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 soridium is currently considered to be close to Me-

Finnish Forest Research Institute, P. 0. Box 18, lampsorella and Pucciniastrum in the Pucciniastraceae
FIN 01301 Vantaa, Finland (Cummins and Hiratsuka 1983).
Melampsoridium betulinum (Pers.) Kleb. on birch is
Mart Hanso
common in northern hemisphere (Gaumann 1959).
Estonian Agricultural University, Faculty of Forestry,
F.R Kreutzwaldi 5, EE-2400 Tartu, Estonia In northern Europe it infects leaves of three species
(Betula pendula Roth., B. pubescens Ehrh., and B.
Jarkko Hantula nanaL.) (e.g., Liro 1908). Melampsoridiumisrareon
Finnish Forest Research Institute, P.O. Box 18, alder (Alnus) and it has been recorded only once in
FIN 01301 Vantaa, Finland Finland (J0rstad and Nannfeldt 1958). Elsewhere,
two alder rust fungi, M. alni (Thiimen) Dietel and
M. hiratsukanum Ito have been described (Gaumann
Abstract: Melampsoridium rust epidemics on alder 1959, Wilson and Henderson 1966). Melampsoridium
occurred in Estonia in 1996-1998, and in Finland in hiratsukanum differs from other Melampsoridium spe-
1997-1998. In this study we show that the length and cies as it has uniformly echinulated, relatively small
roundness of urediniospores and the PCR-amplified urediniospores (Wilson and Henderson 1966),
internal transcribed spacer (ITS) sequences of rusts whereas those of M. betulinum and M. alni lack echi-
occurring on Alnus and Betula are different. Mor- nulation in the broader end (Arthur 1962, Kuprevich
phologically the rust on alder belongs to M. hirat- and Tranzschel1957, Gaumann 1959). According to
sukanum, and based on our results is not conspecific Kaneko and Hiratsuka (1981) there are a similar
with M. betulinum. The sequence comparisons indi- number of germ pores in urediniospores of M. be-
cated that 5.8S rDNA was similar in both Melampso- tulinum and M. hiratsukanum and they are bizonate,
ridium species and identical to several Cronartium se- while the urediniospores of M. alni have two germ
quences. The 5.8S rDNA of Melampsoridium was also
pores, one at each end of the spore. Roll-Hansen and
more similar to Pucciniastrum, Chrysomyxa and Puc-
Roll-Hansen (1981) were able to infect alders with
cinia than to Melampsora species. The ITS sequences
urediniospores obtained from both birch and alder
were distinct from all other rusts, but relatively simi-
leaves, and therefore concluded that Melampsoridium
lar to Cronartium, Pucciniastrum and Chrysomyxa se-
rusts on both hosts represent the same species, name-
quences.
ly M. betulinum.
Key Words: 5.8S rDNA, Alnus, Betula, DGGE,
In Finland and Estonia Melampsoridium rust is ob-
ITS, Uredinales, urediniospore
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
INTRODUCTION fungi are conspecific there should either be a differ-
ence in susceptibility between birch and alder, or the
The heteroecious rust genus, Melampsoridium, is
composed of uredinial and telial stages occurring on infection capability of rust fungus strains on these
Betulaceae (Arthur 1962, Kaneko and Hiratsuka two hosts should be different. Both of these possibil-
1981) and Magnolia campbellii (Magnoliaceae) ities are supported from the literature. Several stud-
(Singh and Pandley 1972). The aecial stage of some ies have shown that there are differences in infection
of these rusts is known to occur on Larix spp. (e.g., capability between M. betulinum originating from dif-
Kuprevich and Tranzschel 1957). Melampsoridium ferent birch species (Klebahn 1903, 1905, Liro 1907,
was segregated from Melampsora based on the pres- Poteri 1992) and also that the degree of resistance of
ence of a peridermium in both aecia and uredinia. different birch species varies considerably (Kechel
Morphologically, the taxonomic position of Melamp- and Boden 1984, Poteri 1992, Poteri and Rousi 1996,
Poteri et al 1997). However, the lack of rust on alder
Accepted for publication July 14, 1999. during times of heavy infection on birch does not
1 Email: timo.kurkela@metla.fi
support the Roll-Hansens' (1981) idea of conspeci-

987

Published online 04 Jun 2019

988 MYCOLOGIA

ficity. We therefore address this assumption using glutinosa, nine A. incana, eight B. pendula, and eight B.
modern methods of molecular biology and fresh ma- pubescens trees, using one sample per tree. The samples
terial from recent alder rust epidemics in Estonia originated from two locations (Tuusula and Nummi-Pusula)
(Poldmaa 1997) and Finland. in southern Finland.
Different species usually have distinct sequences in DNA isolations.-Total DNA was isolated from the leaf piec-
their internal transcribed spacer (ITS) regions (Gar- es with uredinia using a modification of the method de-
des et al 1990). The ITS region (including the 5.8S scribed by Vainio et al (1998). The protocol included cell
rRNA gene) is thus one of the most popular markers disruption (using quartz sand), three phenol: chloroform
in mycological studies. The sequence has been used (1: 1) extractions, a chloroform: isoamyl alcohol (24: 1) ex-
traction, precipitation with polyethylene glycol, and drying.
in taxonomic studies (Jasalavich et al 1995, Hamelin
The DNA was resuspended in 10 mM Tris-HCl buffer (pH
and Rail 1997) and as a tool to indicate species com-
8.0) containing 1 mM EDTA.
position of mycorrhizal communities directly from
rootlets (Gardes et al 1991, Erland et al 1994). PCR-reactions.-The fungal ITS region (including 5.8S
The denaturing gradient gel electrophoresis rDNA) was amplified by polymerase chain reaction (PCR)
(DGGE) is an electrophoretic technique which effi- using basidiomycete specific primer ITS4-B and universal
fungal primer ITS1-F (Gardes and Bruns 1993). A 40 bp
ciently separates DNA-molecules according to their
GC-clamp was added to the 5'-end of the ITS1-F-primer to
size, as well as sequence differences (Myers et al1986, allow efficient separation of the amplification products of
Lessa 1992). When the electrophoresis parameters ITS in DGGE or CDGE. The sequences of the primers used
are well set, the electrophoresis can be run in a con- were as follows: CGC CCG CCG CGC GCG GCG GGC GGG
stant denaturing agent concentration without loosing GCG GGG GCA CGG GGG GCT TGG TCA m AGA GGA
separation power (i.e., constant denaturing gel elec- AGT AA (CG-clamped ITS1-F), and CAG GAG ACT TGT
trophoresis or CDGE). ACA CGG TCC AG (ITS4-B). The PCR-reactions were car-
In this study we tested whether or not the rust fun- ried out as suggested by the manufacturer of the Dynazyme
gi observed on birch and alder can be separated by thermostable polymerase (Finnzymes Ltd, Finland). The re-
morphological and/ or PCR-based methods, and de- action cycles were the same as given by Gardes and Bruns
termined the taxonomic relationship of Melampsori- (1993).
dium to other rust genera using 5.8S rDNA and ITS CDGE analysis of the amplification products.-The PCR re-
sequences. 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
MATERIALS AND METHODS in TAE-buffer (40 mM Tris-Acetate pH 8.0, 1 mM EDTA).
The lengths of the amplification products were estimated
Morphology.-Rust infected leaves of Alnus incana (L.)
by comparing them to a 100 bp DNA ladder (Gibco BRL).
Moench., A. glutinosa (L.) Gaertn., Betula pendula, and B.
The constant denaturing gel electrophoresis was run ac-
pubescens, were collected from Tuusula (representing each
cording to the instructions of the manufacturer of the D-
of the four hosts) and Helsinki (Suutarila, birch samples
GENE denaturing gel system (BioRad). The denaturant
only) in southern Finland and from Tartu in Estonia (rep-
(urea and formamide) concentration used was 30%. In
resenting A. incana, A. glutinosa and B. pubescens). Ure-
both types of electrophoreses the amplification products
diniospores were stained with cotton blue in lactophenol
were visualized using ethidium bromide under UV-light.
and measured using an image analysis system (Cambridge
500 by Leica). The numbers of spores representing the rust Sequence analysis.-Amplified ITS-fragments from PCR-re-
on each host mentioned above were 316, 240, 209, and 634, actions were purified and ligated into pCR®2.1 vector using
respectively. Data were obtained from the length, width, the TOPO TA Cloning kit (Invitrogen, California). There-
and roundness of spores. Roundness is a shape factor which sulting recombinant plasmids were used to transform Esche-
gives a minimum unity (1) for a circle. This is calculated richia coli TOP10 cells as recommended by the manufac-
from the ratio of perimeter squared to area, turer of the kit. The selected cloned inserts were sequenced
by A.L.F. DNA sequencer® (Pharmacia Biotech, Uppsala,
Perimeter 2
Roundness = - - - - - - - - - Sweden) using M13 reverse and forward primers and Ther-
4 X 1T X Area X 1.064 mo Sequenase fluorescent labeled primer cycle sequencing
The adjustment factor of 1.064 corrects the perimeter for kit (Amersham Pharmacia Biotech, England). As the two
the effect of the corners produced by the digitization of the sequences overlapped for only a few hundred base pairs,
image (Anonymous 1993). Statistical analysis was made with additional sequence analyses were performed using primers
SYSTAT software. CAA GGT GCG TIC AAA GAT TC and GAA TCT TTG
AAC GCA CCT TG, which start priming from the 5.8S
Samples for ITS-analysis. -Samples of approximately 1 mm2 rDNA gene to both directions. The sequences were aligned
leaf pieces containing single uredinia were aseptically cut manually and neighbor joining (NJ) dendrograms were
from A. glutinosa, A. incana, B. pendula, and B. pubescens. constructed using the MEGA software (Kumar et al 1993)
The PCR-reactions were performed on samples from five A. and sequences obtained from GenBank. The Phylogeny In-
 KURKELA ET AI..: MELAMPSORJDIUM RUSTS INFECTING BIRCH AND ALDER 989

FIG. 1. Urediniospores of Melampsoridium betulinum

from Betula pendula (from Tuusula, Finland). Arrows in-
dicate the bizonate position of germ pores. Bar = 20 JLm.
FIG. 2. Urediniospores of Melampsoridium sp. Alnus in-
cana (from Tuusula, Finland). Arrows indicate 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). 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-
RESULTS
ing measures were 14.0 j.Lm on A. incana and 13.9
No significant morphological differences were ob- j.Lm on A. glutinosa, respectively. In the combined
served between the rust fungi originating from the data, difference in width between alder and birch
two birch species (except width) and generally, the rust fungi was insignificant and the number of spores
spores produced on B. pubecens were larger than distributed similarly when classified according to
those produced on B. pendula. The spores produced their width in categories of 1 j.Lm (FIGS. 3, 4). The
on both alder species were similar. In birch rust roundness of the spores was also a significant (P <
spores the broader end was smooth, but in alder rust 0.001) differentiating character between the alder
fungus the surface of urediniospores was uniformly and birch samples. The average roundness index var-
echinulated (FIGS. 1, 2). The bizonate position and ied in alder rust fungi from 1.35 (A. incana) to 1.39
number of germ pores was similar in both birch and (A. glutinosa). The average roundness indexes were
alder rust fungi. 1.52 and 1.53 in the samples from B. pendula and B.
The rust occurring on birch had significantly long- pubescens, respectively.
er (P < 0.001) urediniospores than the rust collected The differentiating power of the measured mor-
from alder. The average length of the urediniospores phological characters of spores was tested using the
from birch were 31.6 IJ.m on B. pendula and 33.6 IJ.m collected material. In a discriminant analysis where
on B. pubescens, and in alder rust fungus the corre- only the length and roundness of spores were used
sponding measures were 25.0 IJ.m on A. incana and to classify the urediniospores the host origin was pre-
26.6 j.Lm on A. glutinosa, respectively (FIGS. 3, 4). The dicted correctly for 91% of the spores (TABLE I). The
average width of the urediniospores from birch were 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 50
Width and length, f..lm h) suggesting that there may have been some intra-
FIG. 3. The width and length distribution of uredinio- specific variation not properly resolved by CDGE.
spores in M. betulinum on birch and Melampsoridium sp. However, this was not tested by sequencing.
on alder in Estonian (EST) samples. 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,
ysis did not increase the correctness of the predic- respectively. Although the two sequences were of
tion. identical length six 1 bp insertions/ deletions and two
The PCR amplification from uredinia containing transversions were observed in the ITS1 region. In
birch or alder leaf pieces resulted in one major 900 the ITS2 region no length mutations were observed
bp band and sometimes in faint, smaller molecular but there were seven transversions. The 5.8S rDNA
weight bands in agarose gel electrophoresis (not gene was identical in both species and similar to that
shown). We concluded that the major band corre- of Cronartium comandrae, C. quercuum, C. compto-
sponded to the ITS region whereas the fainter bands niae, C. conigenum, and C. strobilinum. In NJ analysis
were probably due to nonspecific priming or other of 5.8S rDNA sequences Melampsoridium clustered
PCR artifacts. No differences in the sizes of the major closely to Cronartium, Pucciniastrum, and Chrysomyxa
900 bp bands were observed between the samples. (FIG. 6). Puccinia thlaspeoswas grouped into the same
In constant denaturing gel electrophoresis the am- cluster although in a separate branch, whereas the
plified ITS bands from the birch rust migrated faster two Melampsora species formed a distinct cluster sup-
than those from the alder rust (FIG. 5). All 14 sam- ported by a high bootstrap value. The same grouping
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

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 991

j Melampsoridium
28
land. Alder rust has also been recorded by several
~ Cro-um cornandrae
authors in Britain (see Wilson and Henderson 1966),
53 : ..__ Pucciniastrum goeppertianum
but its correct diagnosis has remained uncertain.
I r Cronartium flaccidum Henderson and Bennell (1979), Kaneko and Hiratsu-
85
[__ Chrysomyxa arctostaphy1i
ka ( 1981), and Roll-Hansen and Roll-Hansen ( 1981)
regarded British alder rust fungi as M. betulinum.
Pucdnia thlaspeos
The sequence analyses of 5.8S rDNA and ITS re-
L_

Melampsora medusae
gions confirmed that the taxonomic position of ge-
100 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
FIG. 6. Neighbor joining tree of 5.8S rDNA sequences (Cullings and Vogler 1998). As 5.8S rDNA and ITS
from several rust fungus species and Heterobasidion anna- sequences are not yet available for all rust genera, it
sum. The bootstrap values are given in branches. Bar is ca will be interesting to see if future studies shed further
1% difference in sequence.
light to the taxonomy of genus Melampsoridium.
In conclusion, we have shown in this study that
was also supported by the parsimony analysis (not Melampsoridium rust fungi on birch and alder rep-
shown). In these analyses Heterobasidion annosumwas resent two separate species in Finland and in Estonia.
used as an outgroup to identify the root of the rust The species on birch is M. betulinum, but the species
dendrogram. on alder is not M. alni, but M. hiratsukanum, as sug-
The ITS1 and ITS2 sequences of the two Melamp- gested also by P6ldmaa ( 1997) who reported obser-
soridium species were most similar to each other, and vations on a similar rust on A. incana in Estonia in
more similar to those of Cronartium, Pucciniastrum 1996. These studies represent the first reports of M.
and Chrysomyxa than to those of Puccinia and Me- hiratsukanum in Fennoscandia and Estonia.
lampsora, which were only distantly related and ho-
mologous nucleotides could not be identified. As the
alignments were uncertain between different genera, ACKNOWLEDGMENTS
ITS1 and ITS2 sequences were not subjected to a Ms. Laura Paavolainen is acknowledged for her critical com-
clustering analysis. ments. This work is part of the Finnish Biodiversity Re-
search Programme FIBRE (1997-2002).
DISCUSSION

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