Characterisation of Armillaria species based on pectic isozyme analyses

Fungal Diversity

Characterisation of Armillaria species based on pectic isozyme analyses



Coetzee, M.P.A.1, Maphosa, L.1, Mwenje, E.2, Wingfield, M.J.1 and Wingfield, B.D.1*

1Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria,
0002, South Africa
2Department of Applied Biology and Biochemistry, National University of Science and Technology, P.O. Box AC 939
Ascot, Bulawayo, Zimbabwe

Coetzee, M.P.A., Maphosa, L., Wingfield, B.D., Mwenje, E. and Wingfield, M.J. (2009). Characterisation of Armillaria
species based on pectic isozyme analyses. Fungal Diversity 36: 9-16.

Armillaria spp. are the causal agents of Armillaria root rot on a wide variety of mainly woody plants. Identification of
these fungi using morphological characteristics is complicated by the fact that fruiting structures are uncommon and
often ephemeral. Pectic isozyme analysis has been successfully applied in taxonomic studies of Armillaria species. This
technique, however, has never been used in a study that included a collection of species from across the world. In the
present study, 36 Armillaria isolates, representing 17 Armillaria spp. from different hosts and geographic regions were
characterised using isozyme patterns for pectin lyase (PL), pectin methylesterase (PME) and polygalacturonase (PG).
Isozyme patterns were determined directly from culture filtrates through electrophoresis in polyacrylamide gels stained
in ruthenium red. The majority of species could clearly be separated from one another and isolates belonging to the
same species had similar banding patterns, grouping them together after cluster analysis. Results from this study
showed that pectic enzyme analysis can be an effective tool in the identification of Armillaria species. Furthermore the
isozyme analysis supports previous observations regarding the relationships between Armillaria species.

Key words: Basidiomycetes, isozymes, Armillaria root rot, taxonomy


Article Information
Received 25 May 2008
Accepted 15 February 2009
Published online 31 May 2009
*Corresponding author: Martin Coetzee; e-mail: martin.coetzee@fabi.up.ac.za

Introduction
species (Hintikka, 1973; Korhonen, 1978;

Anderson and Ullrich, 1979). The tests,
Species of Armillaria are basidiomy-
however, are time consuming, the results are
cetous root pathogens of a wide range of
often ambiguous and are only applicable to
woody plants. These fungi have a wide global
heterothallic and sexual species.
distribution and include some of the most
Qualitative DNA based methods
important pathogens of trees. In this regard
including comparisons of sequences of the
they are especially important in forests and
IGS-1 and ITS regions of the ribosomal DNA
fruit crops.
(Anderson and Stasovski, 1992; Chillali et al.,
Various techniques have been used to
1997, 1998; Coetzee et al., 2000, 2001, 2003;
identify and group Armillaria species. Earlier
Sekizaki et al., 2008), RFLPs (Jahnke et al.,
work relied exclusively on pairing tests and
1987; Anderson et al., 1989; Smith and
morphology. Morphological identification is
Anderson, 1989; Sekizaki et al., 2008;
based primarily on fruiting body characteristics
Wingfield et al., 2009) and AFLPs (Pérez-
(e.g. Watling et al., 1982; Bérubé and Sierra et al., 2004; Wingfield et al., 2009) have
Dessureault, 1988, 1989). However, the recently been increasingly used to identify
seasonal nature and short life span of fruiting
Armillaria species. Protein based techniques
bodies limits the use of this method for
have also been employed in Armillaria
identification. Mating tests, while not taxonomy (Morrison, 1982; Morrison et al.,
dependant on fruiting structures, rely on 1985; Lin et al., 1989; Wahlström et al., 1991;
biological compatibility of isolates of the same
Mwenje and Ride, 1996; Mwenje et al., 2006).
9

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Despite limited resolution of protein-based
comparison of the pectic enzyme profiles of a
methods in comparison to DNA techniques,
collection of the well-known species is not
they can potentially provide additional available.
molecular characters for the identification and
The aim of this study was to analyse the
delineation of species, including those in relationships between a wide range of
Armillaria.
Armillaria spp. using pectin lyases, pectin
Isozymes are multiple forms of the same
methylesterases and polygalacturonases iso-
enzyme and which differ in molecular weight,
zyme profiles. These relationships were then
regulation, isoelectric points and electro-
compared with those of previously published
phoretic mobilities (D’Ovidio et al., 2004).
molecular based comparisons. In this way, the
Isozymes arise as a result of the presence of
value of the enzyme profiles in the taxonomy
multiple genes coding for a protein or as a
of a global collection of Armillaria species
result of post-translational modification of the
could be assessed.
enzymes (D’Ovidio et al., 2004). Pectic
isozymes have been widely used in fungal
Materials and methods
taxonomy (Johansson, 1988; Karlsson and
Stenlid, 1991; Chang and Mills, 1992). The
Origin of Isolates
technique has also been applied successfully
In total, 17 species of Armillaria
for the identification of Armillaria spp.
represented by 36 isolates from many different
Morrison et al. (1985) used esterase and
origins were considered in this study (Fig. 1).
polyphenol oxidases to study isolates from
Armillaria mellea was represented by eight
British Columbia and separated the isolates
isolates; two from each of the four geo-
into A. bulbosa [= A. gallica], North American
graphical clades as indicated by Coetzee et al.
Biological species (NABS) IX [= A. nabsnona],
(2000), while one to three isolates were
NABS X, and group F clustered with NABS V
included for the other Armillaria species. The
[= A. sinapina]. Esterase patterns were also
isolates are maintained in the culture collection
used to differentiate four North American
of the Forestry and Agricultural Biotechnology
Biological Species of Armillaria by Lin et al.
Institute (FABI). A representative set of
(1989). Whalström et al. (1991) analysed the
isolates has also been deposited with the
pectic esterase and polygalacturonases isozyme
Centraalbureau voor Schimmelcultures,
patterns of five European species and found
Utrecht, Netherlands.
that the patterns differed among the species.

Armillaria mellea had two specific Cell wall preparation
polygalacturonase bands, which were absent in
In order to produce cell walls, fresh stem
the other species. Armillaria ostoyae and A.
segments of Msasa (Brachystegia spiciformis)
borealis had very similar profiles. In Japan,
were ground into fine sawdust using a mill as
researchers differentiated Japanese biological
described by Mwenje and Ride (1996). This
species of Armillaria based on isozyme sawdust was soaked for one hour in 100%
patterns from selected enzymes (Cha and ethanol, filtered through Whatman filter paper
Igarashi, 1995; Matsushita et al., 1996). (#1) and rinsed in 100% ethanol for 10 minutes.
Armillaria isolates from Zimbabwe have been
This was followed by washing twice in acetone
shown to reside in three groups using isozymes
(10 minutes per wash) after which it was air
(Mwenje and Ride, 1996). Mwenje and Ride
dried. The crude extract was then stored at
(1997) also identified four taxonomic groups in
room temperature until further use.
Africa based on pectin lyase and pectin
methylesterase isozyme patterns. More recently,
Enzyme production
Mwenje et al. (2006) employed isozyme
Isolates were grown in duplicate at 25°C
patterns from these enzymes together with
under stationary conditions in 250 mL conical
DNA sequence data to elucidate the Armillaria
flasks containing 50 mL Vogel’s medium
species causing disease on tea in Kenya.
(Vogel, 1956) amended with one gram cell
Despite the usefulness of pectic enzymes in
walls. After 30-35 days of incubation in the
distinguishing some species of Armillaria, a
dark, the cultures were harvested by filtration
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Fungal Diversity

Fig. 1. Diagram showing the type and number of pectin lyase (PL), pectin methylesterase (PME) and polygalacturonase
(PG) patterns as well as a dendrogram generated from the isozyme profiles after cluster analysis for the Armillaria
isolates. Each column represents an isozyme and a black block represents the presence of a corresponding isozyme in an
isolate. The country of origin is indicated within the brackets for each isolate. NH and SH next to the closed circles
indicate the nodes that are shared by the species occurring in the Northern hemisphere and those from the Southern
hemisphere, respectively. Dissimilarity values are shown on the scale.

using Whatman filter paper (#1). Filtrate (30-
stacking gels, to enable the detection of pectic
40 mL) was concentrated overnight by dialysis
enzymes. Equal amounts of double strength
to approximately one mL using 12.5% (w/v)
buffer and enzyme solution were mixed prior
polyethylene glycol dissolved in sodium to loading the gel.
acetate buffer (pH 5.5). The concentrate was

stored at –20ºC.
Detection of Pectin Lyases (PL) and Pectin

Methylesterases (PME)
Native gel electrophoresis
The method used by Mwenje and Ride
Electrophoresis was performed in 10%
(1996) was employed to detect PLs and PMEs.
polyacrylamide resolving gels, using a high pH,
Gels were incubated for 10 minutes at 5°C in
non-dissociating discontinuous system (Hames,
10 mM CaCl2 after electrophoresis. They were
1987). Pectin was incorporated at 0.5% (w/v)
then incubated for 40 minutes at room
into resolving and plug gels, but not in the
temperature in 20 mM Tris-HCl (pH 8.5)
11

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containing 10 mM CaCl2. Gels were stained
but were also detected in A. tabescens, A.
overnight in 0.03 (w/v) ruthenium red. Excess
luteobubalina, A. ostoyae, A. borealis and the
ruthenium red was removed using at least three
unnamed Armillaria sp. from New Zealand
changes of distilled water. Pectin lyase bands
(CMW5597).
appeared as white bands, while PME bands
Four different PME bands, designated
appeared as dark, red/ purple bands.
PME1 – PME4, were found in this study (Fig.

1). PME1 was detected only in the isolate of an
Detection of Polygalacturonases (PGs) and
unnamed Armillaria sp. from New Zealand
Pectin Methylesterases (PMEs)
(CMW5597). Band PME2 was absent in A.
After electrophoresis, gels were washed
tabescens, the unnamed species from New
briefly in water and incubated for 1.5 hour at
Zealand and all A. mellea isolates. Band PME3
25°C in 100 mM malic acid without shaking.
was detected in A. borealis, the unnamed
Gels were rinsed in water for 5 min and stained
species from New Zealand, A. limonea, A.
overnight in ruthenium red (0.03 w/v in water).
luteobubalina, A. novae-zelandiae, A. palli-
Stained gels were washed in water to remove
dula, A. tabescens, A. mellea from Japan, A.
excess ruthenium red. Polygalacturonases were
fumosa and A. ostoyae. Band PME4 was
visualised as white bands and PMEs appeared
present in all isolates from Africa, A. gallica, A.
as dark bands.
fumosa and A. pallidula.

Polygalacturonase isozyme analysis
Numerical analyses
resulted in the production of eight different
Bands that were clearly visible on the
bands (PG1 – PG8) (Fig. 1). All African
zymograms were scored as 1 and 0 repre-
isolates were identical in their PG banding
senting present and absent bands, respectively.
patterns; having PG1, PG2 and PG3. The PG1
The combined data set for polygalacturonase,
band was also present in A. luteobubalina, A.
pectin lyase and pectin methylesterase was
novae-zelandiae, A. limonea, A. tabescens, A.
analysed using the Numerical Taxonomy and
mellea isolates from North America and Japan,
Multivariate Analysis System version 2.1 A. ostoyae and unnamed isolate (CMW5597)
(NTSYSpc) (Exeter Software, New York, from New Zealand. Band PG2 was present in
USA). Euclidean distances were calculated and
the African isolates and A. pallidula, and PG3
a dendrogram generated using an unweighted
was exclusive to the African isolates. Band
pair group method with arithmetic mean PG4 was detected in A. cepistipes, A. borealis,
(UPGMA).
A. nabsnona, A. ostoyae, A. gemina and A.

gallica and band PG5 was present in A. gallica,
Results
A. gemina, A. nabsnona, and A. cepistipes.

Bands PG6 and PG7 were present in A.
Isozyme patterns for various pectic enzymes
borealis, A. tabescens, A. ostoyae, A. cepistipes,
A total of eight different PL isozymes,
A. nabsnona, A. gemina, A. gallica and all A.
designated PL1 – PL8, were detected (Fig. 1).
mellea isolates. Band PG8 was present in
European A. mellea strains (CMW4615 and
unnamed species from New Zealand, A. gallica,
CMW11265) yielded six PL bands, and thus
A. limonea, A. luteobubalina, A. tabescens and
the largest number of PL isozymes. PL1 and
North American and Japanese A. mellea.
PL2 bands were present in these isolates, as

well as in A. novae-zelandiae and A. gallica
Numerical analyses and grouping of isolates
isolates. PL3 was present only in the species
All isolates included in this study could
from Africa. Band PL4 was detected only in
be grouped according to the Armillaria spp.
Zimbabwean group III isolates (CMW9954 and
that they represented in the dendrogram
CMW10115). All A. mellea isolates except one
generated based on combined data of pectin
from Japan had band PL5, which they shared
lyase, polygalacturonases and pectin methyl-
with A. novae-zelandiae and A. tabescens
esterases (Fig. 1). Isolates also resided in two
isolates. Band PL6 was common in all A.
major groups. One of these included all
mellea isolates and was also detected in A.
Armillaria spp. found only in the Northern
novae-zelandiae and A. tabescens. Bands PL7
hemisphere and the second group included all
and PL8 were common in all A. mellea isolates
species from the Southern hemisphere.
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Fungal Diversity

Armillaria borealis grouped very close to
Isozyme comparisons in this study, confirm
A. ostoyae, forming one group within the
this view.
Northern hemisphere collection of isolates.
Isolates from Zimbabwe representing
This group was connected to a cluster that
Armillaria group II (Mwenje and Ride, 1996;
included A. gallica, A. gemina, A. nabsnona
Mwenje et al., 2003) and A. fuscipes had
and A. cepistipes. A second major cluster
identical banding patterns. These isolates also
within the Northern hemisphere group was
clustered together in the dendrogram. This
formed by A. mellea and A. tabescens.
result is in contrast to IGS-1 sequence data
Armillaria mellea isolates formed clusters,
(Mwenje et al., 2003), which separates these
comprising of isolates from Japan, Europe,
isolates into different groups. Isolates of
eastern and western North America, African Armillaria group III (Mwenje and Ride,
respecttively. Isolates representing A. tabescens
1996; Mwenje et al., 2003) differed slightly in
were connected to the cluster comprising of
their enzyme profile from groups I and III. This
Japanese A. mellea isolates.
is also consistent with analyses of IGS-1
The Southern hemisphere group included
sequence data, where these isolates have been
isolates representing the unnamed isolate from
shown to represent different, but closely related
New Zealand, A. limonea, A. luteobubalina, A.
groups (Mwenje et al., 2003).
novae-zelandiae, A. pallidula, A. fumosa, A.
Armillaria fumosa and A. pallidula differ
fuscipes and the Zimbabwean groups II and III
at only one isozyme and thus grouped closely
as defined by Mwenje and Ride (1996). Two
in cluster analysis. Previous reports have
major groups were observed within the indicated that these two species are phyloge-
Southern hemisphere cluster. One of the groups
netically closely related. For example, Coetzee
included A. novae-zelandiae, A. limonea, A.
et al. (2001) could not differentiate between the
luteobubalina, and the unknown species from
two species based on sequences of the ITS
New Zealand. Within this group, the unnamed
region. The species could also not be separated
species was closely related to A. luteobubalina
based on EF 1-? sequence data in a previous
and together formed a cluster closely related to
study (Maphosa et al., 2006). However, A.
A. limonea. The other group included the
fumosa and A. pallidula were previously shown
African taxa together with A. pallidula and A.
to be distinct species based on morphology and
fumosa to form the second major cluster.
mating type tests (Kile and Watling, 1988).
Within this group, A. pallidula and A. fumosa
Armillaria luteobubalina and A. limonea
were closely related and the African taxa
grouped in the same cluster and had slightly
formed a distinct group.
different banding patterns. Armillaria luteobu-

balina is of Australian origin and A. limonea
Discussion
originates from New Zealand. Using compati-

bility tests, Kile and Watling (1988) concluded
In this study, we have shown that the
that these represent different biological species.
pectic enzymes PL, PG and PME can be used
The grouping of these species based on pectic
to separate most species of Armillaria. These
enzymes in this study is consistent with the
included 17 species and the majority of those
findings of Coetzee et al. (2003), who showed
that are commonly encountered. We have
that these two species are phylogenetically
further been able to show that Northern closely related based on their ITS sequence
hemisphere isolates are completely different to
data. Also present in this cluster was an isolate
those from the Southern hemisphere. Southern
of an unnamed species from New Zealand.
hemisphere species have been found to group
The fact that this isolate grouped with isolates
basally in a DNA based phylogeny, hence
representing A. luteobubalina indicated that it
ancestral to those from the Northern Hemi-
might be related to A. luteobubalina. Coetzee
sphere (Coetzee et al., 2001; Dunne et al.,
et al. (2003) showed that this isolate has ITS
2002). DNA sequence data have shown that
sequences that are not identical, but that it is
species from the Southern hemisphere are more
phylogenetically related to A. luteobubalina.
closely related to each other and very distantly
This unnamed isolate also has distinct EF 1-?
to those from the Northern hemisphere. sequences (Maphosa et al., 2006) providing
13

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further evidence that it represents a previously
(Harrington and Wingfield, 1995) and they
undescibed species.
have previously been shown to be closely
Armillaria novae-zelandiae isolates from
related based on their isozyme profiles
Chile, New Zealand and Australia grouped
(Wahlström et al., 1991). Furthermore,
together regardless of geographic origin. This
Anderson et al. (1989) also concluded that
result is consistent with the report of Kile and
these two species are closely related using
Watling (1983) who showed that A. novae-
rDNA operon data and Anderson and Stasovski
zelandiae from Australia and New Zealand are
(1992) showed that the intergenic region
sexually compatible and belong to the same
sequences for A. borealis and A. ostoyae are
biological species. Using ITS sequence data,
very similar.
Coetzee et al. (2001) showed that these isolates
Armillaria gemina, A. nabsnona and A.
grouped in a single clade further confirming
cepistipes had identical banding profiles and
that they represent a single species.
these species clustered together in the dendro-
Armillaria mellea isolates had differing
gram. They shared most of the bands with A.
isozyme banding patterns and formed four sub-
gallica, although the latter species was clearly
clusters corresponding to their biogeographic
different. The grouping of A. gallica, A.
distributions. Thus, isolates from Japan, Europe,
nabsnona and A. cepistipes but not A. gemina
western and eastern North America formed
concurs with previous studies indicating that
separate sub-clusters, which are consistent with
these species are phylogenetically closely
those emerging from DNA sequence data. The
related (Anderson and Stasovski, 1992; Miller
separation of A. mellea isolates according to
et al., 1994; Chillali et al., 1998). The grouping
their geographic origin is supported by a
of A. gemina within this group was unexpected
number of studies. Anderson et al. (1989)
as it has been shown to be more closely related
using RFLP data from the rRNA operon
to A. ostoyae in IGS-1 DNA sequence and
showed that A. mellea from Europe and North
rDNA data (Smith and Anderson, 1989;
America have different restriction patterns.
Anderson and Stasovski 1992), RFLP patterns
Harrington and Wingfield (1995) also showed
(Harrington and Wingfield, 1995) and
that North American and European isolates of
morphology (Bérubé and Dessureault, 1989).
A. mellea have different IGS-1 RFLP profiles
Results of this study have demonstrated
after digestion with AluI. Likewise, Coetzee et
that combined pectin lyase, polygalacturonase
al. (2000) using ITS and IGS-1 data separated
and pectin methylesterase data can be used to
A. mellea isolates according to geographic
differentiate between many Armillaria species.
origin.
The technique, however, failed to separate
Isolates representing A. tabescens had
some closely related species, reflecting
banding patterns different, yet closely related
findings from earlier studies based on
to those of A. mellea from western North
morphology and DNA sequence data. The
America. This grouping is consistent with the
relationships among species based on the
finding of Coetzee et al. (2000) who showed
combined isozyme profiles is in agreement
that A. tabescens resides in a clade basal to
with results from previous phylogenetic studies.
those of A. mellea. It has similarly been shown,
Results from this study also support previous
using DNA re-association data, that A.
suggestions that species from the Northern and
tabescens is very closely related to A. mellea
Southern hemispheres reside in two distinct
(Miller et al., 1994). Although these species
groups.
are clearly different, we have added evidence

that A. tabescens and A. mellea are closely
Acknowledgements
related species.

Armillaria ostoyae had a unique banding
We thank the members of the Tree Protection Co-
pattern. The profile of this species was most
operative Programme (TPCP), the National Research
closely related to that of
Foundation (NRF), the THRIP initiative of the
A. borealis and they
Department of Trade and Industry and the DST/NRF
clustered closely together on the dendrogram
Centre of Excellence in Tree Health Biotechnology
obtained in this study. The IGS-1 RFLP
(CTHB) South Africa for financial assistance.
patterns for these two species are very similar

14

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Fungal Diversity

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