Journal of Plant Biology Research 2012, 1(2): 51-56

ISSN: 2233-0275
http://www.inast.org/jpbr.html

REGULAR ARTICLE

Molecular and xylanolytic variation identified among isolates of Fusarium species

Y. Bakri, M. Jawhar and M.I. E. Arabi*
Department of Molecular Biology and Biotechnology, AECS, P. O. Box 6091, Damascus, Syria

ABSTRACT
Twenty-four isolates of Fusarium sp. (9 F. verticillioides, 8 F. culmorum, 4 F. solani and 3 F. equiseti) were studied
in relation to xylanase production and analysis of genomic DNA through inter-retrotransposon amplified
polymorphism (IRAP). The data demonstrated that variation occurred in xylanase activity and molecular level
among isolates. The molecular parameter used showed that Fusarium isolates reside in four phylogenetic groups. A
neighbour-joining diagram based on Nei's genetic distances, result some clades specific to xylanase production.
Hence, the described approach presented here constitutes no prior assumption about the characterization of
Fusarium sp. differing in xylanase production.
Keywords: Fusarium spp., IRAP, xylanase, solid state fermentation

INTRODUCTION
Xylanolytic enzymes are produced by a
wide variety of microorganisms, among
which the filamentous fungi are especially
interesting as they secrete these enzymes into
the medium and their xylanase activities are
much higher than those found in yeast and
bacteria (6). As plant pathogens, the
Fusarium species primarily direct their attack
on the plant cell wall by producing key
enzymes associated with its hydrolysis (11).
Identification of species of the genus
Fusarium is complicated on several aspects.
Morphological characteristics such as the
shape and size of the macroconidia, the
presence or absence of microconidia and
chlamydospores, and colony morphology are
often insufficient to allow identification at the
species level. In addition, these observations
need some practices and are difficult for the
*

Corresponding author; M.I. E. Arabi

e-mail: ascientific@aec.org.sy,

non-specialist (23, 4). Although pathogenicity

phenotypes can be useful for assessing
genetic variation in fungal pathogens;
however, pathogenicity markers are often
limited in number and subject to host
selection (13).
The solo long terminal repeats (LTRs)
found both in fungi and plant genomes
suggest that excision of the element is
possible by intraelement LTR to LTR
recombination. Retrotransposon markers
can be exploited by the IRAP (InterRetrotransposon Amplified Polymorphism)
technique that amplifies bands from two
nearby LTRs using outward-facing primers
annealing to LTR target sequences (8, 7).
IRAP method has recently been exploited to
study genetic diversity and phylogenetic in
plant species [9, 22] and fungal pathogens
(19).

J. Plant Bio. Res. 1(2): 51-56, 2012

Fusarium is a species rich genus which

considered important from the industrial
viewpoint due to extracellular release of
xylanases and they can be cultivated very easily
(11). However, xylanases are produced by
either solid state or submerged fermentation.
Enzyme productivity in solid state fermentation
(SSF) is usually much higher than that of
submerged fermentation (7). Therefore, solid
state fermentation has gained interest from
researchers in recent years and has often been
employed for the production of xylanases
because of economic and engineering
advantages (18). The objectives of the current
research were (i) to investigate variability
within the Fusarium sp. using IRAP marker,
and (ii) to determine a relationship between the
genetic diversity of these pathogenic isolates
and xylanase production in SSF.
MATERIALS AND METHODS
Microorganism
The 24 isolates of Fusarium species (9 F.
verticillioides, 8 F. culmorum, 4 F. solani and
3 F. equiseti) are described by Alazem (1).
They isolated from infected wheat seeds
showing disease symptoms, and screened
among 105 isolates for their various in
xylanase production and host-pathogen
reactions (10). The isolates were grown
separately in 9 cm Petri dishes containing
potato dextrose agar (PDA, DIFCO, Detroit,
MI. USA) and incubated for 10 days, at 23
1C in the dark to allow mycelia growth. All
isolates were identified morphologically
according to Nelson et al. (17). The Fusarium
isolates are listed in Table 1. The cultures
were maintained on silica gel at 4 C until
needed.
Enzyme production
Enzyme production by the selected isolates
was carried out in 250 ml Erlenmeyer flasks
containing 5 g of solid substrate and nutrients
(based on 100 ml of liquid medium) plus

Table 1. Xylanase activities produced by Fusarium

spp. grown in
Isolate
Activity
(IU/g of substrate)
F.culmorum
SY1
20.3
2
96.36
3
163.69
4
12.16
6
131.93
12
115.92
13
90.64
14
19.52
F. verticillioides
SY5
16.02
9
138.72
10
19.52
15
61.92
16
18.5
17
16.56
18
129.92
19
108.56
21
102.3
F.solani
SY7
1465.8
8
125.6
11
112.16
20
234.96
F.equiseti
SY22
93.2
23
84.64
24
122.43
LSD 5%
LSD: Least Significant Difference at P<0.05

distilled water to adjust the moisture content

to 80%. The fermentation medium consisted
of: (g/L) Na2HPO4 .2H2O 10; KCl 0.5;
MgSO4.7H2O 0.15, and Yeast extract 5, as a
nitrogen source. The pH was adjusted to 5
before sterilization. This pH value was found
to be the optimal one for xylanase production
in our experiments (data not included). Fresh
fungal spores have been used as inoculums
and 1mL spore suspension (containing around
106 spores/ mL) was added to sterilized
medium and incubated at 30 C. Flasks were

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J. Plant Bio. Res. 1(2): 51-56, 2012

removed after cultivation and the enzyme was

extracted by adding distilled water containing
0.1% Triton x 100 to make the volume in
flask equivalent to 100 mL. The flasks
contents were stirred for 1.5 hours on a
magnetic stirrer. The clear supernatant was
obtained by centrifugation (5000 x g for 15
min) followed by filtration (Whatman no. 1.
paper).
Enzyme assays
Xylanase activity was assayed by the
optimized method described by Bailey et al.
(3), using 1% birchwood xylan as substrate.
The solution of xylan and the enzyme at an
appropriate dilution were incubated at 55 C
for 5 minutes and the reducing sugars were
determined by the dinitrosalicylic acid
procedure (16), with xylose as standard. The
released
xylose
was
measured
spectrophotometrically at 540 nm. One unit
(U) of enzyme activity is defined as the
amount of enzyme releasing 1 mol
xylose/ml per minute under the described
assay conditions. The experiments were
repeated twice, and all the results represent
the mean values. Statistical analyses were
performed using the STAT-ITCF program (2)
to test the differences in xylanase production
among isolates tested.
DNA extraction
Twentyfour isolates were grown on PDA
medium for 2 weeks at 21 1C and stored at
4 C for further study. Mycelium was
harvested and DNA was extracted according
to standard protocols (12), resuspended in TE
buffer (10 mM Tris HCl, pH 8.0; 1mM
EDTA) and stored at -20C.
IRAP analysis
The IRAP method was performed using the
LTR primers derived from barley LTRs (8, 9).
Polymerase chain reaction (PCR) was carried
out in a reactions of 20 l contained: 0.075 M
Tris-HCl pH 8.8, 0.02 M (NH4)2SO4, 1.5 mM

MgCl2, 0.01% Tween-20, 0.2 mM dNTPs, 0.2

M each primer, 1.5 U Taq polymerase, and
10 ng DNA template. PCR was performed in
a Thermocycler (BIO-RAD system, USA).
Initial denaturation of 95 C for 2 min was
followed by 35 cycles [1 cycle consists of
denaturation for 1 min at 95C, annealing for
1 min at 45C and extension for 2 min at 72
C]. A final extension of 72 C for 5 min was
incorporated into the program, followed by
cooling to 4 C until recovery of the samples.
Amplified products were electrophoresed in a
2% agarose gel using 1 TrisborateEDTA
buffer (100 mmol TrisHCl/L, pH 8.3, 83
mmol boric acid/L, 1 mmol EDTA/L) at
100V. The gels were stained with ethidium
bromide (0.5 g/mL) solution and visualized
under ultraviolet illumination. Sizes of the
amplified products were determined relative
to a 100-bp DNA ladder (Q.BIOgene,
Heidelberg, Germany).
Each amplified fragment was treated
as a unit character and scored as a binary code
1 and 0 for presence and absence,
respectively, using 1Dscan EX 3.1software
(Scanalytics, Inc.). The experiments were
repeated twice for each isolate to confirm the
repeatability and the monomorphic bands
were removed from the analysis. The data
were analyzed using Treecon software (21).
A phylogenetic tree was constructed using the
neighbour-joining algorithm (20) by the
PHYLIP package ver 3.5c (5). Bootstrap
analysis with 1000 replicates was performed
to estimate the robustness of the evolutionary
tree.
RESULTS AND DISCUSSION
Table 1 shows that all the Fusarium species
were capable of producing xylanase activity.
However, significant differences (P<0.05) in
the mean yield values were detected among
isolates, with high values being consistently
higher in the isolates F. solani SY7 and SY20
with mean value 1465.8 and 234.69 U/g,
respectively. These isolates could be a good

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J. Plant Bio. Res. 1(2): 51-56, 2012

candidate for biotechnological applications.

On the other hand, low enzyme of 12.16 and
16.56 U/g was detected for F. culmorum SY4
and F. verticillioides SY17, respectively
(Table1).
PCR
consistently produced
many
distinctively amplified fragments ranging in
size from 100 to 1800 bp (Fig. 1). Similar
results have been reported for other organisms
(7, 14). Four clear IRAP groups comprising
Fusarium species can be observed (Fig. 2),
strongly supported by high bootstrap values.
Group1 supported by a bootstrap score of
94 %, contained F. culmorum isolates,
whereas the F. solani isolates were placed in
group 2 with a bootstrap value of 41% and
70%. F. equiseti isolates were placed in group
3 with a bootstrap value of 87%. Finally, F.
verticillioides isolates were placed in group 4
with a bootstrap value of 68%.
However, comparison of the IRAP
clustering and the enzyme production profiles
shows some resolution between clustering
isolates and their xylanase production. The
isolates F. solani SY7 and SY20 with higher
mean value 1465.8 and 234.69 U/g,
respectively were closely placed in one clade.
Similarly, the isolates F. culmorum SY1 and
SY4 with lower mean value 20.3 and 12.16
U/g, respectively were closely placed in one
clade (Fig. 2). The reason for the differences
in the production of inducible xylanase when
compared to the other isolates from different
Fusarium species, have yet to be clarified.
According to Kalendar et al. (9) more
IRAP bands are associated with the presence
of more retrotransposon insertion sites and
subsequently with higher evolutionary
position. On the other hand, Manicom et al.
(15) reported that genetic relationships among
Fusarium species could be attributed either to
a higher evolutionary rate in these species
(fast evolutionary clock) or to an earlier
branching in the Pyrenomycetes. However,
the studied species played a prominent role in
plant diseases of diverse host, therefore a

more comprehensive study of each group is

warranted.

Figure 1. Electrophoretic pattern generated by IRAP

primers sukkula and 5'LRT2 for 10 Fusarium isolates
(F. culmorum; 1 and 13), (F. equiseti; 22, 23 and 24),
(F. verticillioides; 5 and 19) and (F. solani; 8, 11 and
20). Lane M represents 100-bp DNA marker (HinFI,
MBI Fermentas, York, UK).

*
*

**
**

0.1

Figure 2. Unrooted phylogenetic tree of 24 Fusarium

sp. isolates (F. culmorum;1, 4, 12, 6, 2, 3, 13, and
14), (F. solani; 8, 11, 7, 20, (F.equiseti; 22, 23 and
24) and (F.verticillioids 5, 10, 15, 16, 17, 18, 19, 21
and 9) based on IRAP data . Percentages from 1000
bootstrap replications are given on tree branches. * and
** The isolates produced high and low xylanase
activity, respectively.

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J. Plant Bio. Res. 1(2): 51-56, 2012

The present study reveals that F. solani

SY7 isolates is very promising as it produced
a high level of xylanase under SSF. IRAP
markers provide a rich source of molecular
markers which are rapid and suitable way to
group Fusarium isolates differing in their
xylanase activity. Correlation could be
established between the groups and the levels
of xylanase produced. The results provide a
vast amount of information that can guide
hypothesis-driven research to elucidate the
molecular mechanisms involved in xylanase
activity and fungal plant pathogens.
ACKNOWLEDGEMENTS
The authors thank the Director General of
AECS and the Head of the Molecular Biology
and Biotechnology Department for their
continuous support throughout this work.
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