© Benaki Phytopathological Institute
Al-Doude
et al.
72
established method forquantifying the rela-
tive expression level of a particular transcript
and determines its expression after be-
ing exposed to a specific alteration, such as
pathogen infection (Kralik and Ricchi 2017).
In the present work, we studied the defense
responses of two barley genotypes Banteng
and WI 2291, which are integrated in inter-
national breeding programs aimed at devel-
oping NB resistant barley genotypes. Ban-
teng was described as a highly resistant to
P.
teres
(Arabi
et al.,
2003), i.e. exhibited a lower
level (compared with WI2291) of NB symp-
tom development. We thus hypothesized
that SA-triggered defenses could drive con-
trasted levels of resistance in Banteng and
WI2291, inoculated by the same pathogen
isolate. Thus, the aim of the current study
was to evaluate the changes in SA content
and induction of
PR2
gene expression in two
barley cultivars with different resistance to
P. teres.
Materials and Methods
Plant materials and pathogen
inoculation
Τhe German genotype Banteng has
proved to be the most resistant genotype
to all NB isolates available so far under field
and greenhouse conditions for over fifteen
years (Arabi
et al
., 2003). For this reason, it
was chosen and used in this study. A uni-
versal susceptible control genotype (cv.
WI2291) from Australia was also included in
the experiments. The
P. teres
single conidi-
um isolate (NB4) tested was the most Syrian
virulent pathotype to all barley genotypes
available up to now (Arabi
et al
., 2003). The
fungus was incubated in Petri dishes con-
taining potato dextrose agar (PDA, DIFCO,
Detroit, MI, USA) for 8 days at 20-22°C in the
dark. Conidia were collected in 10 mL ster-
ile distilled water and the suspension was
adjusted to 2 x 10
4
conidia/mL using he-
macytometer. A surfactant (polyoxyethyl-
ene-20-sorbitan monolaurate) was added
(100 μL/L) to the conidial suspension to fa-
cilitate dispersion of the inoculum over leaf
surfaces.
Pyrenophora teres
inoculum prep-
aration, inoculation, and post-inoculation
were similar to those described by Abu Qa-
mar
et al.
(2008). Barley plants were grown in
the greenhouse and inoculated at the two-
to three-leaf stage with the second leaf ful-
ly expanded.
SA quantification
Pooled samples containing the fourth
leaf of 20 independent plants/genotype
where used for SA quantification. Pooled
samples were prepared from leaves tak-
en 24, 48 and 72 hpi, respectively. For each
time case studied, six pooled sample repli-
cates were used for quantification. SA was
extracted from approximately 200 mg of
freshly ground leaves in 1.5 ml tubes fol-
lowing the method described by Trapp
et al.
(2014), with minor modifications. Briefly, 100
mg of plant material were dried overnight in
a freeze drier at −42°C. The extraction was
achieved by adding 1.0 mL of ethyl acetate,
dichloromethane, isopropanol, MeOH:H
2
O
into each tube containing dry plant materi-
al. Samples were shaken for 30 min and cen-
trifuged at 16,000 g and 4°C for 5 min. The
supernatant was transferred into a new 1.5
micro-centrifuge tube and dried in a speed
vac. After drying, 100 μL of MeOH was add-
ed to each sample, homogenized under vor-
tex and centrifuged at 16,000 g and 4°C for
10 min. The supernatant was analyzed by a
high-performance liquid chromatography
coupled mass spectrophotometer (HPLC-
MS/MS) system (Agilent Technologies,
Böblingen, Germany). Changes in SA con-
tent were compared to the control for each
time point. Six independent repetitions
were performed for each time point. Data
were analyzed using the standard deviation
and t-test methods.
RNA isolation and cDNA synthesis
Primary leaves from three individual bi-
ological replicates were collected at 24, 48
and 72 hpi, and homogenized with a tube
pestle in liquid nitrogen. mRNA was extract-
ed with the Nucleotrap mRNA mini kit (Ma-
cherey-Nagel, MN, Germany) following the
