Morphological and molecular identification of Fusarium oxysporum f. sp. lycopersici associated with Olea europaea var. sylvestris decline phenomenon in Tunisia

Abstract

Decline phenomena and mortality of Olea europaea var. sylvestris (oleaster) have been
reported throughout the forest of Henchir Kort (northeastern of Tunisia). The affected plants show progressive dieback of shoots, twig blight symptoms and trunk canker. The fungi appear to have the most significant potential threat to the disease. However, it has been less well-studied in Tunisia. A survey on the causal agents of O. europeae decline attacked twigs with symptoms of wilting and vascular necrosis were collected. The causal agent of the syndrome was identified as Fusarium oxysporum f. sp. lycopersici based on morphological characteristics and molecular identification performed by sequencing the ITS region of the ribosomal DNA. Fusarium species are among the most aggressive telluric fungi, causing diebacks of many plant species, especially on Olea europaea. To the best of our knowledge, this is the first record on the occurrence of Fusarium oxysporum f. sp. lycopersici on O. europaea in the word and in Tunisia.

Full text

Submitted 5 December 2020, Accepted 26 April 2021, Published 1 June 2021
Corresponding Author: Sawssen Hlaiem – e-mail – sawssenhlaiem@gmail.com 15
Morphological and molecular identification of Fusarium oxysporum f.
sp. lycopersici associated with Olea europaea var. sylvestris decline
phenomenon in Tunisia
Hlaiem S1,2*, Della Rocca G 3, Barberini S3, Danti R3 and Ben Jamâa ML1
1University of Carthage, National Institute for Research in Rural Engineering Water and Forest (INRGREF), LR11
INRGREF01 Laboratory of Management and Valorization of Forest Resources, Bp. 10, 2080, Ariana, Tunisia
2University of Carthage, National Agronomic Institute of Tunisia (INAT), Department of Phytiatry, 43 street Charles
Nicolle, 1082, Tunis Mahrajene, Tunisia
3National Research Council, Institute for Sustainable Plant Protection of Italy (IPSP-CNR), Unit of Plant Protection,
Via Madonna del Piano10, 50019, Sesto Fiorentino, Firenze (Florence), Italy
Hlaiem S, Della Rocca G, Barberini S, Danti R, Ben Jamâa ML 2021 – Morphological and
molecular identification of Fusarium oxysporum f. sp. lycopersici associated with Olea europaea
var. sylvestris decline phenomenon in Tunisia. Plant Pathology & Quarantine 11(1), 15–22,
Doi 10.5943/ppq/11/1/3
Abstract
Decline phenomena and mortality of Olea europaea var. sylvestris (oleaster) have been
reported throughout the forest of Henchir Kort (northeastern of Tunisia). The affected plants show
progressive dieback of shoots, twig blight symptoms and trunk canker. The fungi appear to have
the most significant potential threat to the disease. However, it has been less well-studied in
Tunisia. A survey on the causal agents of O. europeae decline attacked twigs with symptoms of
wilting and vascular necrosis were collected. The causal agent of the syndrome was identified as
Fusarium oxysporum f. sp. lycopersici based on morphological characteristics and molecular
identification performed by sequencing the ITS region of the ribosomal DNA. Fusarium species are
among the most aggressive telluric fungi, causing diebacks of many plant species, especially on
Olea europaea. To the best of our knowledge, this is the first record on the occurrence of Fusarium
oxysporum f. sp. lycopersici on O. europaea in the word and in Tunisia.
Keywords – Dieback – Fungi – Identification – ITS region
Introduction
The olive tree is present on six continents: Europe, North America, South America, Africa,
Asia and Oceania. It’s a very ancient Mediterranean species. It belongs to the family of Oleaceae,
genus Olea, species europaea. Two subspecies distinguish it: Olea europaea var. sylvestris
(oleaster) and Olea europaea var. sativa (cultivated olive tree) (Villa 2003). Its wild form Olea
europaea var. sylvestris is mainly found in Mediterranean countries: Portugal, Spain, Italy, Turkey,
Greece, Morocco, Syria and Tunisia (Villa 2003). Furthermore, O. europaea var. sylvestris seems
to be the ancestor of the cultivated olive tree (Belaj et al. 2010). The economic and ecological
interest of the oleaster is major. It is used as a firewall to maintain soils and limit erosion (Breton et
al. 2006).
Plant Pathology & Quarantine 11(1): 15–22 (2021) ISSN 2229-2217
www.ppqjournal.org Article
Doi 10.5943/ppq/11/1/3

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However, this evergreen specie is withering in countries of the Mediterranean basin,
including Tunisia (Lazzizera et al. 2008, Moral et al. 2009), due to the interaction of several abiotic
factors (anthropic action, drought, water stress, and fires), and biotics (insect pests and pathogenic
fungi) (Ben Jamâa & Hasnaoui 1996). Most fungal species causing dieback of oleaster are common
saprophytes or secondary invaders usually penetrating through injuries made by biotic or abiotic
factors (Lazzizera et al. 2008). Moreover, infections caused by fungi can cause very worrying
diebacks, such as those caused by telluric fungi including Fusarium spp. which have been shown to
cause symptoms of wilting and partial or total dieback of the olive (Jardak et al. 2007, Trabelsi et
al. 2017) and caused extensive damage in several countries in the Mediterranean basin (Porras-
Soriano et al. 2003). The Fusarium genus is one of the most complex and adaptive species in the
Nectriaceae. The Fusarium oxysporum (Fo) species complex includes plant, animal and human
pathogens and a diverse range of non-pathogens (Gordon 2017). This fungal pathogen is widely
represented with a predominance of Fusarium oxysporum (Chliyeh et al. 2017), which are
responsible for two distinct types of symptoms: wilting of the aerial part of the plant and root
and/or collar rots (Gordon 1965). Members of Fusarium species are ubiquitous soil-borne
pathogens of a wide range of horticultural and food crops which cause destructive vascular wilts,
rots, and damping-off diseases (Bodah 2017). In particularly, Fusarium oxysporum f. sp.
lycopersici first described in Europe at the end of the 19th century; it is present in dozens of
countries on every continent (Blancard et al. 2009). Based on its economic importance and
scientific interest, this species has been ranked among the “top 10” of plant pathogenic fungi (Dean
et al. 2012).
In this context, this work consists of (i) the isolation of the pathogen (Fusarium) from
symptomatic oleaster branches, (ii) its morphological identification (macroscopic and microscopic)
and (iii) its molecular identification.
Materials & Methods
Study area
The study was carried out in 2017, in the forest of Henchir Kort (36.30'.406" N;
10.38'.780"E) in Cap-Bon in the northeastern of Tunisia (Fig. 1). The vegetation is a mixture of
pine trees with Mediterranean scrub composed mainly of Olea europaea var. sylvestris.
Fig. 1 – Localization of studied site (Henchira Kort forest is indicated with a red star).
Phytosanitary status
Damage degree was estimated based on visual appreciation of disease symptoms and a
decline class of each O. europaea plant was assessed following the methodology described by

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Franceschini et al. (2005), with: (C0) no attack /absence of symptoms; (C1) = 1 to 25%; (C2) = 26
to 50%; (C3) = 51 to 75% and (C4) = 76 to 100%.
Collection of samples and isolation
In December 2017, typical disease symptoms were collected from stems of O. europaea and
transferred to the laboratory. First, all samples are superficially disinfected using the surface
sterilization method described by Alves et al. (2013). Small pieces of necrotic tissue (3× 3 mm)
were taken from the margin of infected tissues were placed in Petri dishes containing potato
dextrose agar (PDA) added with streptomycin sulfate (0.05 g/l) antibiotic according to the
technique used by Franceshini et al. (2005) and incubated in the dark at 25°C for 3 days. Pure
cultures were obtained by plating a small piece of mycelium from the margin of each colony grown
on PDA and incubating them under the same conditions described above.
Morphological and molecular identification
Fusarium oxysporum was identified based on its cultural traits, conidial morphological
characteristics by referring to identification keys (Rayner 1970) and forest mycology guide (Lanier
et al. 1976). Colony morphology, including color, shape, and growth rate, was determined after 7
days of incubation on PDA at 25°C in darkness. Microscopic characters were studied according to
the technique explained by Arzanlou et al. (2007). Dimension of microscopic structures were
calculated based on 30 measurements for conidia morphology (shape, color, and cell number), size
(length and width). The percentage of isolation frequency (IF) was calculated using the formula of
IF (%) Franceschini et al. (2005): IF = Ni/Nt x 100 with Ni (number of fragments colonized by the
fungus) and Nt (total number of plated fragments).
Regarding the molecular identification, fungal DNA was directly extracted from mycelia
growing on plates, using a commercial Kit Macherey-Nagel- 07/2014, Rev.09.PCR reactions were
carried out with ITS1 and ITS4 primers (White et al. 1990) to amplify the ITS region of the
ribosomal RNA as described by Alves et al. (2004). Products from PCR reactions were
electrophoresed on a 1.5% agarose gel, then stained with GelRed, and visualized with UV
transilluminator. The size of PCR products was estimated by comparison with a DNA ladder 100
bp plus, Transgen Biotech. All PCR amplifiers were sent for sequencing to the Interdepartmental
Center for Chemical and Industrial Agricultural Biotechnology Services (Italy) laboratory. The
representative sequence was deposed in GenBank.
Phylogenetic analysis
ITS sequences were used to conduct a phylogenetic analysis. Sequences of Fusarium species
were retrieved from GenBank and aligned with sequence of the isolate (TN.24) obtained in this
study. Sequences were aligned with ClustalX v. 1.83 (Thompson et al. 1997), using alignment
parameters according to Linaldeddu et al. (2015). Phylogenetic tree was generated under Maximum
Likelihood (ML) and analyzed to build the tree topology by the Neighbour-Joining method using
MEGA 6.0 software (Tamura et al. 2013).
Results
Incidence
The Henchir kort forest investigation revealed that 40% of the examined oleaster plants
showed dead twigs with necrotic lesions (Fig. 2). Subjects of decline class (C1) dominate with a
rate of 50% followed by (C2) subjects with a rate of 34% and 16% for (C3).
Morphological characteristic
Morphological identification was based on an observation combining colony morphology and
microscopic spore observation; it was taken place after 7 days of incubation. A collection of 20

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isolates was obtained from infected samples collected from the oleaster plants at the Henchir kort
forest. Preliminary identification of the genus was carried out by analyzing morphological traits
based on the general appearance of colonies and the aspect of conidia. On PDA, colonies have a
whitish, cottony and dense medium-growing mycelium (Fig. 3). Microscopic observation revealed
the presence of very short conidiophore even invisible, septate macroconidia (3 to 7 septate) more
or less curved fusiform measuring 2.9 to 4.9 μm by 23 to 53 μm. The apical cell is tapered and
curved, and the basal cell is pedicelled. Microconidia are ellipsoidal and slightly curved, measuring
2 to 3.5 μm by 4.5 to 11 μm (Fig. 2).
In this study, Fusarium species were isolated from all sampled O. europaea plants showing
disease symptoms and 45% of isolates were identical to Fusarium oxysporum f. sp. lycopersici.
Fig. 2 – Necrotic lesion on stem observed in naturally infected Olea europeae plant.
Fig. 3 – Fusarium oxysporum f. sp. lycopersici: Macroscopic appearance of the colony incubated
on PDA at 25°C for 5 days (right) and microscopic appearance of conidia (40X) (left).

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Phylogenetic analyses
A representative isolate was selected for molecular identification and the identity of the
species was confirmed by DNA sequence analysis of the ITS regions. The BLAST research of the
isolate TN.24 selected for molecular identification showed 98% homology with Fusarium
oxysporum f. sp. lycopersici (FSOT) (KY100124) and the representative sequence was deposited in
GenBank under the accession number (MN843963). The phylogenetic tree, resulting from the PCR
amplification sequence of the ADNr nuclear operon using the ITS1 and ITS4 primers of the isolate
TN.24 obtained in this study reveals that our isolate is grouped with other F. oxysporum f. sp.
lycopersici (FOL) (Snyder & Hansen 1940) downloaded from the GenBank database and separate
from the group of F. equiseti (Fig. 4).
*
Fig. 4 – Phylogenetic tree obtained from ITS sequence data from the TN.24 isolate. Bootstrap
support values (%) from 1000 replications are shown at the nodes. The tree is rooted in
Colletotrichum acutatum. The scale bar shows 0.005 substitutions per site.
Discussion
Surveys in the Henchir Kort forest revealed the presence of dieback and necrosis on the twigs
of olive shrubs resulting in defoliation and drying of new shoots. The pathogen was isolated from
samples of the infected branches. Based on macroscopic and microscopic morphological criteria,
the isolate TN.24 obtained in this study was identified as Fusarium sp. which is consistent with the
results obtained by Isebaert et al. (2005). Moreover, the amplification of ribosomal DNA by the
two universal primers (ITS1/ITS4) made it possible to distinguish the exact isolated species.
According, Nasraoui & Lepoivre (2003) asserted that ITS regions are widely used for species
identification. As a result, morphological and molecular phytopathological analyses have confirmed
the presence of Fusarium oxysprum f. sp. lycopersici. Thus, O. europaea dieback is caused by this
pathogen. The results obtained in this study are consistent with those of Cristobal-Alejo et al.
(2016) in Mexico, which confirmed that Fusarium oxysporum f. sp. lycopersici (FSOT) caused
significant damage to the stems of the Saccharum officinarum. Others researches, also such as
those of Al-Ahmad (1984) in Syria and Sanchez et al. (1998) in Spain, have shown that telluric
fungi causing threat to olive trees. In fact, the genus Fusarium includes many plant pathogenic
species that can induce disease in many plants. Furthermore, symptoms of dieback caused by the
genus Fusarium have been observed on young olive trees planted in Morocco (Chliyeh et al. 2014).
As well, two species F. oxysporum and F. solani were isolated from crown and olive stems in
Algeria (Merzoug et al. 2018). In Saudi Arabia, AL-Shebel et al. (2005) identified two olive
dieback agents, Fusarium spp. et Verticillium dahliae. In the other hand, Messiaen & Cassini
(1968) confirmed that Fusarium oxysporum f. sp. radicis lycopersici attacks the root parts and

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Fusarium oxysporum f. sp. lycopersici (Sacc.) WC Snyder & HN Hansen attacks the aerial parts of
the plant. In addition, Asha et al. 2011 reported that Fusarium oxysporum f. sp. lycopersici (Sacc.)
WC Snyder & HN Hansen (FOL) caused vascular wilt of tomato disease and reduced the yield to
the maximum extent.
Conclusion
This finding was the first record of Fusarium oxysporum f. sp. lycopersici as fungal pathogen
associated with Olea europea var. sylvestris dieback in Tunisia. Despite the economic losses it
causes, control of this pathogen is still limited to prophylactic measures, disinfection of the soil is
never complete due to the difficulty of its production and to resistant strains (Benhamou et al.
1997). Therefore, and before the implementation of a strategy to control this phytopathogen, it is
necessary to better understand the epidemiology and the mechanisms of fusarium oxysporum f. sp.
lycopersici infection to assess its aggressiveness by artificial inoculations on the branches of
oleaster. The assessment of the pathogenicity of this pathogen is being appraised.
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