Morphological Variability Within and Among Verticillium
Species Collected in Tunisia
Hayfa Jabnoun-Khiareddine, Institut Supérieur Agronomique de Chott-Mariem,
4042, Chott-Mariem, Sousse, Tunisia, Mejda Daami-Remadi, Centre Régional des
Recherches en Horticulture et Agriculture Biologique, 4042, Chott-Mariem, Sousse,
Tunisia, Dez J. Barbara, Warwick HRI, University of Warwick, Wellesbourne,
Warwickshire, CV35 9EF, UK, and Mohamed El Mahjoub, Institut Supérieur
Agronomique de Chott-Mariem, 4042, Chott-Mariem, Sousse, Tunisia.
__________________________________________________________________________
ABSTRACT
Jabnoun-Khiareddine, H., Daami-Remadi, M., Barbara, D.J., and El Mahjoub, M. 2010.
Morphological variability within and among Verticillium species collected in Tunisia. Tunisian
Journal of Plant Protection 5: 19-38.
Verticillium wilt, caused by Verticillium dahliae or V. albo-atrum, is an important disease of many
crop species worldwide. In Tunisia, 160 isolates of Verticillium spp. were obtained from diseased
vegetables in different localities. Out of these isolates, 120 were identified as V. dahliae, 26 as V.
tricorpus, while 9 were identified as V. albo-atrum and 5 as V. nigrescens (syn. Gibellulopsis
nigrescens), based on morphological, cultural and biometrical characteristics. When cultured on PDA
medium, huge morphologic variation was noted within and among Verticillium species. Many
morphotypes have been recognized within V. dahliae, V. albo-atrum and V. tricorpus based on colony
color and texture and on the abundance of the resting structures. For V. dahliae, morphotypes were
related only to the microsclerotial forming types (MF types), whereas, in V. tricorpus, a relationship
was noted between morphotypes, original host and geographical origin. However, for these two
species, no relationship was found between morphotypes and their aggressiveness on tomato, cv.
Ventura. V. albo-atrum morphotypes isolates were not associated neither with original host and
geographical origin, nor with the aggressiveness on tomato.
Keywords: Aggressiveness, characterization, geographical origin, host, morphotypes, relationship,
resting structures, Verticillium
_________________________________________________________________________
The genus Verticillium, as recently
defined (11) includes the virulent plant
pathogenic species V. dahliae and V.
albo-atrum, which have low saprotrophic
abilities, V. nubilum a saprotroph which is
only weakly pathogenic, and V. tricorpus
which has intermediate saprotrophic
ability but is pathogenic on a limited
number of crops (10, 28, 36, 37, 41). V.
nigrescens, another saprotroph which is
only weakly pathogenic, was recently
taken out of the genus and placed in
Gibellulopsis (43).
In Tunisia, during the last few years,
many Verticillium isolates have been
obtained from crops exhibiting vascular
wilt symptoms including, tomato, potato,
eggplant and melon (8, 24, 25). These
isolates were identified as one of V. alboatrum, V. dahliae or V. tricorpus based on
cultural and morphological characteristics
Corresponding author: Hayfa Jabnoun-Khiareddine
Email: [email protected]
Accepted for publication 27 February 2010
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
(16, 24, 25). However, as the Verticillium
collection grew, great variability in these
characteristics was noted among isolates
belonging to the same Verticillium sp. It
has been previously reported that fungi
belonging to the genus Verticillium
display great variation in morphology (4,
26).
The
presence
of
different
physiological, ecological, biochemical,
pathogenic and molecular variations
within Verticillium isolates has also been
reported (5, 9, 21, 26, 37, 39). For
example, Qin et al. (39) reported that
isolates of V. dahliae recovered from one
host, lettuce, have shown great
morphological,
physiological,
and
pathogenic differences.
In Tunisia, Verticllium isolates have
been recovered from several host plants,
but
little
is
known
regarding
morphological and cultural variability
within and among Verticillium species
present in soils, and it was felt that
additional insight into wilt problems in
Tunisia might be achieved
via
morphological characterization of local
isolates. In fact, no studies have been
done on whether or not these differences
in morphology are related to the host of
origin, geographical origin or to pathogen
aggressiveness.
Such
studies
are
warranted.
The objectives of the present study
were (i) to characterize the Verticillium
isolates collected from diverse hosts in
several geographical locations on the
basis of morphological, cultural and
biometrical characteristics and (ii) to
determine whether there is a relationship
between variation in morphology,
aggressiveness on tomato, original host
and geographical origin of local isolates.
stems and petioles of plants exhibiting
vascular discoloration to try to ensure the
collection of all pathogens involved in
plant wilting. Plant parts were rinsed
thoroughly in tap water and cut into 0.5
cm2 pieces. After surface-disinfection in
sodium hypochlorite (commercial bleach
10%) for 3 min, the plant pieces were
rinsed three times in sterile distilled
water, dried on sterile filter paper and
plated onto potato dextrose agar (PDA)
medium with streptomycin sulphate (300
mg/l). Four or five pieces were used per
plate for each plant tissue. Plates were
incubated for two weeks at 20°C. Since
problems can occur with general media
such as PDA, which supports growth of
many microorganisms (38), fungal
isolates were sub-cultured on PDA plates,
amended with streptomycin sulphate (300
mg/l), from the edge of actively growing
colonies, until a pure culture of the
pathogen was obtained. All cultures were
then single spored. Isolates were
identified based on published descriptions
(17, 18, 19, 20, 23). Monoconidial
subcultures of all Verticillium spp.
isolates were stored in 25% aqueous
glycerol solution at – 20°C (41).
Colony characteristics (size of
conidia, size and occurrence of resting
structures, and color of colonies) were
determined after three to four weeks of
incubation at 20°C on PDA. The
morphology of phialides, conidia and
resting structures of Tunisian Verticillium
isolates on PDA were compared with
published descriptions of Verticillium
spp.
Lengths and widths of 100 randomly
chosen conidia were measured after 21
days of incubation. Lengths and widths of
microsclerotia and chlamydospores (50
per isolate) were measured after 21-28
days of incubation.
MATERIALS AND METHODS
Isolation
and
identification.
Isolations were performed from roots,
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
Table 1. Morphological characterization of V. dahliae isolates with relation to their host, locality
(geographical origin) and aggressiveness degree on tomato cv. Ventura
Morphotype
Host
MF typec
MF2 MF3 MF4
2
1
2
1
1
5
3
2
5
1
1
2
1
1
1
2
1
1
2
1
1
2
1
1
2
9
10
2
11
8
6
1
4
4
1
3
1
1
1
2
1
1
2
1
1
1
2
12
48
56
Locality
Aggressivenessd
NT NP MA A
2
3
2
MF1
HA
Tomato (6)b
Sousse
3
1
Tomato (2)
Monastir
M1 (10)a
Eggplant (1)
Monastir
1
Pepper (1)
Sousse
1
Tomato (10)
Sousse
2
4
4
Tomato (6)
Nabeul
2
4
Tomato (3)
Monastir
1
1
1
M2 (26)
Eggplant (2)
Sousse
1
1
Potato (4)
Sousse
1
1
2
1
Olive (1)
Sousse
1
Tomato (3)
Sousse
1
1
1
Tomato (1)
Monastir
1
Potato (3)
Sousse
2
1
M3 (11)
Potato (1)
Nabeul
1
Eggplant (1)
Sousse
1
Artichoke (2)
Sousse
2
Tomato (19)
Sousse
1
10
6
2
Tomato (13)
Monastir
8
1
4
Tomato (14)
Nabeul
5
6
3
Potato (1)
Sousse
1
Potato (8)
Nabeul
3
3
2
Potato (1)
Monastir
1
Potato (3)
Jendouba
3
Potato (1)
Bizerte
1
M4 (73)
Eggplant (1)
Monastir
1
Pepper (1)
Sousse
1
Melon (2)
Sousse
1
1
Melon (2)
Mahdia
2
Melon (2)
Monastir
1
1
Olive (1)
Monastir
1
Cucumber (2) Sousse
2
Artichoke (2)
Sousse
2
4
9
4
47
38 22
Total
120
a Number of isolates per morphotype
b Number of isolates in each morphotype/host/locality combination
c MF type: Microsclerotial forming type
d MA: moderately aggressive; A: aggressive; HA: highly aggressive; NP: not pathogenic; NT: not tested
Aggressiveness of all isolates tested
was measured using a conidial suspension
of 107 spores/ml for root dip inoculation
of seedling tomato cv. Ventura (which
does not carry the resistance gene Ve)
(25). The evaluation of isolates
Tunisian Journal of Plant Protection
aggressiveness was assessed 60 days
post-inoculation based on the records of
leaf damage index (LDI) which is
calculated according to 0-4 scale (25).
Based on LDI records, isolates were
classified as follows: weakly aggressive
21
Vol. 5, No. 1, 2010
(0.0-0.9); moderately aggressive (1.01.9);
aggressive
(2.0-2.9);
highly
aggressive (≥ 3.0).
All Verticillium isolates used in this
study, including their designation,
original host, locality and aggressiveness
on tomato cv. Ventura, are listed in tables
1 and 2. The aggressiveness of most
Verticillium isolates had not been
previously published.
For each Verticillium species, the
relationship
between
morphology,
original host, locality and aggressiveness
on tomato cv. Ventura, was analyzed by
chi-square (χ2) test and correspondence
analysis. Significance levels were set at
5%. Statistical analyses were performed
using the SPSS 16.0.
Table 2. Morphological characterization of V. tricorpus and V. albo-atrum
isolates with relation to their host, locality (geographical origin) and
aggressiveness degree on tomato cv. Ventura
Aggressivenessc
Species/Morphotype
Host
Locality
NT
V. tricorpus
M1 (2)a
M2 (4)
M3 (20)
Total
MA
Melon (1)b
Mahdia
1
Melon (1)
Sousse
1
Squash (2)
Sousse
2
Squash (1)
Monastir
1
Artichoke (1)
Sousse
1
Tomato (5)
Sousse
4
1
Potato (13)
Sousse
11
1
1
Potato (2)
Monastir
2
26
-
23
2
1
NT
WA
MA
V. albo-atrum
M1 (2)
WA
Tomato (2)
Sousse
2
Tomato (2)
Sousse
2
Potato (2)
Sousse
1
1
Tomato (2)
Sousse
1
1
Potato (1)
Sousse
1
M2 (4)
M3 (3)
Total
9
-
7
2
a Number of isolates per morphotype
b Number of isolates in each morphotype/host/locality combination
c WA: weakly aggressive; MA: moderately aggressive; NT: not tested
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
and melon plants, isolations made from
roots and collar, sometimes indicated
mixed infections including, in addition to
Verticillium, other soil-borne pathogenic
fungi such as Fusarium solani, F.
oxysporum, Colletotrichum coccodes and
Rhizoctonia solani and/or nematodes
belonging to the genus Meloidogyne. F.
oxysporum was also isolated from roots
and stems of wilted potato plants,
individually or in association with
Verticillium spp. Furthermore, successful
isolations of Verticillium spp. were more
frequent at the beginning and at the
middle of the cropping season than at
plant senescence due to the increased
colonization of tissues by the secondary
fungus C. coccodes.
RESULTS
Isolation and identification of
pathogens involved in plant wilting.
Isolations were performed from roots,
collar, stems and petioles of wilted
vegetable plants. Typical cultures of
Verticillium colonies were obtained.
Microscopic examination revealed the
presence of conidiophores, which were
more or less erect, hyaline, with
verticillate branches, and with 3-4
phialides at each node, that were
consistently isolated from vascular tissues
of symptomatic plants collected from
different Tunisian localities (Fig. 1).
Attempted isolations from stems and
petioles of the basal leaves more often led
to pure pathogen cultures as compared to
attempts from roots. For tomato, potato
A
C
B
D
Fig. 1. Isolation of Verticillium sp. from several solanaceous plants on
PDA at 20°C. A: tomato stem, B: tomato petiole, C: eggplant petiole, D:
potato stem.
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
One hundred and sixty isolates of
Verticillium spp. were obtained from the
different vegetable producing localities
surveyed. All Verticillium isolates
produced verticilliate conidiophores, but
based on several other morphological,
biometrical and cultural characteristics,
four
Verticillium
(including
G.
nigrescens) species were identified.
produced at the phialide tips. They were
hyaline, ovoid to elongate. Conidia were
2.5-5 µm × 1.25-2.5 µm. Melanized
microsclerotia were the sole resting
structures observed. Dark mycelium and
chlamydospores were absent. On PDA,
these microsclerotia were dark brown to
black and were produced on single
hyaline hyphae (Fig. 3C). They were very
irregular in shape, subglobose to elongate,
and 17-47 µm in diameter.
Based on these morphological and
biometrical characteristics, 120 of the
isolates were identified as V. dahliae (19).
V. dahliae. Isolates had more or less
erect, totally hyaline and verticilliate
conidiophores with two to four phialides
at each node (Fig. 2A). Conidia were also
c
b
b
c
a
a
B
A
c
c
b
b
a
C
a
D
Fig. 2. Morphology of conidiophores (a), phialides (b) and conidia (c) of V. dahliae (A),
V. albo-atrum (B), V. tricorpus (C) and V. nigrescens (D) after 21 days of incubation on
PDA at 20°C (× 400).
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
B
A
C
D
F
E
Dm
D
m
m
m
m
c
C
C
c
G
H
Fig. 3. Resting structures of V. albo-atrum (A and B), V. dahliae (C), V. nigrescens (D) and V.
tricorpus (E-H) after 21 days of incubation on PDA at 20°C. A (×100); B-H (× 400); Dark
mycelia (Dm), microsclerotia (m) and chlamydospores (c).
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
Colonies formed on PDA were
generally hyaline or white to gray.
However, the Tunisian V. dahliae isolates
on PDA medium could be divided into
four significantly different morphotypes
based on colony color and texture (Fig.
4):
Morphotype 1: colony with milkywhite and usually smooth textured
surface and flocculose mycelium (Fig.
4A);
Morphotype 2: colony with grayishwhite surface and relatively dense
mycelium (Fig. 4B);
Morphotype 3: colony with white
and dense mycelium in the center and
with
blackish
margins
(strong
sclerotinization) (Fig. 4C);
Morphotype 4: colony with grayishblack felty surface (Fig. 4D).
Morphotype 4 was the most
frequently encountered among the
Tunisian V. dahliae isolates collected; it
represented
60.8%
of
isolates.
Morphotypes 1 and 3 were the less
frequent (8.3 and 9.2%, respectively) and
morphotype 2 was intermediate (21.7%).
A
B
C
D
Fig. 4. Cultural characteristics of different V. dahliae morphotypes cultured on PDA during 21
to 30 days at 20°C.
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
In addition to the color and texture
differences, other highly variable
characteristics of V. dahliae colonies
were noted during the characterization of
this pathogen. Although all isolates
produced microsclerotia, the pattern in
which these microsclerotia are formed
and the intensity of sclerotinization on
PDA medium were variable. Based on the
abundance and the distribution of
microsclerotia through the culture
medium, Tunisian isolates of V. dahliae
could
be
classified
into
four
microsclerotial forming types (MF types):
MF type 1: colony with few
scattered microsclerotia with no clear
pattern (Fig. 5A);
MF type 2: colony with more or less
abundant
and
radially
formed
microsclerotia (Fig. 5B);
MF type 3: colony with abundant
and radially formed microsclerotia (Fig.
5C);
MF type 4: colony with very
abundant microsclerotia formed in
“strand end” pattern (Fig. 5D).
MF types 3 and 4 represented 40.8
and 46.7% of the isolates, respectively.
MF type 2 represented 9.2% while MF
type 1 represented only 3.3% of the
isolates.
A
B
C
D
Fig. 5. Cultural characteristics of different V. dahliae MF types cultured on PDA during 21
to 30 days at 20°C.
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
contrasted the isolates of morphotype 2
and MF type 2 and isolates of
morphotype 1 and MF type 1 (Fig. 6).
However, these morphotypes and
MF types are not related neither to their
geographical origin and host of origin nor
to their aggressiveness on tomato cv.
Ventura, as the chi-square tests were not
significant at 5%.
V. dahliae morphotypes seem to be
related to MF types according to a
significant chi-square test (χ2= 36.18,
degree of freedom = 9, P = 0.000).
Correspondence analysis yielded two
main axes accounting for 73.3 and 24.8%
of the total inertia, respectively. The first
axis contrasted the isolates of morphotype
1 and MF type 1 and isolates of
morphotype 4 and MF type 4. The axis 2
Fig. 6. Correspondence analysis of data showing two dimensional
representations of V. dahliae morphotypes (M1, M2, M3 and M4) and MFtypes
(MF1, MF2, MF3 and MF4) and their associations.
V. albo-atrum. Isolates were
characterized by hyaline, abundant and
large conidiophores which are ramified
and more or less erect. Conidiophores
were verticilliate with two to four
phialides at each node. Each phialide
carried at its tip a mucilaginous slime
where conidia are released successively
Tunisian Journal of Plant Protection
(Fig. 2B). Conidia were hyaline,
ellipsoidal to sub-cylindrical, 2.5-5 ×
1.25-2.5 µm.
V. albo-atrum isolates formed dark
thickened
mycelia
(dark
resting
mycelium, DRM) as the sole resting
structure, but no microsclerotia or
chlamydospores (Fig. 3A and B). DRM
28
Vol. 5, No. 1, 2010
appears after 5 to 12 days of culture on
PDA at 20°C. These characteristics were
used to identify 9 isolates as V. alboatrum (20).
Individual colonies of V. albo-atrum
isolates were white, gray or black.
Although the number of V. albo-atrum
isolates was small, these nine Tunisian
isolates were tentatively subdivided into
three morphotypes based on colony color,
texture and the abundance of DRM (Fig.
7):
Morphotype 1: colony with felty
white to grayish mycelium and with little
DRM (2 isolates) (Fig. 7A and B);
Morphotype 2: colony with white,
dense and smooth textured mycelium and
with more or less abundant DRM (4
isolates) (Fig. 7C and D);
Morphotype 3: colony with black
thin and no aerial mycelium and with
very abundant DRM (3 isolates) (Fig. 7E
and F).
A
B
C
D
E
F
Fig. 7. Cultural characteristics of V. albo-atrum morphotypes cultured
on PDA during 21 to 30 days at 20°C.
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Vol. 5, No. 1, 2010
No relationships were noted
between V. albo-atrum morphotypes,
their original host or geographical origin
and their aggressiveness on tomato cv.
Ventura, according to non significant chisquare tests at 5%.
Chlamydospores were generally less
abundant than microsclerotia or DRM and
had variable shape (Fig. 3H). On PDA,
isolates formed large and irregularly
shaped microsclerotia, elongate to almost
spherical (mean size 55 to 67 µm in
diameter), usually with melanized hyphae
growing from them.
Most of the isolates produce a
yellow pigment that diffused into the
culture medium (Fig. 8). Based on these
morphological characteristics, twenty-six
Tunisian Verticillium isolates were
identified as V. tricorpus (17). It is
important to note that the intensity of
yellow pigmentation, typical for V.
tricorpus, was variable depending on
isolates and was absent from some of
them (Fig. 8D). The color of the colony
changed from white to gray or black.
V. tricorpus. The isolates produced
hyaline and erect conidiophores with two
to four phialides arising verticillately at
each node (Fig. 2C). Conidia were
hyaline, ellipsoidal to sub-cylindrical,
produced at the apices of phialides,
formed in a false head (a in Fig. 2C).
Conidia were 3.75- 6.25 × 2.5 µm.
These isolates produced three types
of resting structures (Fig. 3E-H): DRM
(Fig. 3E), microsclerotia (Fig. 3F) and
chlamydospores (Fig. 3H). The relative
abundance of these structures was
variable depending on isolate.
A
B
C
D
Fig. 8. Colony of V. tricorpus showing yellow pigmentation with variable
intensity depending on isolates: intense (A), medium (B), little (C) and
absent (D).
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Vol. 5, No. 1, 2010
These Tunisian isolates of V.
tricorpus produced, on PDA at 20°C,
morphologically distinguishable colonies.
This morphological variability was noted
at both macroscopic and microscopic
levels and three morphotypes were
distinguished (Fig. 9):
Morphotype 1: colony with white
cottony mycelium at the center and with
grayish-white felty margin (Fig. 9A);
Morphotype 2: colony with white
wrinkled cottony mycelium at the center
and with a blackish narrow margin (Fig.
9B);
Morphotype 3: colony with a
grayish-white felty surface (Fig. 9C);
The morphotype 3 was the most
encountered (76.9% of isolates) with
morphotypes 2 and 1 representing 15.4
and 7.7% of the isolates, respectively.
A
B
C
Fig. 9. Cultural characteristics of V. tricorpus morphotypes cultured on PDA during 21
to 30 days at 20°C.
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Vol. 5, No. 1, 2010
A relationship was found between V.
tricorpus morphotypes and their hosts of
origin according to a chi-square test (χ2=
52.00, degree of freedom = 8, P = 0.000).
In fact, morphotype 1 isolates were
associated with one host which is melon.
Morphotype 2 isolates came from squash
and artichoke while morphotypes 3
isolates were isolated from potato and
tomato. In addition, these morphotypes
were related with their geographical
origin (χ2= 13.29, degree of freedom = 4,
P = 0.01). In fact, the majority of V.
tricorpus isolates were related to Sousse
locality. Morphotype 2 and morphotype 1
isolates originated from Monastir and
Mahdia localities, respectively.
V. tricorpus morphotypes were,
however, not associated with their
aggressiveness on tomato cv. Ventura, as
the chi-square test was not significant
(χ2= 1.017, degree of freedom = 4, P =
0.907).
Based on microscopic observations
and especially on the relative abundance
of their resting structures, Tunisian V.
tricorpus isolates are divided into three
resting structure types (RS types):
RS type 1: colony with abundant
dark mycelia and few microsclerotia (Fig.
10A);
RS type 2: colony with abundant
microsclerotia and dark mycelia (Fig.
10B);
RS type 3: colony with abundant
microsclerotia and few dark mycelia (Fig.
10C and D);
For all three RS types recognized,
chlamydospores were the least abundant
of the resting structures.
A
B
C
D
Fig. 10. Microscopic variations of V. tricorpus RS types: RS type 1: A (× 400); RS type 2: B
(× 100); RS type 3: C (× 400) and D (× 100).
Tunisian Journal of Plant Protection
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Vol. 5, No. 1, 2010
V. nigrescens (syn. G. nigrescens).
Isolates formed conidiophores abundantly
in young colonies (7-day-old cultures),
but these become sparse in old cultures
(21to
30-day-old
cultures).
Conidiophores were hyaline, more or less
erect and verticillately branched with one
to two phialides arising at each node (Fig.
2D). Hyaline, ovoid conidia arose singly
at the apices of the phialides (c in Fig.
2D). Conidia were 5-7.5 × 2.5 µm.
These isolates produced only
chlamydospores as resting structures (Fig.
3D) which became very abundant after 7
to 15 days of culture at 20°C and are
generally single or grouped, being
occasionally in chains of 2 to 5.
Five
Tunisian
isolates
were
identified
morphologically
as
V.
nigrescens (18). V. nigrescens colonies
were characterized by a whitish aerial
mycelium which turns to brown or black
due to the production of chlamydospores.
No morphotypes were identified within V.
nigrescens.
among 1770
Verticillium isolates
collected from a wide range of vegetable
crops, 84.3% were V. dahliae, 5.5% V.
albo-atrum and 10.2% V. nigrescens.
Similarly, Govorova and Govorov (14)
reported that Verticillium wilt of pepper,
tomato, eggplant and strawberry, in the
Southern localities of the former Soviet
Union, was caused mainly by V. dahliae
and only occasionally by V. albo-atrum.
In Korea, Kim et al. (27) found that out of
103 isolates of Verticillium obtained from
wilted tomato plants, 75 were V. dahliae
while 28 were V. albo-atrum. In South
Africa, Millard (34) recorded that among
the 93 isolates of Verticillium spp.,
collected from diseased potato plants,
60% were identified as V. dahliae and 8%
as V. nigrescens.
Verticillium isolates collected in
Tunisia were identified on the basis of
morphological
and
cultural
characteristics. However, many of these
traits used to differentiate the species may
be highly variable on different media and
under different incubation conditions and
this may sometimes handicap the rapid
and practical identification of Verticillium
species (12, 13, 37). Here we have shown
that even when cultured on the same
medium (PDA), huge morphological
variability was noted within Tunisian
Verticillium species. Similar findings
have been given by Katan (26). Tunisian
V. dahliae isolates developed different
types of colonies on PDA with regard to
the color and the texture as well as the
presence
and
distribution
of
microsclerotia. In fact, the occurrence of
microsclerotia is variable ranging from
scarce to abundant. Moreover, the
mycelium aspect varies from the cottony
and dense to the thin and felty. These
differences were also noted by other
authors (6, 26, 31). Blanco-Lopez et al.
(3) reported considerable morphological
DISCUSSION
The present study concentrated on
the causal agents of Verticillium wilt
detected in different vegetable producing
areas in Tunisia. The results showed the
association of four Verticillium species
with wilt symptoms: V. dahliae, the
predominant species, V. tricorpus, V.
albo-atrum and V. nigrescens (syn. G.
nigrescens). V. nigrescens is reported for
the first time in Tunisia in the present
paper whereas V. albo-atrum and V.
tricorpus have been already reported (16,
24, 25). Out of the 160 Verticillium
isolates, 120 were identified as V.
dahliae, 26 as V. tricorpus, 9 as V. alboatrum and 5 as V. nigrescens. These
results suggesting the predominance of V.
dahliae are in accordance with earlier
reports. McKeen (33) reported that
Tunisian Journal of Plant Protection
33
Vol. 5, No. 1, 2010
shown not to be related to the
geographical origin of the isolates. In
fact, as 50% of V. dahliae isolates came
from Sousse locality, the likelihood of
obtaining isolates with different colony
morphologies is quite high. All the
recognized morphotypes were recorded in
this area. Similarly, no relationship was
found between the different V. dahliae
morphotypes, their original host or their
aggressiveness on tomato cv. Ventura. As
shown in table 1, isolates belonging to the
same morphotype, could come from
different hosts and show variable
aggressiveness against the same tomato
cultivar. Morphotypes 4 isolates, for
example, came from tomato, potato,
eggplant, melon, artichoke and olive.
Moreover, these isolates, even when from
the same host, could be moderately to
highly aggressive when inoculated to
tomato. Thus, morphotype 4 is a
heterogeneous group both in host range
and aggressiveness.
Some other studies have reported
that the morphology of some Verticillium
species on culture media may be related
to virulence. On agar media, cotton
defoliating isolates of V. dahliae
produced colonies with a mixture of
elongate and round microsclerotia while
the non-defoliating types formed only
around microsclerotia (2). However,
many other did not find any relationship
between Verticillium isolates, their
original host, geographical origin or
pathogenicity (1, 15, 32, 35).
For V. tricorpus, a relationship was
found between the different morphotypes
and their original host indicating that each
morphotype isolates was associated with
one or two hosts. However, as most of
these isolates were not tested on tomato
cv. Ventura, no relationship could be
established between these morphotypes
and their aggressiveness. V. tricorpus
variation in microsclerotia of V. dahliae
isolates obtained from the same soil
sample.
Many morphological differences
were also noted within V. albo-atrum and
V. tricorpus recovered in Tunisia. Some
V. tricorpus isolates, described for the
first time in the present study, did not
produce the typical yellow-orange
pigment characteristic of this species.
Similar pigmentless V. tricorpus isolates
were described by Qin et al. (40), as
atypical isolates of the species. Most V.
tricorpus isolates, in contrast, secrete the
yellow-orange pigment but with variable
intensity. The variability within V.
tricorpus has also concerned the
pigmentation and the aspect of mycelium.
Differences in morphology have also
been noted by other authors (29, 40). For
V. albo-atrum, Tunisian isolates were
divided into different morphotypes based
on the abundance of the aerial mycelium
and the dark mycelia. In fact, the
variation in the amount of dark mycelia
among isolates has also been noted by
Christen and French (7). Moreover,
isolates of V. albo-atrum from alfalfa
originated from Europe and USA
produced a range of colony morphologies
on prune lactose yeast agar (PLYA) (22).
The present study showed that a
relationship exists between V. dahliae
morphotypes, based either on colony
color and texture, and the different V.
dahliae MF types, based on the
abundance and the distribution of
microsclerotia through the culture
medium. In fact, the association between
these two morphological groups may
indicate that the high density of V.
dahliae mycelium in culture medium
could be associated with low abundance
of microsclerotia.
This
morphological
variability
observed within V. dahliae has been
Tunisian Journal of Plant Protection
34
Vol. 5, No. 1, 2010
morphotypes were also related to their
tricorpus, morphotypic variation seems to
geographical origin. However, this could
be related to the original host and
be insignificant as 85% of morphotypes
geographical origin.
isolates originated from Sousse locality.
Nevertheless, if more genetic,
For V. albo-atrum, no relationships were
physiological, and biochemical traits were
found between morphotypes and original
known, Verticillium isolates might be
host,
geographical
origin
or
placed in more meaningful groups. In
aggressiveness on cv. Ventura. In fact, the
fact, if the collection of Verticillium
limited number of isolates seems to be the
isolates could be enlarged to include
origin of absence of such relationships.
many other isolates from different
In conclusion, Verticillium isolates
Tunisian areas and host plants, the
have been identified based on the type of
morphological,
pathogenic
and
resting structures they produce on PDA.
physiological
differences
may
be
However, the application of appropriate
associated with different vegetative
molecular
methods
for
strain
compatibility groups (VCGs), as in potato
identification is needed. The Verticillium
and cotton (26, 30, 31). Further studies
species inventoried in Tunisia exhibit
are needed to understand the importance
high variability of important traits (e.g.
of morphological variation within these
morphology, host range, aggressiveness,
species in relation to their molecular and
etc.) and that they are mainly
biochemical characteristics and most
characterized by the predominance of one
importantly their pathogenic potential.
or two morphotypes. The variation in
ACKNOWLEDGEMENTS
morphology among V. dahliae, V.
Authors
gratefully
acknowledge
the
tricorpus and V. albo-atrum is not related
assistance of Besma Souissi (Institut Supérieur de
with the most important trait which is the
Gestion, Sousse) in the statistical analyses.
aggressiveness on a particular host. In V.
_________________________________________________________________________
RESUME
Jabnoun-Khiareddine H., Daami-Remadi M., Barbara D.J. et El Mahjoub, M. 2010. Variabilité
morphologique au sein et entre les espèces de Verticillium collectées en Tunisie. Tunisian Journal
of Plant Protection 5: 19-38.
La flétrissure verticillienne causée par Verticillium dahliae ou V. albo-atrum, est une maladie
importante de nombreuses cultures, partout dans le monde. En Tunisie, 160 isolats de Verticillium spp.
ont été obtenus à partir de légumes infectées dans différentes régions. Parmi ces isolats, 120 ont été
identifiés comme V. dahliae, 26 comme V. tricorpus, alors que 9 sont identifiés comme V. albo-atrum
et 5 comme V. nigrescens (syn. Gibellulopsis nigrescens) et ce, en se basant sur des caractéristiques
morphologiques, culturales et biométriques. Lorsqu'ils sont cultivés sur milieu PDA, une énorme
variation morphologique a été notée au sein et parmi les espèces de Verticillium. Plusieurs
morphotypes ont été reconnus au sein de V. dahliae, V. albo-atrum et V. tricorpus en se basant sur la
couleur et la texture de la colonie et sur l'abondance des structures de conservation. Chez V. dahliae,
les morphotypes sont relatés uniquement aux types MF, alors que chez V. tricorpus, une relation a été
notée entre les morphotypes, l'hôte d'origine et l'origine géographique. Par contre, pour ces deux
espèces, aucune relation n'a été trouvée entre les morphotypes et leur agressivité sur tomate cv.
Ventura. Les isolats des morphotypes de V. albo-atrum ne sont associés ni avec l'hôte d'origine et
l'origine géographique ni avec l'agressivité sur tomate.
Tunisian Journal of Plant Protection
35
Vol. 5, No. 1, 2010
Mots clés: Agressivité, caractérisation, relation, hôte, morphotypes, origine géographique, structures de
conservation, Verticillium
__________________________________________________________________________
+ ,‫ ا* ار‬.2010 .‫دي وداز !ر!را و ا ب‬%‫ا‬-‫ هء و
ة ا‬،‫ر ا‬-‫
ن‬
Tunisian Journal of Plant Protection 5: 19-38.
.0.1 , 23ّ Verticillium ‫اع‬.‫و ! أ‬
‫ ض ه!م ا‬V. albo-atrum ‫ أو‬Verticillium dahliae ‫ ال ا
اي ا‬
*4! Verticillium spp. ‫ أ)اع ا‬23 160 ‫ل‬.‫ ' ا‬،0) , .'!‫ آ* أ)!ء ا‬, ‫!!ت‬%&‫ا‬
26‫ و‬،V. dahliae ‫! ا&ع‬B)‫ أ‬120 =6> ' ،29<9‫;ت ا‬3‫ ا‬.256 78!& , 2!.9‫ ا‬:6‫ا‬
(syn. Gibellulopsis V. nigrescens !B)‫ أ‬5 ‫ و‬V. albo-atrum !B)‫ أ‬9 ‫ و‬،V. tricorpus !B)‫ أ‬
NL‫;ت ا‬3‫ ا‬9) & .2‫ ا‬2L!M‫ وا‬2‫ر‬39‫ وا‬2J,‫ر‬9‫= ا‬I!.6‫!دا ا‬95‫ ا‬G‫ و ذ‬nigrescens)
‫!ط‬9)T‫= ا ا‬6> ' .!B& !9,‫ و‬Verticillium ‫ أ)اع‬9R *I!‫ ه‬J,‫! ر‬% PQ ،PDA I‫ا‬O‫ا‬
2,!X‫! و آ‬B‫ا‬W‫ة و‬95
9‫!دا ن ا‬95‫ ا‬V. dahliae ‫ و‬V. albo-atrum ‫ و‬V. tricorpus 9R 2J,‫ر‬9‫ا‬
،NM, ‫<ات‬5‫[و‬9 2<5&9‫!ط ا‬9)T! 2W\ 2J,‫ر‬9‫!ط ا‬9)Z ‫ أن‬% ،V. dahliae ‫ ـ‬2%
&! .2&‫!ت ا
!آ‬%‫آ‬5‫ا‬
.,‫ا‬O<‫_ ا‬W9‫* وا‬I!‫!ت ا‬%&‫ وا‬2J,‫ر‬9‫!ط ا‬9)T‫ ا‬N‫ ا‬2]Q\ ^9 ،V. tricorpus ‫ ـ‬2%
&! !9&
`&4 !B5)‫ ووا‬2J,‫ر‬9‫!ط ا‬9)T‫ ا‬2W\ ‫ أي‬%5 ' ،‫ ا& ا‬B 2%
&! G‫ ذ‬0[
‫!ت‬%&! ; 2W\ !B ^
V. albo-atrum 2J,‫ر‬9‫!ط ا‬9)T‫ أن ا‬%‫ و‬.(Ventura) ‫رة‬5&, ‫&ورة‬%‫ا‬/'8!9‫ا‬
.'8!9‫! ا‬B5)‫ و; وا‬,‫ا‬O<‫_ ا‬W9‫* وا‬I!‫ا‬
Verticillium ،,‫ا‬OJ _W ،J,‫ ر‬N9) ،2W\ ،2)‫وا‬، *I! ،`4 ،2&‫!آ‬L ‫!ت‬%‫ آ‬:2! ‫!ت‬9‫آ‬
__________________________________________________________________________
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