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614
Mycologia,
96(3), 2004, pp. 614–626.
q2004 by The Mycological Society of America, Lawrence, KS 66044-8897
Tuberculina
: rust relatives attack rusts
1
Matthias Lutz
2
Robert Bauer
Dominik Begerow
Franz Oberwinkler
Universita¨t Tu¨bingen, Botanisches Institut, Lehrstuhl
Spezielle Botanik und Mykologie, Auf der Morgenstelle
1, 72076 Tu¨bingen, Germany
Dagmar Triebel
Botanische Staatssammlung Mu¨nchen, Menzinger
Straße 67, 80638 Mu¨nchen, Germany
Abstract:
Molecular sequence data together with ul-
trastructural features were used to infer the phylo-
genetic position of
Tuberculina
species. Additional ul-
trastructural characteristics were used to determine
their mode of nutrition. We investigated ultrastruc-
tural morphology of the type species
Tuberculina per-
sicina
and determined base sequences from the D1/
D2 region of the nuclear large-subunit ribosomal
DNA of the three commonly distinguished
Tubercu-
lina
species,
T. maxima, T. persicina
and
T. sbrozzii.
Analyses of sequence data by means of a Bayesian
method of phylogenetic inference using a Markov
Chain Monte Carlo technique reveal the basidiomy-
cetous nature of
Tuberculina.
Within the Uredini-
omycetes,
Tuberculina
clusters as a sister group of
Hel-
icobasidium,
closely related to the rusts (Uredinales).
This phylogenetic position is supported by the ure-
dinalean architecture of septal pores in
Tuberculina.
In addition, we present aspects of the ultrastructural
morphology of the cellular interaction of
Tuberculina
and rusts showing a unique interaction with large fu-
sion pores, revealing the mycoparasitic nature of
Tub-
erculina
on its close relatives, the rusts.
Key words:
cellular interaction, molecular phy-
logeny, mycoparasitism, nuc-LSU rDNA, septal pore
morphology, systematics, ultrastructure, Urediniomy-
cetes
INTRODUCTION
In 1817, a member of the genus
Tuberculina
Sacc.
first was described accurately by Ditmar (1817) as
Accepted for publication September 29, 2003.
1
Part 212 in the series
Studies in Heterobasidiomycetes
from the Bo-
tanical Institute, University of Tu¨ bingen.
2
Corresponding author. E-mail: matthias.lutz@uni-tuebingen.de
Tubercularia persicina.
His morphological character-
ization, with some additions, remains valid: Members
of the genus
Tuberculina
are characterized by the for-
mation of hemispherical lilac to violet sporodochia.
They consist of palisade-like arranged, short, mod-
erately thick conidiogenous cells, each of which bears
one globose, smooth conidium at the tip. The spo-
rodochia break through the surface of higher plants
and emit a powdery mass of conidia. Sometimes
spherical sclerotium-like structures are formed. In
addition,
Tuberculina
is known to exist only in asso-
ciation with rusts as first postulated by Saccardo
(1880) and later elaborated by Tubeuf (1901) and
others. Saccardo (1880) excluded
Tubercularia persi-
cina
Ditmar from the genus
Tubercularia
Tode:Fr.,
which should have pleurogenous conidiogenesis and
thread-like conidiogenous cells, and described the
new genus
Tuberculina
Sacc. for species with acroge-
nous conidiogenesis, short and broad conidiogenous
cells which are parasitic on the aecial stage of rust
fungi.
The three
Tuberculina
species,
T. maxima, T. per-
sicina
and
T. sbrozzii,
commonly are recognized (e.g.,
von Arx 1981, Ellis and Ellis 1988). They are distrib-
uted worldwide, living in association with more than
150 rust species from at least 15 genera. However, up
to 45 species were described with the authors follow-
ing strikingly different species concepts. Adopting a
concept based on morphological characters, plant
parasites (e.g.,
T. solanicola
Ellis parasitic on fruits of
Solanum melongena
L. [Ellis 1893]) and parasites of
non-rust fungi (e.g.,
T. ovalispora
Pat. parasitic on
Darluca filum
[Biv.] Castagne [Patouillard and Gail-
lard 1888]) were included in the genus. Other au-
thors used a species concept based on host specific-
ities, distinguishing
Tuberculina
species on different
rust hosts (Spegazzini 1880, 1884) or even plant hosts
(Gobi 1885).
After controversial discussions whether
Tuberculi-
na
-like fungi should be treated as smuts, rusts, asco-
mycetes or hymenomycetes, the genus presently is as-
signed mostly to the Fungi Imperfecti because no
stages of sexual reproduction are known.
Research on
Tuberculina
was motivated by two
main factors:
a
-taxonomy (e.g., Cooke 1888, Patouil-
lard and Gaillard 1888, Spegazzini 1880, 1884, 1911)
and the use of
Tuberculina
as a biological agent in
615L
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UBERCULINA
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UST RELATIVES ATTACK RUSTS
T
ABLE
I. List of studied species, reference material, host, and GenBank accession number.
Species Reference material
a
Host fungus/plant
GenBank acc.
no.
Helicobasidium longisporum
Wakef.
(syn.
H. compactum
Boedijn)
USA. (CBS 296.50)—culture
Coffea
sp. AY222046
Tuberculina maxima
Rostr. Canada, British Columbia, Wap Lake, 12.
9. 1965. (CBS 136.66)—culture
Cronartium ribicola
J. C. Fisch.
AY222044
Tuberculina persicina
(Ditmar) Sacc. Germany, Baden-Wu¨ rttemberg, Tu¨ bingen,
Hagelloch, 6. 11. 2000. (M. Lutz 799,
TUB 011529)
Puccinia silvatica
J.
Schro¨t.
AY222049
Tuberculina persicina
(Ditmar) Sacc. Germany, Baden-Wu¨ rttemberg, Nu¨ rtingen,
Raidwangen, 25. 4. 2001. (M. Lutz 851,
TUB 011530)
Tranzschelia pruni-
spinosae
(Pers.)
Dietel
AY222043
Tuberculina sbrozzii
Cavara & Sacc. England, Berkshire, East Burnham, 31. 3.
2000. (K (M): 76122)
Puccinia vincae
(DC.) Berk.
AY222045
Puccinia silvatica
J. Schro¨t. Germany, Baden-Wu¨ rttemberg, Tu¨ bingen,
Hagelloch, 13. 10. 2000. (M. Lutz 737,
TUB 011528)
Taraxacum officinale
agg. F. H. Wigg.
AY222048
Puccinia vincae
(DC.) Berk. Germany, Baden-Wu¨ rttemberg, Tu¨ bingen,
20. 9. 2002. (M. Lutz 1422, TUB
011527)
Vinca major
L. AY222047
a
Source acronyms: CBS—Centraalbureau voor Schimmelculures, AG Baarn, The Netherlands; K—Herbarium of the Royal
Botanic Gardens, Kew, England; TUB—Herbarium of the Spezielle Botanik/Mykologie, Eberhard-Karls-Universita¨t Tu¨ bingen,
Germany.
rust control (see review by Wicker 1981). As a result,
aspects of the biology, such as hibernation (Wicker
and Wells 1968), dispersal (Tubeuf 1901), conditions
for germination of conidia (Cornu 1883, Gobi 1885,
Lechmere 1914, Mielke 1933), mode and time of in-
fection (Weissenberg and Kurkela 1979, Wicker and
Kimmey 1967, Wicker and Wells 1970), host specific-
ities (Barkai-Golan 1959, Hubert 1935) or conditions
for artificial cultivation (Vladimirskaya 1939) were
clarified. However, fundamental questions concern-
ing the biology of the genus remain unanswered.
Thus, the relationship among plants, rusts and
Tub-
erculina
remains unresolved.
Tuberculina
species have
been interpreted as mycoparasites specific to rusts
(Tubeuf 1901, Zambettakis et al 1985), as nonspecific
parasites on several substrates (Petrak 1956, Schroe-
ter 1889) or even as specialized parasites on rust-in-
fected plant tissues (Hulea 1939, Wicker and Woo
1969, 1973). Also, the mode of nutrition and inter-
action, respectively, is unidentified. Finally, the evo-
lution and systematic position of the genus is totally
obscure, including questions on delimitation of spe-
cies and of the genus itself.
Ultrastructural characters of septal pore morphol-
ogy played an important role in the arrangement of
basidiomycetes (Bandoni 1984, Bauer et al 1997,
Bauer and Oberwinkler 1994, Ober winkler and
Bauer 1989, Wells 1994), and they correspond well
to phylogenetic hypotheses generated from molecu-
lar data (e.g., Bauer et al 2001, Swann et al 2001).
In this report, we present both molecular and ul-
trastructural data that reveal the basidiomycetous na-
ture of
Tuberculina
and show that it is related closely
to
Helicobasidium
Pat., therefore belonging to the
rust group. The actual mycoparasitic nature of the
genus is indicated on an ultrastructural level by a re-
markable cellular interaction between
Tuberculina
and rust hyphae.
MATERIALS AND METHODS
Materials.
—Specimens and the origins of the sequences
used in the molecular analyses are listed in T
ABLE
I. All
three commonly distinguished
Tuberculina
species,
T. max-
ima, T. persicina
and
T. sbrozzii,
and the rust hosts of the
respective
Tuberculina
specimens were included in the mo-
lecular analyses.
Molecular methods.
—We isolated genomic DNA from five
herbarium specimens and from two cultures on artificial
media (T
ABLE
I) of
Tuberculina, Puccinia
and
Helicobasi-
dium,
respectively. The fungal material was isolated from
the herbarium specimens by five times picking up spores
from the surfaces of either
Tuberculina
sporodochia or rust
sori with a fine needle and depositing the spores directly in
1.5 mL tubes. Dry spores were crushed at room tempera-
ture by shaking the samples 3 min at 30 Hz (Mixer Mill MM
300, Retsch, Haan, Germany) in the tubes together with
616 M
YCOLOGIA
one tungsten carbide ball (3 mm diam). To extract DNA,
we used the DNeasy Plant Mini Kit (Quiagen, Hilden, Ger-
many) following the manufacturer’s protocol.
To infer the phylogenetic position of
Tuberculina
within
the Basidiomycota, we amplified the 59- end (about 625 bp)
of the nuclear large-subunit ribosomal DNA (nuc-LSU
rDNA), comprising the domains D1 and D2 (Guadet et al
1989). Amplification was done by PCR (Mullis and Faloona
1987, Saiki et al 1988) using the primer pair NL1 and NL4
(O’Donnell 1992, 1993) or LR6 (Vilgalys and Hester 1990),
respectively. The selected DNA region is especially useful in
resolving relationships over a broad scale of organisms (Be-
gerow et al 1997, Fell et al 2000), and the D2 domain has
proven to have the lowest levels of homoplasy within the
LSU rDNA (Hopple and Vilgalys 1999). Amplification pa-
rameters were as described in Vogler and Bruns (1998), but
we adjusted the annealing temperature to 50 C and re-
duced the extension time of the last nine cycles to 2.5 min.
PCR products were purified with the QIAquickyKit (Qia-
gen, Hilden, Germany) followed by an ethanol precipita-
tion. Both strands of dsDNA were sequenced directly by
cycle sequencing (modified after Sanger et al 1977) with
NL1 and NL4-reverse as forward and NL4 and LR6 as re-
verse primers and the ABI PRISM Big DyeyTerminator
Cycle Sequencing Ready Reaction Kit (PE Applied Biosys-
tems, Warrington, England) according to the manufactur-
er’s protocol. Electrophoresis was performed on an auto-
mated sequencer (ABI 373A Stretch, PE Applied Biosys-
tems, Foster City, California). The sequences of both
strands were combined and proofread with the help of Se-
quenchery4.1 software (Gene Codes Corp., Ann Arbor,
Michigan). DNA sequences determined for this study were
deposited in GenBank. Accession numbers are given in T
A
-
BLE
I. To obtain a reliable hypothesis on the phylogenetic
position of the
Tuberculina
specimens that we sampled, we
also used sequences from GenBank, representing all groups
of Urediniomycetes (including the respective rust hosts of
the analysed
Tuberculina
specimens) as designated by
Swann et al (2001) and some representatives of Ustilagi-
nomycetes and Hymenomycetes (GenBank accession num-
bers are given in parentheses):
Agaricostilbum pulcherrimum
(AJ406402),
Agaricus arvensis
(U11910),
Auricularia auric-
ula-judae
(L20278),
Bensingtonia
sp. (AF444770),
Boletus
rubinellus
(L20279),
Calocera viscosa
(AF011569),
Chionos-
phaera apobasidialis
(AF393470),
Colacogloea peniophorae
(AF189898),
Cronartium ribicola
(AF426240),
Doassansia
epilobii
(AF007523),
Entyloma ficariae
(AY081013),
Eocron-
artium muscicola
(L20280),
Erythrobasidium hasegawianum
(AF189899),
Helicobasidium mompa
(L20281),
Helicogloea
variabilis
(L20282),
Herpobasidium filicinum
(AF426193),
Insolibasidium deformans
(AF522169),
Kondoa myxariophila
(AF189904),
Kriegeria eriophori
(syn.
Zymoxenogloea eriopho-
ri
) (L20288),
Kurtzmanomyces tardus
(AF393467),
Melamp-
sora lini
(L20283),
Microbotryum violaceum
(AF009866),
Mixia osmundae
(AB052840),
Naohidea sebacea
(AF522176),
Pachnocybe ferruginea
(L20284),
Sakaguchia dacryoidea
(AF444723),
Septobasidium carestianum
(L20289),
Sporobol-
omyces dracophylli
(AF189982),
Tranzschelia pruni-spinosae
(AF426224),
Tremella mesenterica
(AF011570),
Urocystis ra-
nunculi
(AF009879),
Ustilago hordei
(L20286),
Ustilentyloma
fluitans
(AF009882).
DNA sequences were aligned with the MEGALIGN mod-
ule of the LASERGENE package (DNASTAR Inc., Madison,
Wisconsin). Further manual alignment was done in Se-Al
version 2.0a10 (A. Rambaut, University of Oxford, Eng-
land). The final alignment (40 sequences; length: 550 bp;
after exclusion of the sites 40–55, 379–396, 404–424, 482–
497: 289 variable sites) and the tree obtained is deposited
in TreeBase (http://treebase.bio.buffalo.edu/treebase/)
with the study accession number S955. Sequence distances
were computed with the MEGALIGN module of the LAS-
ERGENE package. A Bayesian method of phylogenetic in-
ference using a Markov Chain Monte Carlo (MCMC) tech-
nique (Larget and Simon 1999, Mau et al 1999) as imple-
mented in the computer program MrBayes 3.064 (Huelsen-
beck and Ronquist 2001) was used to analyze the dataset.
This method allows estimating the probabilities (a poster-
iori probabilities) for groups of taxa to be monophyletic
given the DNA alignment. The power of this method re-
cently was demonstrated in computer simulation by Alfaro
et al (2003) and yielded good results in current molecular
studies on fungal systematics (e.g., Maier et al 2003). For
bayesian analysis, the data first were analyzed with Mr-
Modeltest 1.0b ( J.A.A. Nylander, Upsala University, Sweden,
Posada and Crandall 1998) to find the most appropriate
model of DNA substitution. Hierarchical likelihood ratio
tests and Akaike information criterion resulted in
GTR1I1G. Thus, four incrementally heated simultaneous
Markov chains were run over 2 000 000 generations using
the general time reversible model of DNA substitution with
gamma distributed substitution rates (Gu et al 1995, Rod-
riguez et al 1990) and estimation of invariant sites, random
starting trees and default starting parameters of the DNA
substitution model (Huelsenbeck and Ronquist 2001).
Trees were sampled every 100 generations, resulting in an
overall sampling of 20 000 trees. From these, the first 1000
trees were discarded (burn in 51000). The trees computed
after the process remained static (19 000 trees) were used
to compute a 50% majority rule consensus tree to obtain
estimates for the a posteriori probabilities of groups of spe-
cies. This Bayesian approach of phylogenetic analysis was
repeated 10 times to test the reproducibility of its results.
The unrooted phylograms from the MCMC analyses were
rooted with the species belonging to the Ustilaginomycetes
as outgroup species, because the trichotomy of the Basid-
iomycota had been demonstrated by several authors (Be-
gerow et al 1997, Berres et al 1995, Swann and Taylor 1993,
1995).
Light and electron microscopy.
—For light (LM) and trans-
mission electron microscopy (TEM),
Tuberculina persicina
on
Tranzschelia pruni-spinosae
was prepared in two different
ways. In one method, samples were fixed with 2% glutar-
aldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at
room temperature overnight. After six transfers in 0.1 M
sodium cacodylate buffer, samples were postfixed in 1% os-
mium tetroxide in the same buffer for 1 h in the dark,
washed in distilled water and stained in 1% aqueous uranyl
acetate for1hinthedark. After five washes in distilled
617L
UTZ ET AL
:
T
UBERCULINA
:R
UST RELATIVES ATTACK RUSTS
water, samples were dehydrated in acetone, with 10 min
changes at 25%, 50%, 70%, 95% and three times in 100%
acetone. Samples were embedded in Spurr’s plastic (Spurr
1969) and sectioned with a diamond knife. Semithin sec-
tions were stained with new fuchsin and crystal violet,
mounted in Entellan and examined by light microscopy.
Ultrathin serial sections were mounted on formvar-coated,
single-slot copper grids, stained with lead citrate at room
temperature for 5 min and washed with distilled water. They
were examined with a transmission electron microscope
(EM 109, Zeiss, Germany) operating at 80 kV.
In the second method, samples were prepared by high-
pressure freezing and freeze substitution. Infected areas of
leaves were removed with a 2 mm cork borer. To remove
air from intercellular spaces, samples were infiltrated with
distilled water containing 6% (v/v) (2.5 M) methanol for
approximately 5 min at room temperature. Single samples
were placed in an aluminum holder and frozen immediate-
ly in the high-pressure freezer HPM 010 (Balzers Union,
Liechtenstein) as described in detail by Mendgen et al
(1991). Substitution medium (1.5 ml per specimen) con-
sisted of 2% osmium tetroxide in acetone, which was dried
over calcium chloride. Freeze substitution was performed
at 290 C, 260 C and 230 C, 8 h for each step, with a
Balzer’s freeze substitution apparatus FSU 010. The tem-
perature was raised to approximately 0 C during a 30 min
period, and samples were washed in dry acetone another
30 min. Infiltration with an Epon/Araldite mixture (Welter
et al 1988) was performed stepwise: 30% resin in acetone
at 4 C for 7 h, 70% and 100% resin at 8 C for 20 h each
and 100% resin at 18 C for approximately 12 h. Samples
then were transferred to fresh medium and polymerized at
60 C for 10 h. Finally, samples were processed as described
above for chemically fixed samples, except that the ultra-
thin sections were additionally stained with 1% aqueous
uranyl acetate for 1 h.
RESULTS
Molecular analyses.
—Compared to sequence data
available via GenBank, all sequences obtained from
Tuberculina
specimens showed highest similarities to
the sequence of
Helicobasidium mompa
(GenBank ac-
cession number L20281) with a divergence from
4.2% (
T. maxima
) to 4.8% (both
T. persicina
speci-
mens). Compared to the sequence of
Helicobasidium
longisporum,
determined in this study, the divergence
ranged from 2.5% (
T. maxima
) to 3.1% (both
T. per-
sicina
specimens). Comparison within
Tuberculina
ranged from identity (the
T. persicina
specimens) to
0.6% divergence (
T. maxima
compared to both
T.
persicina
specimens).
The different runs of Bayesian phylogenetic anal-
ysis that were performed yielded consistent topolo-
gies. We present the consensus tree of one run to
illustrate the results (F
IG
. 1). The phylogenetic hy-
pothesis obtained by analyzing parts of the nuc-LSU
rDNA of an assortment of basidiomycetes together
with
Tuberculina maxima, T. persicina, T. sbrozzii,
and
their respective rust hosts revealed the expected tri-
chotomy of the sampled basidiomycetes with the
monophyla Ustilaginomycetes, Hymenomycetes and
Urediniomycetes. Within the Urediniomycetes, the
Microbotryum
group, rust group,
Agaricostilbum
group and
Erythrobasidium
group were supported
with a posteriori probabilities of 100%. Together with
Mixia osmundae
and
Helicogloea variabilis,
these
groups represent all major groups of Urediniomyce-
tes (after Swann et al 2001). The phylogenetic rela-
tionships among these groups were not resolved.
All specimens of
Tuberculina
clustered together (a
posteriori probability of 100%) representing the sis-
ter taxon (a posteriori probability of 98%) of
Heli-
cobasidium
(a posteriori probability of 100%) and
consequently being a member of the Urediniomyce-
tidae. The relationship of the
Tuberculina
-
Helicobasi-
dium
cluster to the rusts, to
Pachnocybe ferruginea,
to
Septobasidium carestianum,
and to the sampled Pla-
tygloeales sensu stricto (
Insolibasidium deformans,
Herpobasidium filicinum, Eocronartium muscicola
; af-
ter Swann et al 2001) was not resolved. Within
Tub-
erculina, T. maxima
appeared basal, in opposition to
the sister taxa (a posteriori probability of 100%)
T.
sbrozzii
and the cluster of
T. persicina
(a posteriori
probability of 99%).
Septal pore architecture of
Tuberculina persicina
and
Tranzschelia pruni-spinosae.—Septal wall morpholo-
gy and septal pore architecture in
Tuberculina persi-
cina
essentially was identical to that of
Tranzschelia
pruni-spinosae.
In both species, the septa had a trila-
mellate nature and the simple pores were surround-
ed by microbodies in a more or less circular arrange-
ment (F
IGS
. 10–11). Mature pores in both species
were plugged by osmiophilic material. Usually an or-
ganelle-free zone surrounding the septal pores at
both sides was more distinct in
Tranzschelia pruni-
spinosae
than in
Tuberculina persicina
(cf. F
IG
.11and
F
IG
. 10). In addition, sometimes the pore lips in
Tub-
erculina persicina,
but not in
Tranzschelia pruni-spi-
nosae,
were slightly swollen and more or less abruptly
flattened toward the margin.
Association of
Tuberculina persicina
with
Tranzschelia
pruni-spinosae
on
Anemone ranunculoides.—
Tuber-
culina persicina
strictly overgrew aecia of
Tranzschelia
pruni-spinosae
in different developmental stages and
sporulated on the upper surface of the aecia (F
IGS
.
2–3). During differentiation of the sporodochia, the
epidermis of the leaves ruptured and the conidial
mass of
Tuberculina
was exposed (F
IGS
. 2–3). Within
the leaf tissue, hyphae of
Tuberculina
and those of
Tranzschelia
were mixed (F
IGS
. 4–5). Hyphae of both
Tuberculina
and
Tranzschelia
were without clamps but
618 M
YCOLOGIA
F
IG
. 1. Bayesian inference of phylogenetic relationships within selected basidiomycetous species. Markov Chain Monte
Carlo analysis of an alignment of nuc-LSU rDNA sequences from the D1/D2 region using the general time reversible model
of DNA substitution with gamma distributed substitution rates and estimation of invariant sites, random starting trees and
default starting parameters of the substitution model. Majority-rule consensus tree from 19 000 trees that were sampled after
the process remained static. The topology was rooted with the species belonging to the Ustilaginomycetes. Numbers on
branches are estimates for a posteriori probabilities. Branch lengths are mean values over the sampled trees. They are scaled
in terms of expected numbers of nucleotide substitutions per site.
619L
UTZ ET AL
:
T
UBERCULINA
:R
UST RELATIVES ATTACK RUSTS
F
IGS
. 2–3. Cross section through aecium of
Tranzschelia pruni-spinosae
infected with
Tuberculina persicina.
Samples were
prepared by chemical fixation (2) or high pressure freezing and freeze substitution (3) and observed with a light microscope.
2. Sporulation of
Tuberculina persicina
(arrow) at the top of a young aecium of
Tranzschelia pruni-spinosae.
Note that the
lower epidermis (arrowheads) of
Anemone ranunculoides
becomes ruptured. Scale bar 5100 mm. 3. Sporulation of
Tuber-
culina persicina
(arrow) within the peridium of a mature aecium of
Tranzschelia pruni-spinosae.
Scale bar 5100 mm.
could be distinguished from each other by the num-
ber of nuclei per hyphal cell, the diameter of the
nuclei and the thickness of the cell walls (F
IGS
. 4–5).
Hyphae of
Tuberculina
generally were multinucleate,
whereas those of
Tranzschelia
usually were mononu-
cleate (binucleate hyphal rust cells occurred only at
the base of the aecia). Diameter of the nuclei and
thickness of the cell walls of the rust were roughly
twice as large (or more) compared to those of
Tub-
erculina
(F
IGS
. 4–5). Interaction stages between
Tub-
erculina persicina
and
Tranzschelia pruni-spinosae
fre-
quently were found in the leaves in neighboring ar-
eas of the aecia, especially at the base of the aecia.
In these interaction stages, the protoplasts of both,
the
Tuberculina
and the rust hyphal cell, were fused
via a large pore, measuring 0.5–1
m
m diam (F
IGS
.6–
9). By both fixation techniques, the general fusion
pore architecture was recognizable. In high-pressure
frozen samples, however, fusion pore morphology
had a more regular appearance and was more dis-
tinct than after conventional fixation (cf. F
IGS
. 8–9
and F
IGS
. 6–7). Thus, in high-pressure frozen inter-
action stages the plasma membranes of the
Tubercu-
lina
and the rust cell closely followed the contour of
the respective cell wall (F
IGS
. 8–9), whereas in con-
ventionally fixed interaction stages, plasma mem-
branes often were folded irregularly (F
IGS
. 6–7). For
all interaction stages, prepared by high-pressure
freezing and freeze substitution, it clearly was evident
that the membrane of the fusion pore was continu-
ous with the plasma membranes of both the
Tuber-
culina
and the rust cells (F
IG
. 9).
DISCUSSION
Phylogenetic position of
Tuberculina.—Because no
stages and structures of sexual reproduction are
known in
Tuberculina,
other features were used to
determine the phylogenetic position of the genus.
Conflicting classifications were proposed based on
different features. Ditmar (1817) assigned the fungus
that he described to
Tubercularia
Tode : Fr. Saccardo
(1880) confined
Tubercularia
to anamorphs of the
genus
Nectria
(Fr.) Fr. (which is the current concept,
see also Rossman 2000) and
Tuberculina
to ana-
morphs of rust parasites. Few subsequent researchers
regarded
Tuberculina
as anamorphic ascomycetes
(e.g., Frank 1880, Kirk et al 2001). Tulasne (1854)
and Lutrell (1979) even proposed ascomycetous te-
leomorphs (
Sphaeria loepophaga
Tul. and
Anhellia
Ra-
620 M
YCOLOGIA
F
IGS
. 4–5. Cross section through aecium of
Tranzschelia pruni-spinosae
infected with
Tuberculina persicina.
Samples were
prepared by chemical fixation (4) or high pressure freezing and freeze substitution (5) and observed with a transmission
electron microscope. 4. Section through hyphae of
Tranzschelia pruni-spinosae
(R) and
Tuberculina persicina
(t) illustrated
to show the different sizes of the nuclei and cell walls of the two fungi. Scale bar 53mm. 5. Hypha of
Tuberculina persicina
(t) surrounded by hyphae of
Tranzschelia pruni-spinosae
(R). Note that the diameter of the rust nucleus and the thickness
of the rust cell walls are more than twice as large compared with those of
Tuberculina.
Scale bar 52mm.
cib., respectively). Location and mode of sporulation
as well as the morphology of hyphae inspired some
workers to treat
Tuberculina
species as rusts and to
create new species (Corda 1842, Desmazie`res 1847,
Spegazzini 1880) and genera (Mayr 1890). Other re-
searchers even considered
Tuberculina
as a stage of
asexual rust reproduction (Cunningham 1889;
Rav-
enelia sessilis
Berk., Griffiths 1902;
Gymnoconia riddel-
liae
Griffiths, Plowright 1885;
Puccinia vincae,
Spe-
gazzini 1888; ‘‘
Tuberculina paraguayensis
Speg.’’, Vuil-
lemin 1892a;
Aecidiconium barteti
Vuill., 1892b;
En-
dophyllum sempervivi
[Alb. & Schwein.] de Bary).
Gobi (1885) investigated morphology, sporogenesis,
and dispersal and germination of spores and as-
signed the genus to the smuts. His point of view was
followed by the majority of researchers of that time
(e.g., Plowright 1889, Schroeter 1889, Wildeman
1908). Although considering the same features, Mor-
ini (1886) assigned
Tuberculina
to the Tremellineae.
Buddin et al (1927) were the first to observe that
Helicobasidium
produces ‘‘small raised tubercles,
which eventually become pustules of conidia of the
type which is characteristic of the genus
Tuberculi-
na.
’’ For that reason,
Tuberculina
was assigned to
Hel-
icobasidium
by some researchers (von Arx 1981, Car-
michael et al 1980, Kendrick and Watling 1979).
However, the obvious lack of striking features for phy-
logenetic placement of the genus has prompted most
recent researchers (e.g., Hawksworth et al 1995, Sun-
dheim 1986, Wicker 1981) to follow Fuckel (1870)
in treating
Tuberculina
as Fungi Imperfecti.
Our phylogenetic analyses of nuc-LSU rDNA se-
quences, however, demonstrate clearly that
Tubercu-
lina
species are members of the basidiomycetes, po-
sitioned within the rust group as sister to
Helicobasi-
dium.
This phylogenetic hypothesis agrees well with
ultrastructural data. As shown in this study, the trila-
mellate nature of the septa, in which a thin electron-
transparent middle lamella is sandwiched between
thick electron-opaque layers, indicates that
Tubercu-
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UTZ ET AL
:
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UST RELATIVES ATTACK RUSTS
F
IGS
. 6–9. Cellular interaction with large fusion pores between
Tuberculina persicina
(t) and
Tranzschelia pruni-spinosae
(R). Samples were prepared by chemical fixation (6–7) or high pressure freezing and freeze substitution (8–9) and obser ved
with a transmission electron microscope. 6. Interaction stage in overview with two medianly sectioned nuclei of
Tuberculina
persicina
(n) and one medianly sectioned nucleus of
Tranzschelia pruni-spinosae
(N). Note the different sizes of the nuclei.
The fusion pore is visible at arrow. Scale bar 52mm. 7. Detail from F
IG
. 6 illustrating the large fusion pore (arrow). Scale
bar 50.3 mm. 8. High-pressure frozen interaction stage in overview. Fusion pore is visible at arrow. Note that the pore
morphology is more distinct than after conventional fixation (compare with 6). Scale bar 52mm. 9. Detail from F
IG
.8
showing the fusion pore (arrow) and that the plasma membrane of both partners is continuous through the fusion pore
(arrowheads). Scale bar 50.2 mm.
622 M
YCOLOGIA
F
IGS
. 10–11. Septal pore apparatus of
Tuberculina persicina
(10) and
Tranzschelia pruni-spinosae
(11). Samples were
prepared by high-pressure freezing and freeze substitution and observed with a transmission electron microscope. Each pore
shows a non-swollen pore margin and associated microbodies (arrowheads) in a more or less circular arrangement. Scale
bars 50.3 mm.
lina
is basidiomycetous (Kreger-van Rij and Veenhuis
1971). In addition, the septal pore apparatus in
Tub-
erculina persicina
essentially is identical to that of its
host fungus
Tranzschelia pruni-spinosae.
In both spe-
cies, it is composed of a simple pore surrounded by
microbodies in a more or less circular arrangement.
This type of septal pore apparatus is common among
the members of the rust group (see Bauer 1987,
Bauer and Oberwinkler 1994, Boehm and Mc-
Laughlin 1989, Khan and Kimbrough 1982, Little-
field and Heath 1979) and occurs also in
Helicobasi-
dium
(Bourett and McLaughlin 1986). In addition,
both
Tuberculina
and the members of the rust group
have clampless hyphae.
The close phylogenetic proximity of
Tuberculina
and
Helicobasidium
raises questions on the relation
of the genera, especially since we know that cultures
of
Helicobasidium
on artificial media produce
Tuber-
culina
-like conidia. That observation was repeated
several times (Arai et al 1987, Buddin and Wakefield
1927, 1929, Fukushima 1998, Sayama et al 1994, Vald-
er 1958) but without definitive conclusions or further
investigations. However,
Tuberculina
is reported to be
the anamorphic stage of
Helicobasidium,
justified by
the quoted observations in several compendia (Car-
michael et al 1980, Hawksworth et al 1995). This is
in contrast to our molecular analyses, in which all
three commonly distinguished
Tuberculina
species
are included, as well as two of probably three distin-
guishable
Helicobasidium
species (see Reid 1975,
Roberts 1999).
Tuberculina
is separated from
Helico-
basidium,
and there is no record for conidia forma-
tion by
Helicobasidium
in nature, apart from one re-
port (Patouillard 1886), which could not be con-
firmed by subsequent researchers (Buddin and
Wakefield 1927).
Association between
Tuberculina
and rusts.
—Tulasne
(1854) interpreted the exclusive occurrence of
Tub-
erculina
in association with rusts as argument for the
mycoparasitic nature of the genus. His reasoning was
followed by most researchers (Buchenauer 1982, Ki-
rulis 1940, Lindau 1910, Tubeuf 1901, Zambettakis et
al 1985), adding as arguments the heavy impairment
of rust spore production in the presence of
Tuber-
culina
(Spaulding 1929, Tubeuf 1917), infection ex-
periments showing that rust-free plants could not be
infected by
Tuberculina
(Barkai-Golan 1959) and pre-
sumable structures of parasitic interaction (Gruyer
1921, Sappin-Trouffy 1896, Thirumalachar 1941).
Disagreeing with that, Marchal (1902) was the first to
propose a commensal relationship.
Tuberculina
was
interpreted as saprophyte living in rust-damaged
plant tissues. This point of view was encouraged by
the presumable occurrence of
Tuberculina
in rust-
free plant tissues in nature (Gobi 1885) and in arti-
ficial culture (Wicker and Woo 1969, 1973), experi-
ments of dual cultures of
Tuberculina
and rusts where
no interaction could be recognized (Wicker 1979),
and investigations by light microscopy, where no in-
623L
UTZ ET AL
:
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UBERCULINA
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UST RELATIVES ATTACK RUSTS
teraction of rust and
Tuberculina
was found
(D’Oliveira 1941, Hulea 1939) except the digestion
of plant cells (Wicker 1979).
Our ultrastructural observations demonstrate a
specific and morphologically uncommon interaction
between
Tuberculina persicina
and
Tranzschelia pruni-
spinosae.
Interaction results in a large fusion pore be-
tween
Tuberculina
and its host rust with a direct cy-
toplasm-cytoplasm connection. The plasma mem-
branes of
Tuberculina
and its host form a continuum.
Existence of such an unusual structure of interaction
indicates the mycoparasitic nature of
Tuberculina.
Moreover, our ultrastructural observations confirm
those authors who assumed that
Tuberculina
is re-
stricted to the haploid rust stage and the following
stage on the same host plant (e.g., D’Oliveira 1941,
Lechmere 1914). In addition, the mycoparasitic na-
ture and the distinctive cellular interaction of
Tuber-
culina
provide a good taxonomic boundary for the
genus
Tuberculina.
It definitely should be restricted
to rust parasites.
ACKNOWLEDGMENTS
We thank H. Schwarz for patient performance of high-pres-
sure freezing and freeze substitution, W. Maier and C. Lutz
for critical comments on the manuscript, the anonymous
reviewers for their helpful comments, the Royal Botanic
Gardens, Kew, for the loan of specimens, and the Deutsche
Forschungsgemeinschaft for financial support.
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