Mycologia

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Differentiating characteristics between

Melampsoridium rusts infecting birch and alder
leaves

Timo Kurkela, Märt Hanso & Jarkko Hantula

To cite this article: Timo Kurkela, Märt Hanso & Jarkko Hantula (1999) Differentiating
characteristics between Melampsoridium rusts infecting birch and alder leaves, Mycologia, 91:6,
987-992, DOI: 10.1080/00275514.1999.12061108

To link to this article: https://doi.org/10.1080/00275514.1999.12061108

Published online: 04 Jun 2019.

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Mycologia, 91(6), 1999, pp. 987-992.
© 1999 by The Mycological Society of America, Lawrence, KS 66044-8897
Differentiating characteristics between Melampsoridium rusts infecting birch
and alder leaves
Timo Kurkela1
Finnish Forest Research Institute, P. 0. Box 18,
FIN 01301 Vantaa, Finland
Mart Hanso
Estonian Agricultural University, Faculty of Forestry,
F.R Kreutzwaldi 5, EE-2400 Tartu, Estonia
Jarkko Hantula
Finnish Forest Research Institute, P.O. Box 18,
FIN 01301 Vantaa, Finland
Abstract: Melampsoridium rust epidemics on alder
occurred in Estonia in 1996-1998, and in Finland in
1997-1998. In this study we show that the length and
roundness of urediniospores and the PCR-amplified
internal transcribed spacer (ITS) sequences of rusts
occurring on Alnus and Betula are different. Mor-
phologically the rust on alder belongs to M. hirat-
sukanum, and based on our results is not conspecific
with M. betulinum. The sequence comparisons indi-
cated that 5.8S rDNA was similar in both Melampso-
ridium species and identical to several Cronartium se-
quences. The 5.8S rDNA of Melampsoridium was also
more similar to Pucciniastrum, Chrysomyxa and Puc-
cinia than to Melampsora species. The ITS sequences
were distinct from all other rusts, but relatively simi-
lar to Cronartium, Pucciniastrum and Chrysomyxa se-
quences.
Key Words: 5.8S rDNA, Alnus, Betula, DGGE,
ITS, Uredinales, urediniospore
INTRODUCTION
The heteroecious rust genus, Melampsoridium, is
composed of uredinial and telial stages occurring on
Betulaceae (Arthur 1962, Kaneko and Hiratsuka
1981) and Magnolia campbellii (Magnoliaceae)
(Singh and Pandley 1972). The aecial stage of some
of these rusts is known to occur on Larix spp. (e.g.,
Kuprevich and Tranzschel 1957). Melampsoridium
was segregated from Melampsora based on the pres-
ence of a peridermium in both aecia and uredinia.
Morphologically, the taxonomic position of Melamp-
Accepted for publication July 14, 1999.
1 Email: timo.kurkela@metla.fi
987
Published online 04 Jun 2019
soridium is currently considered to be close to Me-
lampsorella and Pucciniastrum in the Pucciniastraceae
(Cummins and Hiratsuka 1983).
Melampsoridium betulinum (Pers.) Kleb. on birch is
common in northern hemisphere (Gaumann 1959).
In northern Europe it infects leaves of three species
(Betula pendula Roth., B. pubescens Ehrh., and B.
nanaL.) (e.g., Liro 1908). Melampsoridiumisrareon
alder (Alnus) and it has been recorded only once in
Finland (J0rstad and Nannfeldt 1958). Elsewhere,
two alder rust fungi, M. alni (Thiimen) Dietel and
M. hiratsukanum Ito have been described (Gaumann
1959, Wilson and Henderson 1966). Melampsoridium
hiratsukanum differs from other Melampsoridium spe-
cies as it has uniformly echinulated, relatively small
urediniospores (Wilson and Henderson 1966),
whereas those of M. betulinum and M. alni lack echi-
nulation in the broader end (Arthur 1962, Kuprevich
and Tranzschel1957, Gaumann 1959). According to
Kaneko and Hiratsuka (1981) there are a similar
number of germ pores in urediniospores of M. be-
tulinum and M. hiratsukanum and they are bizonate,
while the urediniospores of M. alni have two germ
pores, one at each end of the spore. Roll-Hansen and
Roll-Hansen (1981) were able to infect alders with
urediniospores obtained from both birch and alder
leaves, and therefore concluded that Melampsoridium
rusts on both hosts represent the same species, name-
ly M. betulinum.
In Finland and Estonia Melampsoridium rust is ob-
served regularly during each growing season on
birch, while on alder there are only few records in
the whole of Europe. Thus, assuming that the rust
fungi are conspecific there should either be a differ-
ence in susceptibility between birch and alder, or the
infection capability of rust fungus strains on these
two hosts should be different. Both of these possibil-
ities are supported from the literature. Several stud-
ies have shown that there are differences in infection
capability between M. betulinum originating from dif-
ferent birch species (Klebahn 1903, 1905, Liro 1907,
Poteri 1992) and also that the degree of resistance of
different birch species varies considerably (Kechel
and Boden 1984, Poteri 1992, Poteri and Rousi 1996,
Poteri et al 1997). However, the lack of rust on alder
during times of heavy infection on birch does not
support the Roll-Hansens' (1981) idea of conspeci-
988 MYCOLOGIA
ficity. We therefore address this assumption using
modern methods of molecular biology and fresh ma-
terial from recent alder rust epidemics in Estonia
(Poldmaa 1997) and Finland.
Different species usually have distinct sequences in
their internal transcribed spacer (ITS) regions (Gar-
des et al 1990). The ITS region (including the 5.8S
rRNA gene) is thus one of the most popular markers
in mycological studies. The sequence has been used
in taxonomic studies (Jasalavich et al 1995, Hamelin
and Rail 1997) and as a tool to indicate species com-
position of mycorrhizal communities directly from
rootlets (Gardes et al 1991, Erland et al 1994).
The denaturing gradient gel electrophoresis
(DGGE) is an electrophoretic technique which effi-
ciently separates DNA-molecules according to their
size, as well as sequence differences (Myers et al1986,
Lessa 1992). When the electrophoresis parameters
are well set, the electrophoresis can be run in a con-
stant denaturing agent concentration without loosing
separation power (i.e., constant denaturing gel elec-
trophoresis or CDGE).
In this study we tested whether or not the rust fun-
gi observed on birch and alder can be separated by
morphological and/ or PCR-based methods, and de-
termined the taxonomic relationship of Melampsori-
dium to other rust genera using 5.8S rDNA and ITS
sequences.
MATERIALS AND METHODS
Morphology.-Rust infected leaves of Alnus incana (L.)
Moench., A. glutinosa (L.) Gaertn., Betula pendula, and B.
pubescens, were collected from Tuusula (representing each
of the four hosts) and Helsinki (Suutarila, birch samples
only) in southern Finland and from Tartu in Estonia (rep-
resenting A. incana, A. glutinosa and B. pubescens). Ure-
diniospores were stained with cotton blue in lactophenol
and measured using an image analysis system (Cambridge
500 by Leica). The numbers of spores representing the rust
on each host mentioned above were 316, 240, 209, and 634,
respectively. Data were obtained from the length, width,
and roundness of spores. Roundness is a shape factor which
gives a minimum unity (1) for a circle. This is calculated
from the ratio of perimeter squared to area,
Perimeter 2
Roundness = - - - - - - - - -
4 X 1T X Area X 1.064
The adjustment factor of 1.064 corrects the perimeter for
the effect of the corners produced by the digitization of the
image (Anonymous 1993). Statistical analysis was made with
SYSTAT software.
Samples for ITS-analysis. -Samples of approximately 1 mm2
leaf pieces containing single uredinia were aseptically cut
from A. glutinosa, A. incana, B. pendula, and B. pubescens.
The PCR-reactions were performed on samples from five A.
glutinosa, nine A. incana, eight B. pendula, and eight B.
pubescens trees, using one sample per tree. The samples
originated from two locations (Tuusula and Nummi-Pusula)
in southern Finland.
DNA isolations.-Total DNA was isolated from the leaf piec-
es with uredinia using a modification of the method de-
scribed by Vainio et al (1998). The protocol included cell
disruption (using quartz sand), three phenol: chloroform
(1: 1) extractions, a chloroform: isoamyl alcohol (24: 1) ex-
traction, precipitation with polyethylene glycol, and drying.
The DNA was resuspended in 10 mM Tris-HCl buffer (pH
8.0) containing 1 mM EDTA.
PCR-reactions.-The fungal ITS region (including 5.8S
rDNA) was amplified by polymerase chain reaction (PCR)
using basidiomycete specific primer ITS4-B and universal
fungal primer ITS1-F (Gardes and Bruns 1993). A 40 bp
GC-clamp was added to the 5'-end of the ITS1-F-primer to
allow efficient separation of the amplification products of
ITS in DGGE or CDGE. The sequences of the primers used
were as follows: CGC CCG CCG CGC GCG GCG GGC GGG
GCG GGG GCA CGG GGG GCT TGG TCA m AGA GGA
AGT AA (CG-clamped ITS1-F), and CAG GAG ACT TGT
ACA CGG TCC AG (ITS4-B). The PCR-reactions were car-
ried out as suggested by the manufacturer of the Dynazyme
thermostable polymerase (Finnzymes Ltd, Finland). The re-
action cycles were the same as given by Gardes and Bruns
(1993).
CDGE analysis of the amplification products.-The PCR re-
actions were first analyzed by electrophoresis in agarose gels
containing 1.0% SynerGel (Diversified Biotech) and 1.0%
agarose (FMC BioProducts). The electrophoresis was run
in TAE-buffer (40 mM Tris-Acetate pH 8.0, 1 mM EDTA).
The lengths of the amplification products were estimated
by comparing them to a 100 bp DNA ladder (Gibco BRL).
The constant denaturing gel electrophoresis was run ac-
cording to the instructions of the manufacturer of the D-
GENE denaturing gel system (BioRad). The denaturant
(urea and formamide) concentration used was 30%. In
both types of electrophoreses the amplification products
were visualized using ethidium bromide under UV-light.
Sequence analysis.-Amplified ITS-fragments from PCR-re-
actions were purified and ligated into pCR®2.1 vector using
the TOPO TA Cloning kit (Invitrogen, California). There-
sulting recombinant plasmids were used to transform Esche-
richia coli TOP10 cells as recommended by the manufac-
turer of the kit. The selected cloned inserts were sequenced
by A.L.F. DNA sequencer® (Pharmacia Biotech, Uppsala,
Sweden) using M13 reverse and forward primers and Ther-
mo Sequenase fluorescent labeled primer cycle sequencing
kit (Amersham Pharmacia Biotech, England). As the two
sequences overlapped for only a few hundred base pairs,
additional sequence analyses were performed using primers
CAA GGT GCG TIC AAA GAT TC and GAA TCT TTG
AAC GCA CCT TG, which start priming from the 5.8S
rDNA gene to both directions. The sequences were aligned
manually and neighbor joining (NJ) dendrograms were
constructed using the MEGA software (Kumar et al 1993)
and sequences obtained from GenBank. The Phylogeny In-
 KURKELA ET AI..: MELAMPSORJDIUM
FIG. 1. Urediniospores of Melampsoridium betulinum
from Betula pendula (from Tuusula, Finland). Arrows in-
dicate the bizonate position of germ pores. Bar = 20 JLm.
ference Package (PHYLIP) version 3.57c was used for the
parsimony analyses. The sequences determined during this
investigation can be found in GenBank (AF125177 and
AF125178), and the NJ dendrogram in TreeBASE (M528).
RESULTS
No significant morphological differences were ob-
served between the rust fungi originating from the
two birch species (except width) and generally, the
spores produced on B. pubecens were larger than
those produced on B. pendula. The spores produced
on both alder species were similar. In birch rust
spores the broader end was smooth, but in alder rust
fungus the surface of urediniospores was uniformly
echinulated (FIGS. 1, 2). The bizonate position and
number of germ pores was similar in both birch and
alder rust fungi.
The rust occurring on birch had significantly long-
er (P < 0.001) urediniospores than the rust collected
from alder. The average length of the urediniospores
from birch were 31.6 IJ.m on B. pendula and 33.6 IJ.m
on B. pubescens, and in alder rust fungus the corre-
sponding measures were 25.0 IJ.m on A. incana and
26.6 j.Lm on A. glutinosa, respectively (FIGS. 3, 4). The
average width of the urediniospores from birch were
RUSTS INFECTING BIRCH AND ALDER 989
FIG. 2. Urediniospores of Melampsoridium sp. Alnus in-
cana (from Tuusula, Finland). Arrows indicate the bizonate
position of germ pores. Bar = 20 JLm.
13.6 j.Lm on B. pendula and 15.0 IJ.m on B. pubescens,
respectively (the difference was significant with P <
0.001), and in the alder rust fungus the correspond-
ing measures were 14.0 j.Lm on A. incana and 13.9
j.Lm on A. glutinosa, respectively. In the combined
data, difference in width between alder and birch
rust fungi was insignificant and the number of spores
distributed similarly when classified according to
their width in categories of 1 j.Lm (FIGS. 3, 4). The
roundness of the spores was also a significant (P <
0.001) differentiating character between the alder
and birch samples. The average roundness index var-
ied in alder rust fungi from 1.35 (A. incana) to 1.39
(A. glutinosa). The average roundness indexes were
1.52 and 1.53 in the samples from B. pendula and B.
pubescens, respectively.
The differentiating power of the measured mor-
phological characters of spores was tested using the
collected material. In a discriminant analysis where
only the length and roundness of spores were used
to classify the urediniospores the host origin was pre-
dicted correctly for 91% of the spores (TABLE I). The
addition of other morphological variables to the anal-
990 MYCOLOGIA

40 TABLE I. Reclassification of Melampsoridium sp. uredinio-

spores using length and roundness as discriminant variables
M Width (on Betula)
EST
30 •Length - - Correct
Q)
o Width (on Alnus) Alder" Birch• (%)
Cl
111 ::Length - -
"E
Q) 20
Melampsoridium
~
Q)
(on alder) 504 52 91
a. M. betulinum
10 (on birch) 80 763 91
Total 584 815 91

0 a The host origin suggested by the variables.

0 10 20 30 40
Width and length, f..lm
FIG. 3. The width and length distribution of uredinio-
spores in M. betulinum on birch and Melampsoridium sp.
on alder in Estonian (EST) samples.
ysis did not increase the correctness of the predic-
tion.
The PCR amplification from uredinia containing
birch or alder leaf pieces resulted in one major 900
bp band and sometimes in faint, smaller molecular
weight bands in agarose gel electrophoresis (not
shown). We concluded that the major band corre-
sponded to the ITS region whereas the fainter bands
were probably due to nonspecific priming or other
PCR artifacts. No differences in the sizes of the major
900 bp bands were observed between the samples.
In constant denaturing gel electrophoresis the am-
plified ITS bands from the birch rust migrated faster
than those from the alder rust (FIG. 5). All 14 sam-
ples from the two alder species were identical (FIG.
5). Also the migration of amplified ITS samples from
the birch samples was identical in most cases (FIG. 5),
although some slight differences were observed (lane
50
h) suggesting that there may have been some intra-
specific variation not properly resolved by CDGE.
However, this was not tested by sequencing.
The sequencing of the ITS region indicated that
in both species the ITS1 region, 5.8S rRNA gene and
ITS2 region were 248 bp, 157 bp and 221 bp long,
respectively. Although the two sequences were of
identical length six 1 bp insertions/ deletions and two
transversions were observed in the ITS1 region. In
the ITS2 region no length mutations were observed
but there were seven transversions. The 5.8S rDNA
gene was identical in both species and similar to that
of Cronartium comandrae, C. quercuum, C. compto-
niae, C. conigenum, and C. strobilinum. In NJ analysis
of 5.8S rDNA sequences Melampsoridium clustered
closely to Cronartium, Pucciniastrum, and Chrysomyxa
(FIG. 6). Puccinia thlaspeoswas grouped into the same
cluster although in a separate branch, whereas the
two Melampsora species formed a distinct cluster sup-
ported by a high bootstrap value. The same grouping

40
e: Width (on Betula)
FIN • Length, - -
30 o Width (on Alnus)
Q)
Cl ::;; Length, - -
111
~ 20
~
Q)
a.
10

FIG. 5. A constant denaturant gel electrophoresis anal-

0 ysis of PCR-amplified internal transcribed spacer (ITS) se-
0 10 20 30 40 50 quences of Melampsoridium rust fungi from birch and alder.
Width and length, jlm Lanes: a. 100 bp DNA ladder; b, c. ITS of Melampsoridium
from Alnus glutinosa; d, e. ITS of Melampsoridium from A.
FIG. 4. The width and length distribution of uredinio- incana; f, g. ITS of Melampsoridium from Betula pubescens;
spores in M. betulinum on birch and Melampsoridium sp. h, i. ITS of Melampsoridium from B. pendula; and j. 100 bp
on alder in Finnish (FIN) samples. DNA ladder.
 KURKELA ET AL: MELAMPSORIDIUM RUSTS INFECTING BIRCH AND ALDER
j Melampsoridium
28
~ Cro-um cornandrae
53 : ..__ Pucciniastrum goeppertianum
I r Cronartium flaccidum
85
[__ Chrysomyxa arctostaphy1i
Pucdnia thlaspeos
L_
100
FIG. 6. Neighbor joining tree of 5.8S rDNA sequences
from several rust fungus species and Heterobasidion anna-
sum. The bootstrap values are given in branches. Bar is ca
1% difference in sequence.
was also supported by the parsimony analysis (not
shown). In these analyses Heterobasidion annosumwas
used as an outgroup to identify the root of the rust
dendrogram.
The ITS1 and ITS2 sequences of the two Melamp-
soridium species were most similar to each other, and
more similar to those of Cronartium, Pucciniastrum
and Chrysomyxa than to those of Puccinia and Me-
lampsora, which were only distantly related and ho-
mologous nucleotides could not be identified. As the
alignments were uncertain between different genera,
ITS1 and ITS2 sequences were not subjected to a
clustering analysis.
DISCUSSION
The molecular and morphological data presented
here indicates that the Melampsoridium species on
birch and alder in Finland (and based only on mor-
phological measurements, also in Estonia) are differ-
ent. They are also distinguishable from M. alni be-
cause of different germ pore positions (Kaneko and
Hiratsuka 1981). The samples from both alder spe-
cies were morphologically identical and they had sim-
ilar ITS alleles in CDGE. Similarly, most of the sam-
ples from birch had identical ITS alleles, although in
few samples possible variation in ITS was observed.
These observations support the idea that only one
species (M. betulinum) occurs on birch, and another
species on alder in Finland.
Although some spores may have a smooth area at
one end (FIG. 2) most of the rust fungi we observed
on A. glutinosa and A. incana are morphologically
similar to M. hiratsukanum which has been recorded
in Asia and in the Americas (e.g., Kuprevich and
Tranzschel1957, Peterson 1965, Kaneko and Hiratsu-
ka 1981, Kobayashi and de Guzman 1988) and now
for the first time in Estonia (P6ldmaa 1997) and Fin-
991
land. Alder rust has also been recorded by several
authors in Britain (see Wilson and Henderson 1966),
but its correct diagnosis has remained uncertain.
Henderson and Bennell (1979), Kaneko and Hiratsu-
ka ( 1981), and Roll-Hansen and Roll-Hansen ( 1981)
regarded British alder rust fungi as M. betulinum.
The sequence analyses of 5.8S rDNA and ITS re-
Melampsora medusae
gions confirmed that the taxonomic position of ge-
Melampsora ocddentalls nus Melampsoridium is not close to Melampsora. In-
Heterobasidion annosum
stead, it seems to be more closely related to Cronar-
tium, Pucciniastrum and Chrysomyxa supporting the
distinction of Melampsora from other rust genera
(Cullings and Vogler 1998). As 5.8S rDNA and ITS
sequences are not yet available for all rust genera, it
will be interesting to see if future studies shed further
light to the taxonomy of genus Melampsoridium.
In conclusion, we have shown in this study that
Melampsoridium rust fungi on birch and alder rep-
resent two separate species in Finland and in Estonia.
The species on birch is M. betulinum, but the species
on alder is not M. alni, but M. hiratsukanum, as sug-
gested also by P6ldmaa ( 1997) who reported obser-
vations on a similar rust on A. incana in Estonia in
1996. These studies represent the first reports of M.
hiratsukanum in Fennoscandia and Estonia.
ACKNOWLEDGMENTS
Ms. Laura Paavolainen is acknowledged for her critical com-
ments. This work is part of the Finnish Biodiversity Re-
search Programme FIBRE (1997-2002).
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