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Indian J Microbiol (October 2010) 50(Suppl 1):S110–S116
DOI: 10.1007/s12088-010-0067-0
Indian J Microbiol (October 2010) 50(Suppl 1):S110–S116
ORIGINAL ARTICLE
Morphological and genetic differentiation among four pigment
producing Indian species of Phoma (Saccardo, 1899)
Ajit Chande · G. J. Kövics · S. S. Sandhu · M. K. Rai
Received: 15 February 2008 / Accepted: 28 May 2008
© Association of Microbiologists of India 2010
Abstract A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was
used for assessing genetic relatedness among isolates of
the genus Phoma. Randomly Amplified Polymorphic
DNA (RAPD) revealed the presence of interspecific genetic variation among the pigment producing isolates
of Phoma and has shown distinct phylogenetic cluster.
The major objective of the study was to study the genetic
variation, if any. Study was aimed to differentiate four pigment producing species of Phoma based on morphological
studies and molecular markers in general and RAPD in
particular. We found that the test species of Phoma can be
very well differentiated using molecular markers. Phoma
sorghina was differentiated from P. exigua, P. fi meti and
P. herbarum. RAPD profiles of P. herbarum and
P. fimeti has shown the maximum similarity, which indicates the genetic relatedness among these two species
which were considered earlier as distinct species based on
morphological observation.
Keywords
RAPD · Phoma · Genetic variation
A. Chande1 · G.J. Kövics2 · S. S. Sandhu3 · M. K. Rai1 ()
Department of Biotechnology,
SGB Amravati University,
Amravati - 444 602, Maharashtra, India
2
Debrecen University, Faculty of Agriculture,
Department of Plant Protection H-4015 Debrecen,
P.O. Box 36, Hungary
3
Department of Bioscience,
RD University Jabalpur, M.P., India
1
E-mail: mkrai123@rediffmail.com
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Introduction
Phoma is a taxonomically difficult genus and is not fully
understood. It belongs to order Sphaeropsidales of Deuteromycotina. It is a unique form of pycnidiales, which occurs
ubiquitously and have been reported from a wide variety
of hosts particularly from plant and soil. It has also been
recovered from aquatic and aerial environment [1], marine
environment [2], entomopathogenic [3] and have been
found to cause disease in human beings [4, 5].
The existing Indian species of Phoma have been erected
on the basis of host alone, and thus the importance of host
specificity for the taxonomy of Phoma has been much emphasized and overestimated. The assumption that each host
genus or species was colonized by a specialized Phoma
species prompted many mycologists to ignore morphological characters when erecting new Phoma species. Usually,
a morphological species may attack various host plants. For
example, P. exigua has been reported by investigators on
different hosts [6]. The criterion of identification should
be in such a way so that it should be possible to identify a
Phoma species in case host identification is difficult particularly when floral parts are lacking, or when the fungus
is grown on artificial media.
Molecular techniques have revolutionized analyses of
the diversity of fungi and studies of the interaction with
their hosts. Particularly, the polymerase chain reaction
(PCR) assay has provided a framework to understand taxonomy and population structure. The assessment of genetic
diversity is required for the correct species identification
and recognition of physiological strains (pathotypes). The
random-amplified polymorphic DNA (RAPD) method has
been successfully used to identify strains [7–9], to characterize races [10] and to analyze virulence variability related to
Indian J Microbiol (October 2010) 50(Suppl 1):S110–S116
genetic polymorphisms [11–15] in phytopathogenic fungi.
It has also been used in the study of inter- and intraspecific
variability among populations from different [16–18] and
from the same geographic regions [19–21].
Since its development, the random-amplified polymorphic DNA (RAPD) protocol has acquired a diversity of
uses, such as: the establishment of the genetic similarity
degree between individuals within a population [22], the
construction of genetic maps as well as the localization of
economically interesting genes [23], the production of a
genomic fingerprint [24], and the study of genetic diversity
along with the identification of fungi [25–30].
We used a RAPD assay to determine the genetic variability among four pigment producing species of Phoma.
Materials and methods
Fungal strains isolation and growth conditions
During the course of the present study four different pigment producing Phoma species were selected. These included P. sorghina, P. exigua, P. herbarum (MTCC 2319)
and P. fimeti (MTCC 2323), out of these P. exigua was isolated from soil and leaf litter, while P. sorghina from petiole
of Carica papaya.
Isolations were made by cutting the infected portions
from the junctions of healthy and diseased region of the
leaves and by surface sterilizing with 70% ethanol and
putting it onto petriplate containing sterilized PDA (Potato
Dextrose Agar) and malt agar. Isolations from soil were
performed by serial dilution.
All the isolates were grown on PDA and malt agar. The
cultures of Phoma were maintained on PDA and malt agar
(Hi-Media, Mumbai) slants at 4ºC.
Cultural studies
The cultural studies were carried on potato dextrose agar
and malt agar media. The culture plates were inoculated by
P. sorghina, P. exigua, P. herbarum and P. fimeti and incubated at 22°C for 7 days.
DNA extraction
For isolation of DNA from Phoma sorghina, P. exigua,
P. herbarum and P. fimeti the test isolates were grown in 40
ml malt broth in 150 ml conical flask in dark and maintained
in a growth chamber adjusted at 22°C for 7-days. Mycelial
mat was then separated and washed with sterile distilled
water twice to remove the traces of media. Mycelium was
then dried on a sterile filter paper. Fungal genomic DNA
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was extracted from dried mycelium using modification of
method given by Vandemark et al. [31]. Two gram of mycelium was macerated in liquid nitrogen using mortar and
pestle. The ground mycelium was hydrated for 10 minutes
at room temperature in 3 ml of extraction buffer [10 mM
Tris-Cl, 250 mM NaCl, 10 mM EDTA, pH 8.5 (Qualigens
Fine Chemicals, Mumbai), and 0.5% w/v Sodium Dodecyl
Sulphate (Sigma Aldrich Chemie, Germany). DNA was
extracted with two volume of phenol: Chloroform: isoamyl
alcohol [25:24:1] (Sigma Chemie, Germany). The nucleic
acid was precipitated from upper aqueous layer with twice
the volume of ice-cold ethyl alcohol (–20°C). After centrifugation for 5 minutes at 4000 rpm, the pellet was washed
with 70% ethyl alcohol twice, air dried in laminar flow
and resuspended in 1X Tris-EDTA buffer [1.0 M Tris-HCl
and 0.1 M EDTA] (Sigma Chemie, Germany)]. Dissolved
nucleic acid was stored at –20°C for long term storage.
Screening for random amplified polymorphic DNAs
(RAPD)
DNA from each fungal isolate was screened for RAPD
markers generated by 22 random decamer primers (Operon
Technologies, Inc. Almeda, CA). Five primers were selected
finally for the RAPD analysis (Table 1). Each reaction mixture (25 µl) for PCR amplifi cation consisted of 10X assay
buffer for Taq DNA polymerase with 15mM MgCl2 (MBI
Fermentas), 1U of Taq DNA polymerase (MBI Fermentas),
200 µM dNTP mix (MBI Fermentas), 0.4 µM decamer
primer (Operon Technologies, USA), and approximately 50
ng genomic DNA template. PCR amplification conditions
were as follows: Initial extended step of denaturation at
94°C for 5min, followed by each 40 cycles of denaturation
at 94°C for 1 min, primer annealing at 36°C for 1 min, and
elongation at 72°C for 2 min. The 40th cycle was followed
by final extension step at 72°C for 7 min and then being
held at 4°C until electrophoresis was done. PCR was carried
out in Whatman Biometra 2000 (Germany) thermocycler.
PCR products were mixed with 5.0 µl of 6X gel loading
dye (MBI Fermentas) and the amplification products were
electrophoresed on 1.5% w/v M.B. grade agarose gel at 50
volts in 1X TAE buffer (MBI Fermentas). Gene rular 500 bp
and Lambda EcoRI/Hind III digest (MBI Fermentas) were
used as molecular size standards. The gels were stained
Table 1
Primers and their sequences used for RAPD analysis
S. No.
Primer
Sequence
1.
2.
3.
4.
5.
OPA-02
OPA-04
OPA-08
OPA-10
OPF-01
TGCCGAGCTG
AATCGGGCTG
GTGACGTAGG
GTGATCGCAG
ACGGATCCTG
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with 0.5 µg/ml of ethidium bromide (Sigma), visualized
under ultra violet light, and recorded with an Alpha Imager
2000 (Alpha Innotech, San Leandro, CA). For all isolates,
bands on RAPD gels were scored as present (1) or absent
zero (0). The results were repeated three times to check the
consistency of the RAPD profile.
Data scoring and statistical analysis
Amplicons were scored for the presence (1) or absence of
bands zero (0) across all the lanes. The binary RAPD data
were analyzed to produce a matrix of similarity values based
on Jaccard’s coefficient of similarity (Jij) [32]. Clustering of
lines was done using unweighed pair-group method based
on arithmetic averages (UPGMA) analysis using the programme Numerical Taxonomy and Multivariate Analysis
System (NTSYS-pc) software version 2.1 [33].
Results
Cultural characteristics of Phoma species
All the four species of Phoma were selected for the study in
order to assess their inter-specific variations and relatedness
using Random Amplified Polymorphic DNAs.
P. exigua was isolated from soil (Leaf litter) by serial dilution method and P. sorghina was isolated from leaf petiole
of Carica papaya. Both the species were grown on potato
dextrose agar (PDA) and malt agar (Hi-Media, Mumbai)
for their morphological and cultural studies and were maintained in slants at 4°C.
The cultures of Phoma were identified by using identification key [34, 35]. Two species were sourced from MTCC
(Microbial Type Culture Collectionr and Gene Bank,
IMTECH, Chandigarh, India).
Table 2
Comparative morphological and cultural studies of four
species of Phoma, viz., P. sorghina, P. exigua, P. herbarum
and P. fimeti are provided in Table 2.
P. herbarum was verified on cultural characteristics.
Colonies were ashy to green with compact aerial mycelium;
pycnidia black, globose to sub-globose; conidia hyaline
1-celled, ovoid, yellow discolouration of the medium was
observed on malt agar. Red pigments were produced which
on application of NaOH turned blue.
P. sorghina was verified on the basis of colour of the
colony (Pinkish-Red), Yellow discolouration of the medium
was observed. The mycelium was profuse, erect and pinkish-red. A typical character of this species is production of
red pigment and chlamydospores.
P. exigua was identified on the basis of morphological
characters and spot-test. Discolouration of agar medium
was found on application of NaOH. Colonies were gray to
black with irregularly scalloped margins attained a diameter
of 6–7 cm in 7 days.
P. fimeti by its very slow rate of growth. Colonies
were ashy to green with compact aerial mycelium, attaining the diameter of 1.0–1.5 cm on malt agar; pycnidia
black, globose to sub-globose; pycnidiospores hyaline,
1-celled, ovoid; dull-yellow discolouration of the medium
on malt agar.
DNA isolation and characterization by RAPD-PCR
Fungal genomic DNA was extracted from juvenile mycelium using modification of method given by Vandemark et al.
[31]. A significant yield was noted using modification of the
protocol. Fig. 1 depicts the DNA isolated from four species.
Twenty-two primers were used for the RAPD analysis
of which only five primers detected polymorphism. A total
of 129 bands were obtained by using five primers ranging
between 200–3000 bp out of which 82 bands were scorable
Colony characteristics of different pigment producing species of Phoma (one week old culture)
Name of
species
Pycnidia colour, shape and size
P. sorghina
Colour
Pigmentation
Herbarium/Host
Black, Flask shaped, globose to sub- 5–7 cm
globose, 75–155 µm
Pink to red
Yellow discolouration of the
medium in acidic condition
below the colony. Yellow
colour changes to red with
NaOH.
Carica papaya
Linn.
P. exigua
Black, Globose to sub-globose
coalesce to form irregular
fructification, 79–296 µm
6–7 cm
Gray to black
Greenish blue dicolouration
changes to reddish on
application of NaOH
Soil
P. herbarum
Black,Flask-shaped, 74–150 µm
4–5 cm
Gray to green
Red pigment turns blue on
application of NaOH
MTCC 2319
P. fimeti
Black,Globose to sub-globose,
pseudoparenchymatous, 74–160 µm
1–1.5 cm
Ashy-green
Dull-yellow discolouration
MTCC 2323
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Colony
diameter
Indian J Microbiol (October 2010) 50(Suppl 1):S110–S116
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and showed polymorphism. This resulted in 63.56% polymorphism. The numbers of amplified products were ranged
from 5 to 11. All the primers produced amplification products; however the extent of polymorphism varied with each
primer. Percent genetic similarity using RAPD markers was
22 using OPA-02. Figs. 2 and 3 depicts a section of the
RAPD profile obtained with primer OPA-02 and OPF-01
respectively. No banding pattern was observed in negative
control
Genetic similarity among isolates
Jaccards pair-wise similarity estimates between species
within the genus Phoma were calculated (Fig. 4). The
average similarity coefficient among four Phoma species
revealed by five RAPD markers found to be 0.22. The
species namely P. herbarum and P. fimeti showed 0.2564
similarity coefficient which was found to be the maximum as compared to all other isolates (Table 3). The least
similarity was found between P. sorghina and P. herbarum
(0.1892) using OPA-02 primer.
Fig. 1 Photograph (Alpha Imager 2000) showing DNA isolated
from Phoma species on 0.7% agarose gel. M, Lambda Eco RI/
Hind III digest (MBI Fermentas); Lane 1, P. sorghina; Lane 2,
P. exigua; Lane 3–5, P. fimeti; Lane 4–6, P. herbarum
Discussion
The importance of morphological characters and conidial
ontogeny for the taxonomy of fungi in general and Phoma
Fig. 2 Photograph (Alpha Imager 2000) showing RAPD
profile of four pigment producing Phoma species obtained by
OPA-02 decamer primer (Operon Technologies, USA) on 1.5%
agarose gel stained with ethidium bromide. M, Gene Rular 500
bp (MBI Fermentas); Lane 1, P. sorghina; Lane 2, P. exigua; Lane
3, P. herbarum; Lane 4, P. fimeti
Fig. 3 Photograph (Alpha Imager 2000) showing RAPD
profile of four pigment producing Phoma species obtained by
OPF-01 decamer primer (Operon Technologies, USA) on 1.5%
agarose gel stained with ethidium bromide. M, Lambda Eco RI/
Hind III digest (MBI Fermentas); Lane 1, P. sorghina; Lane 2, P.
exigua; Lane 3, P. herbarum; Lane 4, P. fimeti ; Lane 5, Negative
control
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Fig. 4 Dendrogram based on Jaccard’s Similarity Coefficient generated by UPGMA analysis of four Phoma species using five RAPD
markers
Table 3 Jaccard’s Similarity Coefficient among four Phoma
species revealed by five RAPD primers
P.
sorghina
P.
exigua
P.
herbarum
P. sorghina
1.0000
P. exigua
0.2286
1.0000
P. herbarum
0.1892
0.2143
1.0000
P. fimeti
0.2059
0.2308
0.2564
P.
fimeti
1.0000
in particular has been strongly overestimated and many
species have been erected from India. If morphology alone
is taken to separate the taxa, it may result in considerable
confusion because sometimes the identification based on
morphology itself proves to be wrong or where a morphological species might show different characters with
changed environmental conditions such as pigment production on agar medium.
For this reason, it is essential to study genetic diversity
or relatedness using molecular markers in order to create a
more realistic and usable classification of Phoma and Phoma related fungi in general and pigment producing species
of Phoma in particular.
RAPD analysis is extremely powerful tool and can separate individuals having intra- and interspecific variability
among the pathogen population as it is based on the entire
genome of an organism.
Using RAPD markers, diversity within the four pigment
producing isolates was studied. Prevalence of genetic diversity among four isolates revealed that they can be very
well differentiated from each other using PCR based
RAPD markers. Cultural characteristics of P. fimeti and
P. herbarum revealed that these species show variety of
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differentiating morphology-based characters. The diameter
of the colony when considered as a differentiating character, it was found that diameter of P. fimeti was 1.0–1.5 cm
while that of P. herbarum was 4.0–4.7 cm and in case of
P. sorghina it was 6.0–7.0 cm after 7 days of incubation
under same growth conditions and on the same growth
medium (PDA). Slow growth of P. fimeti clearly differentiates it from other pigment producing species within
the genus Phoma. Secondly, the conidia of P. fi meti are
ovoid and 1-celled but, in case of P. herbarum conidia
are sometimes bi-celled. Only colour of the colony in
this case is some what similar. Irrespective of morphological characters that differentiates these two species, the
RAPD profiles of these two species have shown maximum
similar bands that has been given by screening with different decamer primers. Genetically these species showed
maximum similarity than with other species such as
P. sorghina and P. exigua.
UPGMA cluster analysis using Jaccards similarity coefficient showed two main clusters. The four pigment producing
species were grouped into three distinct sub-clades. We could
differentiate the species using RAPD markers.The genetic diversity of these isolates studied by RAPD markers is prerequisite for developing a diagnostic tool for the identification
and differentiation of Phoma and other allied genera.
The purpose of this study was to elucidate the genetic
relationship between related pigment producing Phoma isolates because they secrete commercially useful anthraquinones of antimicrobial nature, that are often isolated from
plant material, soil samples and sometimes as opportunistic
pathogens in immunocompromised hosts [4, 34]. An earlier
study showed the usefulness of RAPD analysis in detecting
Indian J Microbiol (October 2010) 50(Suppl 1):S110–S116
significant polymorphisms between Didymella. bryoniae
and Phoma species [36–38]. The present study expands
the previous study by using RAPD analysis on a pigment
producing collection of four isolates to determine their
phylogenetic relationships. RAPD analysis of four pigment
producing Phoma species used in this study sub-divided the
isolates into three unique sets of genetic fingerprints. The
present sampling of a collection of isolates confirms the
previous observation regarding P. fimeti and P. herbarum.
The species that show genetic similarity can be included as
a new variety, but further biochemical marker based studies are required for validation of the data for operational
taxonomic units.
Acknowledgement The authors are thankful to Professor
Bailey Karen, Saskatoon Research Centre, Canada for
supplying reprints, and Dr. Prashant Phirke for providing
chemicals. MKR thanks University Grants Commission,
New Delhi for providing financial assistance.
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