USE OF RAPD, ENZYME ACTIVITY STAINING, AND COLONY SIZE TO DIFFERENTIATE PHYTOPATHOGENIC FUZARIUM OXYSPORUM ISOLATES FROM IRAN

Brazilian Journal of Microbiology (2002) 33:299-303
ISSN 1517-8382
USE OF RAPD, ENZYME ACTIVITY STAINING, AND COLONY SIZE TO DIFFERENTIATE
PHYTOPATHOGENIC FUZARIUM OXYSPORUM ISOLATES FROM IRAN
Motallebi Mostafa*; Zamani Mohammad Reza; Jazayeri Omid; Harighi Mohammad Javad
Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran
Submitted: February 18, 2002; Returned to authors for corrections: July 17, 2002; Approved: December 05, 2002.
ABSTRACT
Fusarium oxysporum is a common soilborn plant pathogen with a worldwide distribution. Fusarium yellows
disease of chickpea (Cicer arientinum) caused by F.oxysporum is one of the most destructive soilborn
disease which is a major production constraint in chickpea-growing regions of Iran. Three laboratory methods
“amplification of genomic DNA using random primers, enzyme activity staining, and colony size determination”
have been used to discriminate between highly virulent (HV) and weakly virulent (WV) isolates of F. oxysporum.
On the basis of colony size (a traditional morphological method) and the ability of isolates to produce pectic
enzymes, five HV isolates were differentiated from three WV isolates. The HV isolates formed large colony
(ranging from 10.1 to 12.6 mm in diameter) and showed the same enzyme pattern,while the WV isolates
produced small colony (ranging from 5.8 to 7.8 mm in diameter) and had not detectable enzyme activity in the
stained overlaying gel. Twelve arbitrary 10-mer primers were tested on these 8 isolates of F. oxysporum by
Polymerase Chain Reaction (PCR). Cluster analysis of the data from the DNA amplification by Random
Amplified Polymorphic DNA (RAPD), differentiated HV from WV isolates. The results obtained from RAPD
test confirmed the classification of these eight isolates based on pathogenicity test, colony size, and enzyme
activity staining into two groups (HV and WV).
Key words: Fusarium oxysporum- RAPD- colony size- enzyme activity staining
INTRODUCTION
because they have the potential to identify a large number of
polymorphisms with good coverage of entire genome.
Fusarium yellows disease of chickpea (Cicer arientinum)
This paper describes three laboratory tests and techniques
caused by the vascular wilt pathogen Fusarium oxysporum is a
that confirm the classification of F. oxysporum isolates based
major production constraint in chickpea-growing regions of Iran
on pathogenicity test, as highly virulent (HV) and weakly virulent
(1). Isolates of F. oxysporum form a major component of the
(WV) isolates. These tests and techniques include colony size,
fungal flora of most cultivated soils and most of these isolates
enzyme activity staining and RAPD.
are not perfectly identifiable using phenotypic characters (6).
Molecular tools have been used to characterize the diversity
MATERIALS AND METHODS
among pathogenic isolates of F. oxysporum (2,4,9,12). Molecular
markers, such as Random Amplified Polymorphic DNA (RAPD)
Fungal isolates and growth conditions
have been used extensively as genetic markers in different
Eight isolates (five highly virulent “HV” and 3 weakly
populations (2,4). RAPD markers have some advantages in that
virulent “WV”) of F. oxysporum from aerial sections of chickpea
they are easy to generate, require only very small amounts of
from different geographical locations in Iran were collected (17)
genomic DNA and do not require the use of radioisotopes.
(Table 1). Isolates were maintained in wheat seed medium (50 g
RAPD markers can overcome the limitation of other markers
autoclaved soaked wheat seed in 500 ml flask) at 4ºC. Isolates
* Corresponding author. Mailling address: Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran. E-mail:
motallebi38@yahoo.com
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M. Mostafa et al.
were grown in shake culture in pectic zymogram (PZ) medium
microscope equipped with an ocular micrometer. Colony
containing 2.64 g, (NH4)2 SO4, 0.34 g KH2PO4, 0.14 g
diameters were measured to the nearest 0.1 mm (6). Dendrogram
MgSO4.7H2O, 10 g Citrus pectin (Sigma), one litter distilled water,
was produced by cluster analysis, using the Unweighted Pair-
pH adjusted to 4.5 (14). After 6 days growth at 26ºC a liquid
Group Method Analysis (UPGMA).
culture filtrate was obtained (as crude enzyme) by Whatman
filter paper No.1 and stored at -20ºC until using for detecting
DNA extraction
the enzyme activity. For colony size measurement, the isolates
Lyophilized mycelium of different isolates was homogenized
were grown on Modified D-medium contained 0.14g MgSO4
and genomic DNA was extracted according to Zamani et al. (16).
7H2O, 2.6g (NH4)2SO4, 0.5g NaCl, and 20g agar supplemented
with 20g L-sorbose in one litter distilled water. To reduce
Amplification of genomic DNA using RAPD primers
carmelization, the L-sorbose was autoclaved separately from
Amplification of DNA fragments was carried out by
the other ingredients.
Polymerase Chain Reaction (PCR) using 10-mer arbitrary primers.
Amplification reactions were performed in 50 µl reaction volumes
Enzyme activity staining
containing one unit of Taq DNA polymerase, 2 µmol/ml each
For rapid characterization of pectic enzyme activity, 10 µl of
of dATP, dCTP, dGTP and dTTP, 1.5 µmol/ml primer, and 60 ng
culture filtrate was mixed with equal volume of sample buffer
of genomic DNA.
containing, a solution of 50 mM Tris-HCl pH 6.8, 2% (w/v)
The reaction mixture was overlaid with sterile mineral oil (50
Sodium Dodecyl Sulphate (SDS), 5% (v/v) of 2-?
µl) to prevent evaporation during PCR cycling. The programe
mercaptoethanol, 10% (v/v) glycerol, and 0.05% w/v)
comprised 34 cycles of denaturation at 94ºC for 2 min, primer
bromophenol blue. The proteins were analyzed by Sodium
annealing at 32ºC for 2 min, and extension of primer at 72ºC for
Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-
2 min. After the cycling steps were completed, the mixture was
PAGE). The separated proteins were assayed for
held at 72ºC for 4 min to allow complete extension of amplified
polygalacturonase activity with an ultrathin pectate agarose
products. A total of 12 Primers were used (Table 2). Amplified
gel by staining with 0.05% ruthenium red for 30 min as described
DNA fragments were analyzed by electrophoresis in 2% agarose
by Ried and Collmer (13).
gel in TBE buffer.
(a) Preparation of pectate agrose overlay gel
RAPD product scoring and Data analysis
Thin pectate overlay gels (0.75mm) for detection of enzyme
Data were compiled as a binary 0/1 matrix by the presence
activity were cast by using a gel support film for agarose gels
(1) or absence (0) of a band at particular position. Only major
(FMC Bio products) on one of the glass plates of vertical
RAPD bands were considered for statistical analysis.
apparatus. The overlay contained, in addition of 1% agarose
Dendrogram were produced by cluster analysis using UPGMA.
(Sigma), 0.1% polygalacturonic acid in 100 mM potassium
acetate buffer, pH 4.5 and 10 mM EDTANa2 (11). The agarose
RESULTS
solution was boiled to dissolve the agarose and cooled to 75ºC.
The gel mold was heated to 60ºC before casting.
Colony size
Fifteen isolates of F. oxysporum derived from different
(b) Detection of polygalacturonase activity in SDS-PAGE
geographical regions have been previously studied based on
After electrophoresis, the gel was washed by three 200 ml
pathogenicity test. From these fifteen isolates five highly virulent
changes of 10 mM potassium acetate buffer, pH 4.5 (5). The
isolates (F15, F18, F23, F47 and F59) and three weakly virulent
pectate agarose overlay was laid directly on top of the gel and
isolates (F2, F21 and F58) were distinguished. (17). These eight
incubated at 37ºC for 120 min in a moisture lunch box. The
isolates were grown on D-medium containing L-sorbose.
overlay was removed and stained with 0.05% (w/v) ruthenium
The growth of different isolates tested on medium, and each
red for 30 min. To enhance recovery of renature enzyme activity,
isolate had a characteristic mean colony diameter (Fig. 1).
bovine serum albumin (BSA) at 10 µg/ml was incorporated into
Isolates F02, F21, and F58 produced small-size colonies, with
separating gels as described by Lacks and Springhorn (10).
mean diameters 7.8, 6.4, and 5.8 mm, respectively (Table 1).
Isolates F15, F18, F23, F47, and F59 formed large colonies with
Colony size
mean diameters: 11.5, 10.1, 12.6, 11.1, and 10.1 mm, respectively
Isolates were grown on PDA plates for 7 days. With a thin
(Table 1). A dendrogram constructed by UPGMA cluster
platinum needle, 20 stab transfers were made from the PDA to a
analysis showed a grouping of these isolates into two clusters.
plate of D-medium. Plates were incubated for 72 hours at 25ºC.
One cluster with five isolates (large colony) were highly virulent
The diameters of 20 colonies of each strain were then measured
and second cluster containing three isolates (small colony) were
from the underside of the plate using a binocular dissecting
weakly virulent (Fig. 2).
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Methods to differentiate phytopathogenic F. oxysporum
RAPD analysis
In order to compare the overall similarity between these
isolates at DNA level, 12 decamer oligonucleotide primers were
used which generated polymorphic bands. Example of
polymorphic bands detected in amplified DNA from different
isolates is shown in Fig. 3.
The data from polymorphic bands were analyzed by UPGMA
method. Comparison of the results from pathogenicity test of
these isolates with the results of cluster analysis of RAPD poly
morphic bands demonstrated that among 12 primers, only the
dendrogram obtained from R2 primer (Table 2) was able to
differentiate the weakly virulent isolates F2, F21, and F58 as a
separate cluster (Fig. 4).
Figure 1. Comparison of large colonies (F15) and small colonies
(F58) of F. oxysporum isolates on sorbose medium incubated
Table 2. Sequence of the primers used in this study.
for 72 hr at 25ºC.
Primer Identification
Sequence
81
5’-ACGGTCTTGG-3’
82
5’-GGCGCTAGCA-3’
Table 1. Source,virulence and colony size of different isolates
171
5’-GAAACAGCGG-3’
of F. oxysporum used in this study.
172
5’-GGAGCCCAC-3’
Isolate
Origin
Virulence
Colony size (mm)
173
5’-GGAGGGTGTT-3’
F15
Orumieh, Iran
H
11.59 ± 0.77 L
174
5’-ACGATCGCGG-3’
F18
Orumieh, Iran
H
10.07 ± 0.42 L
R1
5’-CGGCCACCCT-3’
F23
Unknown
H
12.61 ± 0.96 L
R2
5’-CGCGTGCCAG-3’
F47
Lorestan, Iran
H
11.14 ± 1.15 L
R3
5’-ACGATCGCGG-3’
F59
Tabriz, Iran
H
10.09 ± 0.45 L
PU1
5’-AGATGCAGCC-3’
F58
Tabriz, Iran
W
5.82 ± 0.49 S
PU2
5’-ACGGATCCTG-3’
F21
Orumieh, Iran
W
6.46 ± 0.54 S
PU3
5’ ACTGGGACTC 3’
F02
Tabriz, Iran
W
7.85 ± 0.41 S
H = Highly virulent; W = Weakly virulent; L = Large colony;
S = small colony.
CASE
0
5
10
15
20
25
Label
F18
F59
F47
F15
F23
F21
F58
F2
M = Molecular marker in bp; C = Negative control.
Figure 2. UPGMA clustering analysis of colony size data of
HV and WV isolates of F.oxysporum.
Figure 3. Amplification of genomic DNA using R2 primer.
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M. Mostafa et al.
Enzyme activity staining
genomic DNA using random primers, enzyme activity staining,
In order to study whether pectic enzymes are present in
and colony size determination.
the culture supernatant, an attempt was made to stain the gel
In a previous study (17) five highly virulent and three
for pectic enzyme activity, using pectate agarose overlay. An
weakly virulent isolates have been identified. In the present
equivalent amount of enzyme was loaded onto the gel for all
investigation, these eight isolates were characterized by other
isolates (HV and WV). The obtaining results indicated that all
laboratory methods. In the first test, colonies of the different
highly virulent isolates show the same enzyme activity pattern
isolates of F. oxysporum were grown on a sorbose-containing
(Fig. 5). Comparison of the banding patterns or enzyme activity
medium and their diameters were measured. The second test
for the highly virulent and weakly virulent isolates
assessed the ability of the isolates to produce pectic enzymes,
demonstrated that, a high intensity band is present in the
as tested by enzyme activity staining on the gel. By cluster
highly virulent isolates which is absent in weakly virulent
analysis of the results from these two tests, these eight isolates
isolates (Fig. 5).
have been divided into two distinct clusters. Comparing the
arrangement of isolates in Fig. 2 (based on colony size) and
DISCUSSION
Fig. 5 (based on enzyme activity staining) with the
classification in pathogenic groups (Table 1), one can see that
In this study, we have evaluated three laboratory methods
the classification of the isolates by pathogenicity test is in
for their ability to discriminate between highly virulent and
accordance with the results of these two tests. Further analysis
weakly virulent isolates of F. oxysporum: amplification of
of the results revealed that isolates F15, F18, F23, F47, and
F59, which were highly virulent, formed large colonies (ranging
from 10.1 to 12.6 mm in diameter) and produced detectable
pectic enzyme activity in overlaying gel, while isolates F02,
CASE 0
5
10
15
20
25
F21, and F58, which were weakly virulent, formed small colonies
Label
(ranging from 5.8 to 7.8 mm in diameter) and showed not
detectable pectic enzyme activity in overlaying gel. Correll et
F2
al. (6) reported that virulent isolates of F. oxysporum have a
F58
characteristically large colony size which is in agreement with
F21
our finding.
F47
When comparing the RAPD analysis to classification into
pathogenicity groups, the results using 12 primers did not
F59
present good correlation between amplification patterns and
F23
pathotype classification.
F15
However, primer R2 alone allowed differentiation of these
F18
isolates, which was strongly supported by pathotype, colony
Figure 4. UPGMA clustering analysis of RAPD data of HV and
size and overlaying classification. As evidenced in our study
WV isolates of F.oxysporum.
and in others as well, RAPD markers were effective for detecting
polymorphism in F. oxysporum (3,7,8,15).
Finally, from these results it can be concluded that these
three rapid methods (RAPD, colony size, and enzyme activity
staining) could be employed to distinguish highly virulent
from weakly virulrnt isolates of F. oxysporum. A fast
diagnosis of the highly virulent in an early stage of infection
may provide advantages for phytosanitary procedures.
Nevertheless, standardization of the detection system will
be required.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Afshari-Azad from
Ministry of Agriculture and Mr. H. Younesi from Agricultural
Research Center of Kermanshah for supplying the isolates of F.
Figure 5. Enzyme activity staining gel of polygalacturonase in
oxysporum. This work is supported by Ministry of Agriculture
culture supernatant of HV and WV isolates of F.oxysporum.
of Iran.
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Methods to differentiate phytopathogenic F. oxysporum
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