Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

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Session 1 — S. Hamza, M. Medini, T. Sassi, S. Abdennour, M. Rouassi, A.B. Salah, M. Cherif, R. Strange, and M. Harrabi
Table 2. Experimental code and origin of Mycosphaerella graminicola collected from Morocco
and Tunisia to study genetic variation for virulence.
Isolates collected from Tunisia Isolates collected from Morocco
Origin Origin
Code Location Wheat species Code Location Wheat species
TUN1 Beja DW MAR1 Jemaa Sham BT
TUN2 Mateur DW MAR2 Douyet BT
TUN3 Mornag DW MAR3 Agadir BT
TUN4 El Agba DW MAR4 Ain Orma BT
TUN5 Zaghouane DW MAR5 Azrou BT
TUN6 Siliana DW MAR6 Essaouira BT
TUN7 Mjez El Bab DW MAR7 Tetouan BT
were performed in a volume of 50
µl containing 50 mM KCl, 10 mM
Tris-HCl, pH8.3, 1.5 mM MgCl2,
200 µM of dNTP, 50 pmole of
primer, 2.5 units of Taq
polymerase (Boerhringer-
Manheim) and 25 ng of genomic
DNA. Reactions were run in a
Thermolyne, Temptronic model
thermal cycler for 30 cycles, each
consisting of 15s at 94°C, 15 s at
50°C, and 45 s at 72°C. An
additional 5 min polymerization
step at 72°C ended the
amplification. The products were
analyzed by electrophoresis of 20
µl aliquot of each PCR sample on
0.8% agarose gel.
Specific DNA amplifications
were performed as determined by
Beck and Ligon (1995). ITS1 and
JB446 (5’-
CGAGGCTGGAGTGGTGT-3’)
primers were used for specific
amplification of S. tritici DNA and
JB433 (5’-
ACACTCAGTAGTTTACTACT-3’)
and JB434 (5’-
TGTGCTGCGTTCAATA-3’) were
used to amplify S. nodorum DNA.
PCR was performed as described
above, except the annealing
temperature was 57°C.
ITS sequencing was performed
using 90 ng of 500 bp amplified
product with ITS1 and ITS4. The
sequence was determined by the
dideoxynucleotide chain
termination method using an
automated sequencer (Perkin
Elmer) at the Darwin Building of
University College London
(London, United Kingdom).
Fungal DNA amplification in
resistant and susceptible
cultivars
To evaluate the competition of
plant DNA with fungal DNA using
ITS1 and ITS4 primers,
amplification was performed using
1 µl and 5 µl of plant DNA extract
mixed with several amounts (
0.001, 0.01; 0.1; 1; 10; 100 and 1000
ng) of fungal DNA. The PCR
conditions were the same as
described before with a 57°C
annealing temperature.
The time course amplification
using ITS1 and ITS4 primers was
realized using 2 µl of DNA extract
from one infected leaf at 3, 6, 9, 14,
18, and 22 days after inoculation.
The PCR conditions were the same
as described before, with a 57°C
annealing temperature.
Results and Discussion
Bread wheat derived isolates
almost exclusively produced
pycnidia in the bread cultivars,
whereas pycnidial production by
durum wheat derived isolates was
almost entirely restricted to durum
wheat isolates. The analysis of
variance (Table 3) shows highly
significant differences (p