The Mutagenic Activity of Chitosan and its Effect on the Growth of Trichoderma harzianum and Fusarium oxysporum F. Sp. Sesami

Journal of Applied Sciences Research, 3(6): 450-455, 2007
© 2007, INSInet Publication
The Mutagenic Activity of Chitosan and its Effect on the Growth of
Trichoderma harzianum and Fusarium oxysporum F. Sp. Sesami

1A. Abou Sereih, Neven,

1Abd-El-Aal, S., KH. and 2A.F. Sahab
1Microbial Genetics Department, National Research Centre, Dokky, Cairo, Egypt.
2Department of Plant pathology, National Research Centre, Dokky, Cairo, Egypt.
Abstract: Five concentrations of chitosan; 0.38, 0.75, 1.50, 3.00 and 4.50 mg/ml were used to study its
effect on the growth of Fusarium oxysporum f sp. Sesami and Trichoderma harzianum. Chitosan inhibited
the growth of Fusarium increasingly with the doses; 0.38, 0.75and 1.50 mg/ml, while no growth was
obtained when the two concentrations; 3.00and 4.50 mg/ml were used. In case of Trichoderma, in addition
to growth inhibition with chitosan treatment, two doses; 3.00 and 4.50 mg/ml resulted in changing the
growth color from green to yellow. It was found that chitosan treatment of Trichoderma with the two
doses; 3.00 and 4.50 mg/ml has affected the fungus genetically. The induced mutants were isolated and
characterized. Chitosan Addition to the media enhanced the antagonistic properties of Trichoderma against
Fusarium. Protein fingerprinting and Statistical analysis of the obtained data for the chitosan concentrations
and its control were done.
Key words: Fusarium oxysporum f. sp. sesami, Trichoderma harzianum, Chitosan, Biocontrol agent,
Genetic effect and SDS-PAGE
INTRO DUCTIO N
the causal agent of sesame wilt and the antagonistic
effect of Trichoderma harzianum isolates as a
Control of sesame wilt disease depends on
biocontrol agent.
fungicides as seed or soil treatment[17]. Fungicide
application is expensive and can cause environmental
M ATERIALS AND M ETHO DS
po llution. M oreover, it may induce pathogen
resistance[15]. Recently a potential approach in
Fungal Strains: Two fungal strains were used in this
biological control involves the use of the natural
work; Fusarium oxysporum f. sp. sesami known to be
bioactive substance chitosan, which inhibits fungal
highly virulent on sesame plant and the antagonistic
growth and also activates the biological efficiency of
isolate of Trichoderma harzianum which obtained from
the antagonistic microorganisms[10,27]. Chitosan has a
plant pathology Dept. NRC of Egypt.
fungistatic activity demonstrated against some plant
diseases, i.e., virus infection[3] and pathogenic fungi
[2 8 ,2 1 ].
Chitosan Treatments: Purified Chitosan (obtained
Chitosan also stimulates the plant to defend itself
from Sigma chemical steinham, Germany) was prepared
through the activation of resistant gene(s) for induced
by the method which described by El-Ghaouth et al.[8].
multiple biological reactions[12,5]. Efficiency of
To study the effect of different doses of chitosan on
Trichoderma spp. as a biocontrol for plant pathogenic
the linear growth (mm) of Fusarium oxysporum f. sp.
fungi may be increased by combining different
sesami and Trichoderma harzianum, five concentrations
organisms and/or simple chemical substances. Chitosan
of chitosan were used. A disc of (5 mm-dm) of pure
could promote the plant to defend itself besides; it
cultures of the tested fungi was placed on the center of
interferes with the growth of the Pathogen[18]. Chitosan
PDA plate containing; 0.00, 0.38, 0.75, 1.50, 3.00 and
(Poly-?-1, 4 glucosamine) is prepared commercially by
4.50 mg/ml of chitosan. Plates were incubated at
alkaline decelylation of chitin obtained from the
27±2ºC for seven days and the diameter of the fungal
exoskeletons of marine crustaceans.[21]. T he induction of
growth was recorded twice daily. Four replicates were
mutation and the auxotrophic mutant were studied also
used for each particular treatment.
by Kanemaru and M igamoto[16] and Daminko[6] as a
result of chitosan treatment. The present study deals
M utagenicity Test: Chitosan was applied as mentioned
with the mutagenic activity of chitosan and its effect
before to P DA medium in concentrations of; 0.38,
on the growth of Fusarium oxysporum f. sp. sesame,
0.75, 1.5, 3.00 and 4.5 mg/ml based on active
Corresponding Author: A. Abou Sereih, Neven, Microbial Genetics Department, National Research Centre, Dokky, Cairo, Egypt.
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J. Appl. Sci. Res., 3(6): 450-455, 2007
in g r e d i e n t. T h e s u rv iv in g c o lo nie s a n d th e
morphologically different colonies (Yellow) were
isolated and tested on both minimal and complete
media. The colonies which showed growth on
complete medium but not on minimal medium were
considered as auxotrophs (mutants). Survival and
mutation percentages were determined for each
treatment according to M ckennes and M eton[20] and
Sahab et al.[24].
(a)
(b)
Isolation of Auxotrophs and Their Nutritional
Fig. 1 (a,b): T he effect of chitosan in concentration
Requirements: Nutritional requirements of each mutant
3.00 mg/ml on Trichoderma harzianum;
obtained from different mutagenic treatment were
Fig. (a) showed the green color of
identified by replica plating method on the minimal
Trichoderma before treatment and Fig.
medium supplemented with one or more of the
(b) showed the yello w co lo r of
amino acid (250 mg/l) or vitamins (2.5 mg/l) or
T rich o d erm a after tre a tm e n t with
nitrogen bases (25 mg/l) as described by
chitosan.
Abou Sereih et al.[1].
Antagonistic Screening Test: Screening procedure for
detecting the antifungal ac tivity o f d ifferent
Trichoderma harzievum against F. oxysporium f. sp.
sesami was adapted by Ziedan and Elewa[29]. Five ml
of the pathogenic fungal suspension containing 106
CFU/ml was added to 200 ml of melted PDA medium
(50°C) before pouring into the Petri dishes (9 cm
diameter). The Petri dishes were inoculated with disks
(5 mm diameter) loaded with Trichoderma culture of
(a)
(b)
7 days old and incubated at 25±2°C for 5 days. Zones
Fig. 2 (a,b): The effect of Trichoderma harzianum on
of inhibition were estimated in mm and the degree of
the growth of Fusarium oxysporum f. sp.
antagonism observed using a scale ranges from zero
sesami (a) without chitosan and (b)
(no inhibition) to 3 + (excellent inhibition), Ziedan[28].
combined with chitosan.
Total Protein Extraction: T otal protein extraction and
in positive reaction to chitosan concentration of
b a n d i n g p a t t e r n s S D S - p o l y a c r y l a m i d e g e l
3 mg/ml concentrations. The two higher doses of
electrophoresis was performed according to
chitosan 3.00 and 4.50 mg/ml were lethal with
Sheri et al.[26].
Fusarium while in case of Trichoderma they resulted
in the change in growth color from green to yellow.
Gel Documentation System: The gel documentation
These results were in agreement with Ryan[22] who
system image analysis Gel works 1D advanced
reported that chitosan induced multiple biological
software was used for more accurate analysis and
reactions including induction of phytoalin synthesis of
comparison between the treatments via biochemical
B-1-3 gluconase and chitinase. O n the other hand,
genetic analysis. This method is recommended to
Chung and Choi[5] reported that the Trichoderma
determine the relationship within and between the
harzianum is considered as a biocontrol agent caused
treatments[11].
damage on Fusarium oxysporum f. sp. sesam i.
Concomitant use of Trichoderma harzianum and
RESULTS AND DISCUSSIONS
chitosan im p roved the antagonistic effect of
Trichoderm a against Fusarium. This result was in
Effect of Chitosan on Fungal Growth: In this
agreement with Ziedan and Elewa[29] (Fig. 2. a & b).
work, five doses of chitosan; 0.38, 0.75, 1.50, 3.00,
4.50 mg/ml were used to study the effect of this
Genetic Effect of Chitosan: Trichoderma treatments
material on linear growth (mm) of Fusarium oxysporum
were tested on M inimal Media (M M ) and Complete
f. sp. sesami and Trichoderma harzianum. Data in
M edia (CM ) at two chitosan concentrations; 3.00 and
Table (1) showed that chitosan treatments decreased
4.50 mg/ml of each medium. On contrary of complete
the linear growth of both Fusarium and Trichoderma
medium, no growth was found on M inimal media.
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J. Appl. Sci. Res., 3(6): 450-455, 2007
T able 1: Effect of different chitosan concentration s on the linear
T able 3:
In-vitro test of the antagonistic activity of different m utant
growth (m m ) for Fusarium oxysporum f. sp. sesam i and
strains of Trichoderm a harzianum against Fusarium
Trichoderm a harzianum .
oxysporum f. sp. sesam i.
Fungi
C hitosan
N o. of tested
Average zone
------------------------------------------------------
C oncentrations m g/m l
colonies(*)
inhibition (m m )
Linear growth (m m )
1.5
2
20.1
-----------------------------------------------------
4
22.0
C hitosan
Fusarium oxysporum
Trichoderm a
3.00
5
22.2
concentrations (m g/m l)
f. sp. sesam i
harzianum .
7
25.5
0.00
90.00
93.75
2
26.1
0.38
71.23
80.50
4.50
4
16.5
0.75
62.25
69.00
5
27.1
1.50
50.50
60.25
7
26.6
*3.00
00.00
23.00
5
27.5
4.50
00.00
16.75
W ild type
1
14.5
* Lethal dose
(*) N um ber of tested colonies (m utant strains) of Trichoderm a
harzianum have nearly a sam e size of zone inhibition (m m ).
T able 2: C haracterization of chitosan induced m utant produced from
Trichoderm a harzianum
Chitosan concentration
---------------------------------------------
C olonies tested
1.5
2.00
4.50
Total N o. of colonies
500
500
500
Total N o. of m utant
41
43
44
M utant %
8.2
8.6
8.8
Stable m utant %
0.0
6.97
35.5
U nstable m utant %
100
93.03
64.5
M orphological m utants
0
2
4
B iochem ical m utant
N itrogen requiring m utants
0
0
0
Vitam ins requiring m utants
0
0
0
Am ino acid requiring m utants
1* 1***
2* 1**
1* 3***
* A rgenine - requiring m utants.
** Adenine - requiring m utants.
*** M ethionine - requiring m utants.
For Trichoderma, it was able to grow on both M .M .
and C.M. at the rest of chitosan concentrations. These
results indicated that the two chitosan concentrations
3.00 and 4.50 mg/ml have a genetic effect and induced
mutation. The Trichoderma colonies which grown on
Fig. 3: The SDS-PAG E total protein pattern for the
C.M . and not on M .M . at these concentrations are
effect of five concentrations (mg/ml) of
considered as mutants.
chitosan on Trichoderma harzianum ; 0.38
Table (1) shows that the increase in chitosan
(lane 1), 0.75 (lane 2), 1.50 (lane 3), control
concentration was associated with an increase in
(lane 4),, 3.00 (lane 5), 4.50 (lane 6) and (lane
mutation and decrease in survival. The lethal dose
M ) the marker with molecular weigh ( 12 to
(3 mg/ml) affected Fusarium oxysporum f. sp. sesami
97 KDs ).
dose not make a lethality of Trichoderma harzianum;
these indicate that chitosan has a antimicrobial activity
undifferentiated fungal structures. On the other hand,
[9 ]
in Trichoderma but it effect in a gene responsible for
El-Ghaouth et al. suggested that the induction of
color and make a mutation. These result like results
morphological and cytological alterations in Rhizopus
by[21].
stolonifer.
Data in Table (2) shows that chitosan can induce
Data in T able (3) reveal that the tested isolated of
stable and unstable mutants. Generally, the stable
T. harzianum mutants were significant changed in their
mutants were much less than those of unstable mutants
antagonistic action. The lower value of zone of
in both isolates. In this respect, Beremand[2] isolated,
inhibition (20-22 mm) in the comparison with the wild
identified and chemically characterized three stable
type isolate (14.5 mm). On the other hand, isolates of
mutants.
(3.00, 4.50 mg/ml) chitosan were considered a high
The effect of chitosan on the cell wall of the
antagonistic activity against Fusarium oxysporum f. sp.
pathogenic fungi has been cleared by Ryerson and
sesami.
Heath[23], they found no effect on a non-pathogen. The
Different doses of chitosan which caused increase
wall fragments were derived from differentiated or
or decrease the antagonistic activity by T. harzianum
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J. Appl. Sci. Res., 3(6): 450-455, 2007
Fig. 4:
Dendrogram tree for the effect of five concentrations (mg/ml) of chitosan on Trichoderma harzianum;
0.38 (1), 0.75 (2), 1.50 (3), wt (4), 3.00 (5), 4.50 (6).
may be due to the changes of the genetic structure
have not any unique or specific band, while only 3.00
or the gene regulation system responsible for
(lane 5) and 4.50 (lane 6) have one specific band. This
antagonistic activity[25], or it may be effect on the
specific band of the two chitosan treatments may be
responsible gene for endochitinase which degraded
referring to the occurred change of growth color and
the cell wall of the pathogen[13] and some times
appearance the yellow color of the Trichoderma
antifungal compounds enhances inhibition of spore
harzianum.
germination[19].
Statistical Analysis for SDS-PAGE data: Package
3-Protein Fingerprinting: Total protein extraction and
SPSS system. Significant different were determine at
banding patterns SDS-polyacrylamid gel electrophoresis
p‹0.05. Data from SDS-PAGE were pooled and
for five treatments resulting from using five
transferred into 1 and o, they were interred into the
concentrations of chitosan on Trichoderma harzianum
input of the program as shown in the dendrograms
and its control are illustrated in Fig. (3). There are
below (Fig 4). The statistical analysis data were carried
observable differences in the protein banding pattern
out with the statistical software according to the
for all five treatments and the control. Some minor
method which described by Iruela[14]. The dendrogram
differences in banding patterns between the control and
generated by (Gel works 1D) analysis confirmed the
the treatments which resulting from the chitosan
above pattern of diversity using SDS-PAGE found
concentrations; 0.38 (lane 1), 0.75 (lane 2), 1.50
genetic difference the two chitosan concentration
(lane 3), control (lane 4) were observed, but variation
treatments, 3.00 & 4.50 mg/ml and the rest of
to the same extent was also present between the two
treatments and in additions of the control The total five
treatments; 3.00 (lane 5), 4.50 (lane 6).Data from
chitosan concentration treatments and in additions of
Fig. (3) also reveal that the total bands number for all
the control were classified for the dendrograme into
the five treatments and their control ranged from 12
five pool clusters. The first and the second clusters
bands (0.75, lane 2), to 17 bands (0.38, lane 1).
(A & B1) include two chitosan concentration
The higher total number of bands for the treatments
treatments, 3.00 & 4.50 mg/ml, these two clusters are
of chitosan concentrations (mg/ml); 0.38(lane 1),
so far from other cluster (about 100% dissimilarity)
control (lane 4) and 4.50 (lane 6) were 17,16 and 16,
they have not genetic similarity of approximately 0% .
respectively. The molecular weight ranged from 14, 4
This result was inagreement with obtained data at
to 94 KDs. There are common bands found in all
Fig (3). In the contrary, the first chitosan concentration
strains. The results also indicate that the two
treatment, 0.38mg/ml (No.1) and the control (No. 4)
treatments of chitosan concentrations (mg/ml); 3.00
have a one group ( cluster B4) with very close distance
(lane 5) and 4.50 (lane 6) have one band at
between them (about 98% similarity). The second
molecular weigh 73.05 K D s and two different
chitosan concentration treatment, 0.75 mg/ml (No.2)
specific (monomorphic) bands at molecular weigh
have one cluster (B 3) near the last cluster but it is
46.6 and 44.88 KDs, respectively, while the rest of
little distance from them (88% similarity) and near the
the treatments of chitosan concentrations have not
last cluster but it is a little distance from them and
any monomorphic band. These results could be used
have a common ancestor the clusters B 2. T he third
to distinguish between these treatments for example,
chitosan concentration treatment, 1.50mg/ml (No.3)
each of the treatments of chitosan concentrations
also have one cluster about 40 % similarity with
(mg/ml) on Trichoderma harzianum 0.38 (lane 1),
cluster (B3) and 48% dissimilarity with the two clusters
0.75 (lane 2), 1.50 (lane 3) and control (lane 4),
A & B1.
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J. Appl. Sci. Res., 3(6): 450-455, 2007
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