Journal of Phytopathology and Pest Management 7(1): 54-63, 2020

pISSN:2356-8577 eISSN: 2356-6507

Journal homepage: http://ppmj.net/

∗

Corresponding author:

Naima Boughalleb-M’Hamdi,

E-mail: n.boughalleb@laposte.net

54

Genetic diversity of

Fusarium oxysporum

f.sp.

niveum

responsible of watermelon Fusarium

wilt in Tunisia and Spain

Naima Boughalleb-M’Hamdi

1

*, Ibtissem Ben Salem

1

, Najwa Benfradj

1

, Paloma Abad-Campus

2

1

Department of Biological Sciences and Plant Protection, University of Sousse, UR13AGR3, Higher

Agronomic Institute of Chott Meriem, 4042, Sousse, Tunisia

2

Mediterranean Agroforestry Institute, Polytechnic University of Valencia, Camino de Vera s / n,

46022, Valencia- Spain

Abstract

Keywords: Fusarium oxysporum f. sp. niveum; genetic diversity; ISSR; molecular detection; watermelon.

55

1. Introduction

Watermelon (

Citrullus lanatus

(Thunb.)

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.

niveum

(E.F.

Sm.) Snyder & Han (Boughalleb &

Mahjoub, 2006). This disease is a

production-limiting disease in

watermelon growing regions of the

world.

F. oxysporum

f. sp.

niveum

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.

niveum

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

virulent

F. oxysporum

f. sp.

niveum

race

2 more than

F. oxysporum

f. sp.

niveum

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

F.

oxysporum

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

formae

speciales. Lin et al., (2010) developed

another primer set Fon1/Fon2 that was

more suitable for differentiating

Taiwanese

F. oxysporum

f. sp.

niveum

from

F. oxysporum

formae speciales. The

set Fon1/Fon2 was also able to detect

F.

oxysporum

f. sp.

niveum

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.

niveum

(Zhang et al., 2005). A study from India

looked at SSRs that could be used in

differentiation of

F. oxysporum

pathogen

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.

niveum

is the most species

commonly isolated. Although effective,

selection for traits by conventional

56

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.

oxysporum

f. sp.

niveum

isolates using

specific primers and their genetic

diversity among

F. oxysporum

f. sp.

niveum

Tunisian population by ISSR

markers.

2. Materials and methods

2.1 Fungal isolates origin

Forty-five

F. oxysporum

f. sp.

niveum

(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

Pathology,

Institut Supérieur

Agronomique de chott Mariem

, Sousse,

Tunisia

2.2 DNA extraction and PCR

identification

Six

Fusarium

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

(5'-

TACCACTTGTTGCCTCGGC-3') and

Fn-2

(5'-TTGAGGAACGCGAATTAAC

-3') sequences were amplified with PCR.

Each PCR reaction mixture contained

1.25×PCR buffer, 1.25 Mm MgCl

2

, 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,

CA).

57

Table 1: Characteristics of Fusarium oxysporum f.sp.

niveum isolates used for molecular identification with

specific primers.

Isolates

Origin

Race

Fon1

Spain

0

Fon2

Spain

0

Fon3

Spain

2

Fon4

Spain

2

Fon5

Spain

2

Fon6

Spain

1

Fon7

Spain

1

Fon8

Spain

1

Fon9

Jebniena

-

Fon10

Testour

-

Fon11

Testour

2

Fon12

Sebbala

-

Fon13

Sebbala

-

Fon14

Oued mliz

-

Fon15

Chébika

-

Fon16

Oued Mliz

-

Fon17

Metouia

2

Fon18

Oued Mliz

1

Fon19

Oued Mliz

-

Fon20

Gafsa

-

Fon21

Skhira

2

Fon22

Skhira

0

Fon23

Jebniena

-

Fon24

Chebika

-

Fon25

Skhira

0

Fon26

Gafsa

-

Fon27

Beja

-

Fon28

Chébika

2

Fon29

Médenine

-

Fon30

Mareth

-

Fon31

Gafsa

1

Fon32

Ben Aoun

-

Fon33

Metouia

2

Fon34

Mareth

-

Fon35

Gafsa

2

Fon36

Skhira

1

Fon37

Gafsa

1

Fon38

Skhira

-

Fon39

Mareth

-

Fon40

Jebniena

-

Fon41

Gaafour

-

Fon42

Sebbala

-

Fon43

Testour

-

Fon44

Beja

-

Fon45

Gaafour

-

2.3 Genetic diversity

2.3.1 Random amplified microsatellites

Twenty-six

F. oxysporum

f. sp.

niveum

(FON) isolates has been used for RAMS

test (FON1, FON 2, FON 3, FON 5, FON

6, FON 7, FON 8, FON 9, FON 12, FON

13, FON 16, FON 18, FON 20, FON 23,

FON 25, FON 29, FON 31, FON 32,

FON 33, FON 34, FON 35, FON 37,

FON 40, FON 42, FON 44 and FON 45).

Extracted DNAs were amplified using

58

ISSR primers. Initially, to select primers

which produce polymorphic bands for the

characterization of

F. oxysporum

f. sp.

niveum

isolates, a total of six ISSR

primers were evaluated for their capacity

to produce polymorphic, scored and

reproducible DNA fingerprint patterns.

The primers included were two

dinucleotide, and four trinucleotide

repeats with or without 5’ anchors:

5’DVD (CT)

7

C (Mahuku et al., 2002) ,

5’YHY(GT)

7

G, 5’DHB(CGA)

5

(Hantula

et al. 1996), 5’DDB(CCA)

5

(Hantula et

al. 1997), 5’(GAC)

5

, 5’(GTG)

5

(Pina et

al., 2005) (Tib Molbiol, Berlin). Each

PCR reaction contained 1 PCR buffer,

2.5 mM MgCl

2

, 100 mM each dNTPs,

0.4 mM of each primer, 0.5 U DNA Taq

polymerase (Dominion MBL, Cordoba)

and 0.5–5 ng template DNA were

quantified spectrophotometrically. The

PCR reaction mix was adjusted to a final

volume of 25 ml with water (Chromasolv

Plus, Sigma-Aldrich, Steinheim). PCR

amplifications were performed on a

Peltier Thermal Cycler-200 (MJ

Research, Waltham, Massachusetts). The

program consisted of an initial step of 5

min at 95°C, followed by 34 cycles of

denaturation at 95°C for 1 min, annealing

at 41°C (CT)7, 58°C (GT)7, 64°C

(CCA)5, 61°C (CGA)5, 46°C (GAC)5,

56°C (GTG)5 for 1 min, and an

elongation at 72°C for 2 min. A final

extension was performed at 72°C for 10

min. PCR products were separated in 1.5

% agarose gels (agarose D-1 Low EEO,

Conda, Madrid), stained with ethidium

bromide and visualized under UV light.

Gene Ruler 100 bp DNA ladder plus was

used as a molecular weight marker (MBI

Fermentas). All ISSR assays were

repeated at least three times, and only

clear and reproducible bands were

considered.

2.4 Data analysis

Fragments amplified by the ISSR

primers were visually scored as present

(1) or absent (0); Fragments with the

same size were considered equal. The

genotype profiles produced by amplified

RAMS markers were scored manually.

The data were assembled in a unique

matrix and analyzed. The similarity

matrix was used to construct

dendrogram. Data from the genetic

distance values were used as inputs in

order to generate a dendrogram using the

unweighted pair-group method with

arithmetic averaging (UPGMA) as

implemented by MEGA software.

3. Results

3.1 PCR amplification of Fon isolates

DNA

In this study, the PCR-based

identification technique with the two

specific primers Fn-1/Fn-2 used for some

F. oxysporum

f. sp.

niveum

isolates was

able to amplify a unique DNA fragment

of approximately 800 bp for all

F.

oxysporum

f. sp.

niveum

isolates

originated from Tunisia and from Spain

as shown on Figure 1.

3.2 Genetic diversity

Six ISSR primers were tested

individually using

F. oxysporum

f. sp.

niveum

isolates DNA to determine which

primer exhibiting a profile of light bands

in agarose gels and revealed

polymorphisms level between

F.

oxysporum

f. sp.

niveum

isolates.

Seventy-one bands were amplified by

59

four ISSR primer combinations.

Diversity in the banding patterns

obtained by DNA fingerprinting was

always >50% and allowed us to

distinguish all the isolates tested,

according to number and size of the

fragments, which ranged from 300 to

2800 bp. The most polymorphic loci

were the trinucleotide primer CGA with

22 bands differentiated with molecular

sizes ranging from 400 to 2000 bp,

followed by CCA which produced 20

bands between 300 and 2800 bp.

However, the two other trinucleotides

primers GTG and GAC generated only

15 bp each, with sizes comprised

between 550 and 1500 and from 600 to

2000, respectively.

Figure 1: Agarose gel electrophoresis of PCR-amplified products using the specific

primers Fn-1/Fn-2. M: 100-bp DNA ladder marker.

RAMS banding patterns generated by

primers CGA (A), GTG (B), CCA (C) and

GAC (D) of FON isolates. (1: Fon1; 2:

Fon2; 3: Fon3; 4: Fon5; 5: Fon6; 6: Fon7;

7: Fon8; 8: Fon9; 9: Fon12; 10: Fon13; 11:

Fon16; 12: Fon18; 13: Fon20; 14: Fon23;

15: Fon25; 16: Fon29 ; 17: Fon31 ; 18:

Fon32 ; 19: Fon33 ; 20: Fon34 ; 21:

Fon35; 22: Fon37 ; 23: Fon40; 24: Fon42;

25: Fon44 and 26: Fon 45) ; M: 100-bp

DNA ladder marker. Reproducibility of

amplified bands yielded by these four

primers was confirmed with CGA and

CCA, and the first primer was chosen to

analyze the intraspecific genetic variability

of

F. oxysporum

f. sp.

niveum

by Cluster

analysis based on Nei’s coefficient and

UPGMA method.

The forty-five genotypes

were grouped into six main clusters at a

similarity index value above 0.5 (Figure 3).

Within the groups, the most abundant was

Cluster VI comprising 39 isolates and the

Nei’s coefficient values ranged from 0.28

to 0.97, indeed, both isolates FON6 and

FON7 race 1 from Spain registered the

similarity

coefficient of 0.97.

The highest

similarity value of 0.75 (97%) occurred

between five FON isolates FON33

(Metouia, race 2), FON35 (Gafsa, race 2),

FON37 (Gafsa, race 1), FON40 (Jbeniana)

and FON42 (Sebbala)), and between the

two FON isolates FON13 (Sebbala) and

FON29 (Medenine). Only cluster I

comprised a single genotype FON23

(Jbeniana) which could be considered as an

out grouping sample. While, clusters II

60

consisted of two isolates FON12 (Sebbala)

and FON34 (Mareth), these three isolates

appeared to be distinct from all the other

genotypes (Figure 2).

Figure 2: Dendrogram of 26 isolates of Fusarium oxysporum f.sp. niveum. Data were

generated using unweighted pair group method of arithmetic means (UPGMA) using the

Mega.5.1 software based on genetic distance coefficient.

Obtained results indicated that isolates

FON5 and FON3 from Spain fall among

Tunisian isolates, and may this indicate

for a possible close relationship between

these isolates and Tunisian isolates, while

the rest of Spanish isolates FON1, FON2,

FON4, FON6 formed a distinct cluster

than Tunisian isolates suggested the

existence of a highly variable genetic

population in both country.

61

4. Discussion

The first step in Fusarium wilt

management is an accurate diagnosis and

detection. Control recommendations are

usually made following putative

diagnosis. Additional research on rapid

detection techniques for

F. oxysporum

f.

sp.

niveum

and economical methods for

field level identification of

F. oxysporum

f. sp.

niveum

races will improve

management of this reemerging disease

(Everts et al., 2015). All heritable

information is potentially accessible

using DNA sequencing. Consequently,

DNA sequence analysis is expected to

provide the solution to the problem

associated with the taxonomy and

phylogeny of

Fusarium

species in

general, and

F. oxysporum

in particular

(Lin et al., 2010). In the present study,

the molecular characterization by PCR

using specific primers couple Fn-1/Fn-2

of Tunisian

F. oxysporum

f. sp.

niveum

isolates was successfully accomplished.

And obtained result was in agreement

with those reported by Zhang et al.

(2005) which indicated that these primers

can be beneficial for rapid detection of

F.

oxysporum

f. sp.

niveum

isolates

affecting watermelon, and could also be

helpful in epidemiological and etiological

studies to monitor the behavior of the

pathogen in diseased plants. Genetic

diversity within populations is an

important indicator referring to a

population’s potential adaptation to

environmental changing and to inform on

the appropriate method of control (Rebib

et al., 2014). Genetic control of the

disease is crucial in managing these

pathogens. Identification of single

nucleotide polymorphism (SNP) markers

linked to resistance can be a powerful

tool for the introgression of valuable

genes needed to develop

Fusarium

-

resistant varieties. In

F. oxysporum

,

DNA fingerprinting involving

hybridization with SSR-containing

probes has been used in the classification

of some

formae specialis

(Barve et al.,

2001). In this study, RAMS analysis

revealed a moderate genetic diversity

between

F. oxysporum

f. sp.

niveum

races. This indicated a low degree of

polymorphism. Similar results were also

obtained with

F. oxysporum

f. sp.

erythroxyli

(Nelson et al., 1997). The

limited genetic variability observed

among isolates would be expected for a

pathogen that became widespread

relatively quickly as a result of an

increase in production of the host plant

(i.e. distribution by seed) (Belabid et al.,

2004). The current study suggested that

some Tunisian and Spanish

F.

oxysporum

f. sp.

niveum

isolates were

indeed genetically similar. However,

isolates from other watermelon-growing

regions of the world should be tested to

determine whether all the Tunisian

isolates are related to other genetic

groups from watermelon around the

world. Since most of the markers were

developed using pairs of ISSR primers,

however, the use of ISSR primers in

pairs, rather than individually, may be

more efficient to develop this technique.

By considering the cultivar and

geographic origin, generally we could

not find a relationship between virulence

and cultivar. Hirano and Arie (2009)

confirmed this result, with no stable

correlation between phylogeny and

pathogenic-group. The SSR primers

should be particularly useful because the

fungus is one of the most common

Fusarium

spp. residing in the soil

62

environment and that it includes

pathogens, biological control agents and

saprophytes. Their application should

also enhance understanding relatedness

of formae speciales in the

F. oxysporum

complex.

Acknowledgements

This study was financed by

UR13AGR03, University of Sousse,

Tunisia.

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