First Report of Didymella rabiei on Chickpea Debris in Tunisia - Iresa

<strong>First</strong> <strong>Report</strong> <strong>of</strong> <strong>Didymella</strong> <strong>rabiei</strong> on Chickpea Debris in
Tunisia
Azza Rhaïem and Mohamed Chérif, Laboratoire de Phytopathologie, Moncef
Harrabi, Laboratoire de Génétique de la Résistance; INAT, 43 Avenue Charles
Nicolle, 1082 Cité Mahrajène, Tunis, Tunisia, and Richard Strange, Department <strong>of</strong>
Biology, University College London, Gower Street, London WC1E 6BT, England
ABSTRACT
Rhaïem, A., Chérif, M., Harrabi, M., and Strange, R. 2006. <strong>First</strong> report <strong>of</strong> <strong>Didymella</strong> <strong>rabiei</strong> on
chickpea debris in Tunisia. Tunisian Journal <strong>of</strong> Plant Protection 1: 13-18.
During the 2001-2002 growing season, <strong>Didymella</strong> <strong>rabiei</strong>, the teleomorph <strong>of</strong> Ascochyta <strong>rabiei</strong> was
found for the first time in Tunisia, on chickpea debris overwintering on the soil surface at different
chickpea growing locations: Tunis, Mornag, Korba, Bizerte, Bou-Salem and Beja. D. <strong>rabiei</strong>
pseudothecial formation varied significantly in frequency according to the location and the sampling
time.
Keywords: <strong>Didymella</strong> <strong>rabiei</strong>, Ascochyta <strong>rabiei</strong>, teleomorph, chickpea, Tunisia
Chickpea (Cicer arietinum L.) is a
major leguminous crop in Asia, Southern
Europe, the Middle East, the northwestern
United States and North Africa.
Ascochyta blight, caused by Ascochyta
<strong>rabiei</strong> (Pass.) Labrousse [teleomorph:
<strong>Didymella</strong> <strong>rabiei</strong> (Kovachevski) v. Arx]
is a frequent and damaging disease <strong>of</strong>
chickpea worldwide. It devastates crops
in regions where the growing season is
cool and wet, and has been reported from
West Asia, North Africa, Southern
and Western Europe, the Indian
subcontinent, and the Palouse region <strong>of</strong>
Eastern Washington and Northern Idaho
in the USA (5, 6, 9, 10, 12). Blight
symptoms occur on all above-ground
parts <strong>of</strong> the plant: on the leaflets round
spots with brown margins and gray
centers, where pycnidia are arranged in
Corresponding author: M. Chérif
cherif.mohamed@inat.agrinet.tn
Accepted for publication 22 February 2006.
concentric rings, while on the stems and
petioles, the lesions bearing pycnidia are
obovate or elongate. Round lesions with
concentric rings <strong>of</strong> pycnidia also develop
on the pods, which may fail to develop
any seed (10, 11). Initial infection sites in
the field can be uniformly distributed,
indicating seed-borne inoculum spread. In
that case, the seedlings develop dark
brown lesions at the base <strong>of</strong> the stem
(10); these lesions may be extensive,
resulting in damping-<strong>of</strong>f, or they may
initially remain unnoticed and evolve
later into patches <strong>of</strong> blighted plants. A
less uniform blight distribution may
indicate inoculum spread by wind or by
plant residue, since, in addition to seedborne
inoculum, airborne conidia <strong>of</strong> the
anamorph constitute a major primary
inoculum as well as windblown
ascospores where the teleomorph <strong>of</strong> the
fungus is present. The teleomorph
<strong>Didymella</strong> <strong>rabiei</strong>, originally named
Mycosphaerella <strong>rabiei</strong>, was first observed
in Bulgaria by Kovachevski in 1936.
Tunisian Journal <strong>of</strong> Plant Protection 13
Vol., No. 1, 2006

Fruiting bodies were found only on
chickpea debris overwintering on field
soil. The teleomorph was then reported in
Russia, Greece, Hungary, Spain, Syria,
the United States (3, 15), Canada (1) and
Australia (2). D. <strong>rabiei</strong> is heterothallic,
with a bipolar, biallelic incompatibility
system (16), and arises from two mating
types MAT1-1 and MAT1-2. Although
both mating types have been identified in
Tunisia, in naturally infected chickpea
debris maintained under favorable
conditions in the laboratory (4), the
teleomorph has never been reported in
Tunisia in the field. The main objectives
<strong>of</strong> the present study were (i) to determine
whether the teleomorph <strong>of</strong> the pathogen
and pseudothecial development occur
under field conditions in Tunisia, and (ii)
to assess how pseudothecial and pycnidial
formation vary according to the location
and the sampling date.
MATERIALS AND METHODS
Stem debris <strong>of</strong> chickpea plants
naturally infected with A. <strong>rabiei</strong> was
collected from the Oued Mliz region in
2001. The stems were air-dried and cut
into 10- to 12-cm-long pieces each <strong>of</strong>
which had at least two necrotic lesions
with some pycnidia typical <strong>of</strong> Ascochyta
<strong>rabiei</strong>. Nylon net bags (14x20 cm) with a
1.6 mm mesh containing each twelve
stem pieces were prepared. In November,
these bags were placed on the soil <strong>of</strong> 4x3-
m plots at six different locations (20 bags
per plot). The bags were held in place by
a large net attached to the soil to prevent
the bags from being blown away by the
wind. Plots were located in fields <strong>of</strong> the
Experimental Research Stations at the
following locations: Tunis (Institut
National Agronomique de Tunisie,
INAT), Morneg, Korba, Bizerte, Bou-
Salem, and Beja. For each plot and each
location, the content <strong>of</strong> one bag was
sampled monthly from February to July
2002. Sampled stem pieces were washed
under running tap water for 2 mn and
dried on filter paper. The density <strong>of</strong> the
fungal fruiting bodies was determined on
three stem pieces per bag by calculating
the number <strong>of</strong> pycnidia and pseudothecia
per square cm <strong>of</strong> tissue under a dissecting
microscope at 80X. The fruiting bodies <strong>of</strong>
each <strong>of</strong> these stem pieces were removed,
squashed in lactophenol-acid fuchsin,
examined under a microscope at 400X
and characterised as pycnidia or
pseudothecia in ten microscope fields
(0.278 mm² each). The total number <strong>of</strong>
pycnidia and pseudothecia was calculated
for each sampled stem piece.
RESULTS
<strong>Didymella</strong> <strong>rabiei</strong> pseudothecia
occurred on all chickpea debris from all
six locations in Tunisia. Typical darkbrown
to black pseudothecia with
perceptible beacks and ostioles (Fig. 1. A,
B) containing parallel arranged asci were
observed (Fig. 1. C). Pseudothecial
dimensions ranged from 76-152 x 120-
250 µm,. Asci were cylindrical-calvate,
pedicellate, bitunicate, narrow (Fig. 1. D)
and contained linearly arranged
ascospores (eight) each <strong>of</strong> which was
divided into two unequal cells with a very
clear constriction at the septum (Fig. 1.
D). Ascospore size was 13.5-17.25x6-
6.75 µm and ascus size 48-50 x 10-12
µm. In addition to the typical asci, wide
flask-shaped asci were also observed
(Fig. 1. E).
The fungus grew saprophytically on
the stem debris and formed asexual
fructifications (pycnidia) as well as sexual
fructifications (pseudothecia) within 8 to
12 weeks after placement on the soil.
Before February, no pseudothecia were
found on any stem debris, at any location.
By February, the first pseudothecia
started to appear on debris from Korba
and Bou-Salem. The extent <strong>of</strong>
pseudothecial and pycnidial formations
varied according to locations but both
sexual and asexual fruiting bodies could
be recorded until June and July in all the
studied locations (Fig. 2). The
examination <strong>of</strong> Ascochyta fruiting body
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Vol., No. 1, 2006

Fig. 1. Sexual fruiting bodies <strong>of</strong> <strong>Didymella</strong> <strong>rabiei</strong> A, B, Pseudothecia with beack and ostiole (x400); C,
Pseudothecium with asci (x400); D, Ascus with ascospores (x1000); E, Flask-shaped asci with ascospores (x400).
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Vol., No. 1, 2006

development on stem pieces overwintering
on the soil revealed that the
density <strong>of</strong> fruiting bodies varied
significantly with the location and the
sampling date (Table 1). Pseudothecia
density as a percentage <strong>of</strong> the density <strong>of</strong>
all Ascochyta fruiting bodies formed on
stem debris was 43.4 % in Korba, 46.4 %
in Bizerte, 44.9 % in Tunis, 22.7 % in
Morneg, 35.7 % in Bou-Salem, and 32.9
% in Beja. Fig. 3 further shows that
pseudothecial formation was more
important in Korba, Bizerte and Tunis
than in Morneg, Bou-Salem, and Beja,
whereas in the last three locations more
pycnidia were formed.
D e n s ity p e r cm ²
600
500
400
300
200
100
0
Pycnidia
Pseudothecia
Feb Mar Apr May Jun Jul
Period
Fig. 2. Mean density <strong>of</strong> sexual and asexual fruiting
bodies formed on chickpea debris from February to
July 2002 (I = standard error).
Density per cm ²
600
500
400
300
200
100
0
Korba Bizerte Tunis Morneg Bousalem Beja
Location
Pycnidia
Pseudothecia
Fig. 3. Mean density <strong>of</strong> sexual and asexual fruiting
bodies formed on chickpea debris at six locations in
Tunisia (I = standard error).
DISCUSSION
The morphological characteristics <strong>of</strong>
pseudothecia (Fig. 1. A-C), asci and
ascospores (Figs. 1. C-E) observed in the
six studied locations confirmed the
fungus to be D. <strong>rabiei</strong>. Pseudothecial
dimensions were consistent with those
reported by Nene (9) and Nene and
Reddy (10). The characteristics <strong>of</strong> asci
and ascospores, including shape, size as
well as number and arrangement <strong>of</strong>
ascospores are consistent with those
reported for D. <strong>rabiei</strong> (9, 10, 13). Wide
flask-shaped asci, which were observed in
addition to the typical asci are similar to
those reported by Armstrong et al. (1) in
Canada.
Sexual fruiting bodies <strong>of</strong> the fungus
could be observed in the six Tunisian
locations from February to July. These
results are similar to those reported by
Navas-Cortés et al. (7) who showed the
presence <strong>of</strong> pseudothetia on chickpea
debris until July in different locations in
Spain. Our studies revealed that the
locations <strong>of</strong> Korba, Bizerte and Tunis
were more favorable to pseudothecia
formation than Morneg, Bou-Salem, and
Beja. These differences may be related
mainly to weather conditions prevailing
in each location. In fact, relative humidity
(RH) has been shown in previous studies
to be the most important factor that
influences fungal development (8, 14).
Pycnidia and pseudothecia <strong>of</strong> the
pathogen have been reported to develop
under humid conditions but the
maturation <strong>of</strong> pseudothecia, in particular,
is delayed by extended periods <strong>of</strong>
drought. When RH is not a limiting
factor, the effect <strong>of</strong> temperature on the
further development <strong>of</strong> pseudothecia has
been found to vary with the pseudothetial
developmental stage and with the type <strong>of</strong>
debris (naturally infected or artificially
infected) on which pseudothecia formed
(8). This suggests that in the locations <strong>of</strong>
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Vol., No. 1, 2006

Table 1. Mean squares <strong>of</strong> the density (number/cm 2 ) <strong>of</strong> all Ascochyta fruiting bodies, pseudothecia and pycnidia
developed on naturally infected debris placed at 6 locations in Tunisia and assessed from February to July 2002 at
monthly intervals
Sources <strong>of</strong>
MS
Df
variation
Fruiting bodies Pseudothecia Pycnidia
Location (L) 5 87331,0 43203,20 * 129076,8 **
Time (T) 5 33604,1 72829,17 ** 152556,5***
L x T 25 100011,7 * 30834,43 * 59566,7**
Residue 72 50129,45 17065,46 28643,46
* significant at 0,01

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<strong>Didymella</strong> <strong>rabiei</strong> على
.2006
.Tunisian Journal <strong>of</strong> Plant Protection 1: 13-18
ملخص
رحيّم،‏ عزّة،‏ محمد الشریف،‏ منصف الهرابي وریتشارد سترانج.‏
بقایا الحمص في تونس.‏
أول ملاحظة للفطر
للفطر
خلال الموسم الزراعي 2002-2001، تمكنا من تشخيص الطور الجنسي
للمرة الأولى في تونس.‏ تمت خلال هذه الدراسة معاینة الفطر على بقایا حمص مصاب بالمرض وضعت على
سطح الأرض في ست مناطق زراعية للحمص في تونس وهي مرناق،‏ قربة،‏ بنزرت،‏ بوسالم،‏ باجة وتونس.‏ وقد تفاوت
عدد الثمار الأسكية الحجيریة بصورة معنویة بين المناطق وعبر الزمن خلال فترة الدراسة.‏
Ascochyta
<strong>Didymella</strong> <strong>rabiei</strong>
<strong>rabiei</strong>
آلمات مفاتيح:‏ ،<strong>Didymella</strong> <strong>rabiei</strong> ،Ascochyta <strong>rabiei</strong> طور جنسي،‏ حمص،‏ تونس
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