© Benaki Phytopathological Institute
Al-Shehadah
et al.
48
Materials and Methods
Experimental material
Rhynchosporium secalis
pathotype Rs
22, which is one of the most virulent Syri-
an pathotypes to all barley genotypes avail-
able so far (Arabi
et al.,
2010), was used in
this study. At the end of the growing sea-
son, symptomatic barley leaves, naturally
infected by pathotype Rs22, were collect-
ed from the field. In the laboratory, leaf sec-
tions measuring 1.5 cm x 1 cm bearing well-
developed scald lesions were cut, placed at
18 °C in the dark and wetted twice a day us-
ing a high-pressure sprayer in order to stim-
ulate the production of conidia. Sporulation
on leaf lesions was checked under a light
microscope. After 7 days, produced conid-
ia were transferred from diseased leaf sec-
tions to coverslips (18 x 18 x 0.14 mm) by
lightly pressing the coverslip on the sur-
face of a sporulating lesion. The coverslips
were then placed (conidia facing up) on a
plastic window screen stretched on a light-
weight wooden frame (20 cm
2
). The cover-
slips were fixed on the screen with a small
piece of transparent, double-sided adhesive
tape (Aylor and Sanogo, 1997).
Exposure tests in the field
The experiments were performed un-
der field conditions during the summer of
two consecutive years (2013-2014). Samples
(coverslips carrying the conidia) were ex-
posed either to direct sunlight or to dark-
ness for time periods ranging from 0.5 to
8 h. For each exposure period, two sets of
samples were used: one set was exposed to
direct sunlight and another one to darkness
(control). For exposing the samples to direct
sunlight, the plastic window screen carrying
the coverslips with the conidia was placed in
the field 1.1 m above the ground under full
sunlight conditions. For exposing the sec-
ond set of samples to darkness in the field,
the window screen with the coverslips was
placed in a darkened enclosure with ventila-
tion. Six coverslips per treatment were used
as replicates. At the beginning of each ex-
posure time, conidia were collected from
each replicate coverslip, placed immediate-
ly on 1.5% water agar Petri dishes, incubat-
ed at 20-22ºC in the dark for 24h, and served
as non-exposed controls (G
0
). The tempera-
ture of the coverslips carrying conidia was
determined with a Multi-thermometer ap-
paratus with copper thermocouple wires
fixed to the bottom of two additional cover-
slips without conidia (one coverslip for each
treatment) placed in the same conditions as
those that carried the conidia.
Exposure tests in the laboratory
Conidia produced on barley leaves, using
the method described above, were trans-
ferred to coverslips and were handled as
described for the field tests. The coverslips
with conidia were exposed in the laboratory
at room temperature (22 to 25°C) to short-
wavelength (254 nm) UV light (UV-C light)
emitted from ultraviolet tubes (TUV-30 W/G
T8-UV-C; Philips, The Netherlands) for 0.5, 5,
15, 30, and 60 min. The distance between
the conidia and the UV-C light tubes was
~15 cm. The UV irradiance at the level of the
conidia was 3.2±0.7 Wm
-2
as measured with
a UVX-CR radiometer with a UVX-25 sensor
(Ultraviolet Products, Cambridge, UK). Three
replicate coverslips were used for each ex-
posure period and treatment (with and
without exposure to UV-light). The non-ex-
posed to UV-C light coverslips (control) were
kept under room temperature. The experi-
ment was repeated three times.
Assessment of conidial viability
At the end of each exposure time in both
the field and the laboratory tests, exposed
and non-exposed coverslips with conid-
ia were collected and placed (conidia fac-
ing down) on 1.5% water agar medium con-
tained in 9-cm plastic Petri dishes (6 Petri
dishes per treatment) and incubated at 20-
22ºC in the dark for 24h to allow germina-
tion. Germinated conidia were counted in
random fields at x 100 magnification with a
light microscope. A total of 100 to 200 conid-
ia was examined on each coverslip. A conidi-
umwas considered germinated if the length
of the germ tube was greater than or equal
