to be about 3 tons per ha for
the USA (OSU, 2007). The high levels of yield result
from fertile soil and sufficient rain on the one hand but
also from high inputs of fertilizers, pesticides, and labor
on the other hand. However, regular precipitation favors
the development of fungal populations in the field and
results in high disease pressure in most years (Ceynowa et
al., 1993; Verreet et al., 2000). Since fungicide resistance
probably develops in regions with high application frequencies
and low doses, fungicide resistance monitoring
and resistance management is particularly important in
these regions (Staub, 1991). Since the breakdown of QoI
efficacy, control of S. tritici depends almost solely on
azoles. Resistance management by alternating effective
fungicides with different modes of action is therefore extraordinarily
difficult with this pathogen.
It was the objective of the present study to determine
the recent trend in sensitivity of S. tritici toward selected
systemic azoles, a strobilurin and non-systemic contact
fungicides in a region with high yield and frequent use
of fungicides.
incubation period and the spores were diluted with sterilized
deionized water and transferred to plates containing
malt yeast extract agar (MYA, yeast extract, glucose,
malt extract, each component at 4 g l -1 plus 15 g l -1
agar). Colonies originating from a single spore were
transferred to new MYA plates and the spores produced
on that plate were washed off using 10% [w/v] sterile
skim milk and stored at -70°C until further use. Spores
used in the bioassays described below always originated
directly from the stock solution stored at -70°C. Fungal
strains transferred from plate to plate quickly ceased
spore production and grew as mycelium which was less
suitable for our bioassay due to its heterogeneity in liquid
media compared to the spores. Spores from the
stock solution were transferred to MYA plates and the
liquid was distributed over the medium surface using a
sterile triangle-shaped metal spreader rod. After 4 days
at room temperature, propagation of the spores resulted
in spore clusters that were washed off using sterile
deionized water and the spore concentrations of the solutions
were determined using a Fuchs Rosenthal
haemocytometer and a light microscope. The optical
density of the same solutions was measured (Fluorescence,
absorbance and luminescence reader, model Genios,
Tecan Group, Switzerland) in the absorbance
mode at 595 nm with a shaking period of 5 sec prior to
measurements. Solutions for bioassays were diluted to
approximately 500,000 spores per ml by measuring the
optical density of a subsample of the spore suspension
using the relationship depicted in Fig. 1. The number of
isolates available for our study were 7, 2, and 7 in 1999,
2004, and 2008, respectively.
Bioassays. Fungicides were either purchased from
Sigma-Aldrich, Switzerland (trifloxystrobin), Fein-
MATERIALS AND METHODS
Fungal strains. Wheat plants (susceptible cv. Ritmo)
with typical Septoria tritici necroses were sampled at
growth stages 21-32 (Zadoks et al., 1974) in Northern
Germany (federal state Schleswig-Holstein, area between
53.70 and 54.38°N latitude and 8.83 and 10.88°E
longitude) in 1999, 2004, and 2008. Plants were stored
at -20°C until further analysis. After defrosting, leaves
were inspected for Septoria tritici pycnidia under a
binocular microscope and leaf pieces (approximately 2
cm in length) with pycnidia were incubated in Petri
dishes on water agar (1.5% [w/w]) for 24 h at room
temperature. Pycnidia discharged spore slime during the
Fig. 1. Relationship between Septoria tritici spore concentration
in deionized water and optical density [measured in absorbance
(dimensionless) mode at a wavelength of 595 nm,
with 200 µl in 96 microtiterplate wells]. The vertical dashed
line indicates the optical density of an empty plate.