Journal of Phytopathology and Pest Management 7(1): 54-63, 2020
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage:
Corresponding author:
Naima Boughalleb-M’Hamdi,
Copyright © 2020
Genetic diversity of
Fusarium oxysporum
responsible of watermelon Fusarium
wilt in Tunisia and Spain
Naima Boughalleb-M’Hamdi
*, Ibtissem Ben Salem
, Najwa Benfradj
, Paloma Abad-Campus
Department of Biological Sciences and Plant Protection, University of Sousse, UR13AGR3, Higher
Agronomic Institute of Chott Meriem, 4042, Sousse, Tunisia
Mediterranean Agroforestry Institute, Polytechnic University of Valencia, Camino de Vera s / n,
46022, Valencia- Spain
Keywords: Fusarium oxysporum f. sp. niveum; genetic diversity; ISSR; molecular detection; watermelon.
1. Introduction
Watermelon (
Citrullus lanatus
Matsum & Nakai) is one of the most
important vegetable crops in the world,
with a yield of 109601914.00 tons in
2013 (FAOSTAT, 2016). In Tunisia,
watermelon crop has, also, a high
economic value with a production of
about 500000.0 tons in 2013 (FAOSTAT,
2016). Watermelon Fusarium wilt (FW)
is the most destructive disease to this crop
and is caused by a soil-borne pathogen,
Fusarium oxysporum
f. sp.
Sm.) Snyder & Han (Boughalleb &
Mahjoub, 2006). This disease is a
production-limiting disease in
watermelon growing regions of the
F. oxysporum
f. sp.
has the
ability to colonize the root cortex of
watermelon plants and penetrate into the
xylem resulting in an initial loss of turgor
pressure, wilting causing the whole plant
death (Callaghan et al., 2016).
Management of FW is difficult because
of the long-term survival of the pathogen
in the soil and the evolution of new races
(Lin et al., 2009). There are three races of
F. oxysporum
f. sp.
designated 0,
1 and 2 based on their aggressiveness or
their ability to overcome specific
resistance (Wehner et al., 2008). FW has
also increased in watermelon production
areas infected mainly by the highly
F. oxysporum
f. sp.
2 more than
F. oxysporum
f. sp.
race 1 which was also detected
(Boughalleb & Mahjoub, 2006). A major
reason for this difficulty is the inability to
accurately detect the presence and
identity of the fungal pathogen, especially
in plant tissues and soil (Zhang et al.,
2005). Molecular methods have been
developed to discriminate Fon from other
Fusarium oxysporum
(Lin et al., 2010)
and involve a PCR (Lin et al., 2009)
based on randomly amplified
polymorphic DNA (RAPD) detection
system. Zhang et al. (2005) developed a
rapid diagnostic method using a primer
set Fn1/Fn2 to differentiate Fon from
Didymella bryoniae
and a broad group of
other fungi, including three other
formae speciales (Fo). This
technique was rapid and reliable for their
isolates. This primer set, however, was
unable to differentiate Taiwanese Fon
isolates from other
F. oxysporum
speciales. Lin et al., (2010) developed
another primer set Fon1/Fon2 that was
more suitable for differentiating
F. oxysporum
f. sp.
F. oxysporum
formae speciales. The
set Fon1/Fon2 was also able to detect
f. sp.
in diseased
watermelon tissue at early stages of wilt.
In the other hand, the genetic diversity is
evidently an important character of
species and populations, determining
their response to changes in
environmental conditions, their survival
and evolvement and an answer to any
confusion in morphological identification
(Leong et al., 2010). The Random
Amplified Microsatellite (RAMS)
technique has been shown to be
applicable for
F. oxysporum
f. sp.
(Zhang et al., 2005). A study from India
looked at SSRs that could be used in
differentiation of
F. oxysporum
lineages (Mahfooz et al., 2012). Appel
and Gordon (1995) demonstrated the
relationship between pathogenic and non-
pathogenic isolates of
F. oxysporum
based on the partial sequence of the
intergenic spacer region of the ribosomal
DNA. Severe outbreaks of Fusarium Wilt
have been observed in Tunisia, causing
yield losses estimated at approximately
100% with a yield losses as high as 60%
(Boughalleb & Mahjoub, 2006). A
diagnostic survey was thus undertaken,
and the results showed that
F. oxysporum
f. sp.
is the most species
commonly isolated. Although effective,
selection for traits by conventional
methods is time consuming and resource
intensive. Having markers linked to traits
of interest can greatly accelerate
conventional breeding and allow timely
release of improved cultivars. The aims
of this study were to characterize
f. sp.
isolates using
specific primers and their genetic
diversity among
F. oxysporum
f. sp.
Tunisian population by ISSR
2. Materials and methods
2.1 Fungal isolates origin
F. oxysporum
f. sp.
(FON) isolates originated from different
regions of Tunisia and Spain were used
for this study. Spanish isolates, were
kindly provided by Pr. Abad-Campos P.
(Universidad Politécnica de Valencia)
(Table 1). The isolation, morphological
identification and pathogenicity test of
these isolates were done by Boughalleb
and El Mahjoub (2006) who proved that
these isolates are the causals agents of
watermelon Fusarium wilt in Tunisia.
The isolates were maintained in a
collection at the laboratory of Plant
Institut Supérieur
Agronomique de chott Mariem
, Sousse,
2.2 DNA extraction and PCR
isolates (Fon 10, Fon 11,
Fon 14, Fon 15, Fon 16 and Fon 17) were
grown in 20 ml of potato dextrose agar
(PDA) for 5 days at 28°C. Genomic
DNA was extracted using the E.Z.N.A.
Plant Miniprep Kit (Omega Bio-tek,
Norcross, GA, USA) following
manufacturer’s instructions. The specific
primers Fn-1
-3') sequences were amplified with PCR.
Each PCR reaction mixture contained
1.25×PCR buffer, 1.25 Mm MgCl
, 1 μM
each dNTP, 0.5 μM of each primer, 0.1
U of DNA Taq polymerase (Dominion
MBL, Córdoba, Spain), and 1 μL of
template DNA. The PCR reaction mix
was adjusted to a final volume of 13 μL
with water (Chromasolv Plus, Sigma-
Aldrich, Steinheim, Germany). DNA
amplification was performed using PCR
amplifications on a Peltier Thermal
Cycler-200. The program consisted of an
initial step of 5 min at 94°C, followed by
35 cycles of denaturation at 94°C for 1
min, annealing at 56°C for 1 min, and an
elongation at 72°C for 2 min. A final
extension was performed at 72°C for 7
min. The volume of 5 µl of PCR
products was subjected to electrophoresis
in 0.7% agarose gels (agarose D-1 Low
EEO; Conda). The amplification
products were examined under UV light,
after ethidium bromide staining, and
photographed using Alpha digidoc 1000
system (Alpha Innotech Corporation,
USA) gel documentation system, for
scoring the bands. The 100 bp DNA
ladder (Biotools, Madrid, Spain) was
used as molecular size marker.
Amplifications from each DNA sample
were repeated at least twice. PCR
products were purified using the High
Pure PCR Product Purification kit
(Roche Diagnostics). The PCR products
were visualized in 1.5% agarose gels
(agarose D-1 Low EEO, Conda, Madrid,
Spain) and molecular weights were
estimated using the GeneRuler 100 bp
Plus DNA Ladder (Fermentas, Carlsbad,